Anti-CLDN18.2 antibody and uses thereof

Abstract

Provided are an anti-CLDN18.2 antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the same, an immunoconjugate, bispecific molecule, chimeric antigen receptor and pharmaceutical composition comprising the same. The antibody or antigen-binding fragment thereof is used for preventing and / or treating a tumor. Fig. 1A Fig 1A

Description

Anti-CLDN18.2 antibody and its uses Technical Field

[0001] This invention relates to the fields of disease treatment and immunology, and more specifically, to antibodies against CLDN18.2 or their antigen-binding fragments, nucleic acid molecules encoding them, immunoconjugates containing them, bispecific molecules, chimeric antigen receptors and pharmaceutical compositions, and their use for the prevention and / or treatment of tumors. Background Technology

[0002] Stomach cancer is one of the most common malignant tumors worldwide, with a low 5-year survival rate, ranging from 20% to 40% in most countries. Approximately 700,000 people die from this disease each year. In China, the survival rate for stomach cancer is 35.9%. Statistics show that in 2015, the number of new cases of stomach cancer in China reached 679.1 per 100,000, and the number of deaths was as high as 498.0 per 100,000, making it the second leading cause of cancer incidence and mortality after lung cancer. Stomach cancer is highly malignant, and due to the lack of widespread stomach cancer screening, patients are often diagnosed at an advanced stage, missing the opportunity for radical surgical treatment and only able to receive chemotherapy as the primary treatment.

[0003] With the development of tumor molecular biology, the efficacy of targeted drugs and immunotherapy in hematologic malignancies, breast cancer, and colorectal cancer has been confirmed, but their application in gastric cancer treatment has been relatively slow. Currently, internationally approved targets for gastric cancer targeted therapy include human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF), while the approved target for gastric cancer immunotherapy is programmed lethal protein 1 (PD-1). In September 2017, based on the large-scale phase III clinical trial ATTRACTION-2, the Japanese Ministry of Health, Labour and Welfare approved the PD-1 antibody Opdivo for the treatment of chemotherapy-resistant advanced gastric cancer patients: compared with placebo, Opdivo reduced mortality by 37%, with an efficacy rate of 11.2%. Based on the phase II clinical trial Keynote 059, the US FDA granted accelerated approval to the PD-1 antibody Keytruda for the treatment of chemotherapy-resistant PD-L1-positive advanced gastric cancer patients: with an efficacy rate of 15.5%. In terms of targeted therapy, vascular endothelial growth factor (VEGF) not only promotes tumor angiogenesis but also binds to receptors on the surface of tumor cells, activating downstream signaling pathways and directly participating in the formation, development, and migration of tumor stem cells. VEGFR2 inhibitors apatinib and ramucirumab have both been clinically proven to be safe and effective as second-line and later-line treatments for advanced gastric cancer. Approximately 6%-35% of new gastric cancer cases each year show HER2 gene amplification or protein overexpression, making HER2 a crucial target in anticancer therapy. The TOGA Phase III clinical trial was the first to demonstrate the advantages of trastuzumab in first-line treatment of HER2(+) advanced gastric cancer. The overall survival (OS) in the trastuzumab plus chemotherapy group was significantly longer than that in the chemotherapy-only group (13.8 months vs. 11 months), and secondary endpoints including PFS, ORR (objective response rate), and TTP (time to disease progression) also showed significant improvements. However, the proportion of HER2-positive patients in my country is only about 10%, making the exploration of new therapeutic targets for gastric cancer imperative.

[0004] Claudin is a transmembrane protein complex located in the tight junctions between epithelial and endothelial cells, situated on the apical side of the intercellular space. Its distribution exhibits tissue- and organ-specific characteristics, and its main functions include cell adhesion, maintaining cell polarity, regulating paracellular permeability, and participating in the regulation of cell proliferation and differentiation. Claudin 18 is a four-transmembrane protein with four transmembrane hydrophobic regions and two extracellular loops, existing as two different splice variants, CLDN18.1 and CLDN18.2. CLDN18.1 and CLDN18.2 differ in sequence at the intracellular N-terminus and the first extracellular loop, while the primary sequences of the other parts are identical. Tissue distribution studies of CLDN18 show that CLDN18.1 is selectively expressed only in lung cells, while CLDN18.2 is expressed only in gastric cells, and CLDN18.2 expression in normal stomach cells is limited to differentiated, short-lived gastric epithelial cells. CLDN18.2 is frequently retained during the malignant transformation of gastric cells, and is therefore frequently displayed on the surface of human gastric cancer cells. 60-80% of gastrointestinal adenomas show CLDN18.2 positivity (Clinical Cancer Research 2008, 14(23):7624–34). In addition, CLDN18.2 is highly expressed in pancreatic duct cancer and metastatic pancreatic cancer, with a positive rate as high as 60-70%, and can be used as a diagnostic marker and therapeutic target for pancreatic duct / pancreatic cancer (Journal of Clinical Pathology 2012, 65:431-436; World Journal of Gastroenterology 2014, 20(31):10813-10824; International Journal of Cancer 2014, 134:731–739). There is no systematic study on the ectopic activation of CLDN18.2 in other tumors. The literature available so far shows that, in addition to the ectopic activation in pancreatic cancer mentioned above, this protein has also been found to be expressed in esophageal cancer, bronchial cancer, non-small cell lung cancer (NSCLC), breast cancer, ENT tumors, ovarian cancer, colon cancer, liver cancer and their metastatic cancers, especially gastric cancer metastases such as Krukenberg tumors, peritoneal metastases and lymph node metastases (Clinical Cancer Research 2008, 14(23):7624–34; International Journal of Cancer 2014, 134:731–739; Cancer Letters 2017, 403:66-73; International Journal of Cancer 2014, 135(9):2206-2214).CLDN18.2 is a tumor target with both preventative and therapeutic value. Its differential expression between cancer cells and normal cells, its membrane localization, its absence in most toxic normal tissues, and its limited expression in the stomach to differentiated gastric cells that can be supplemented by target-negative stem cells (or positionally inaccessible stem cells) make CLDN18.2 an attractive target for cancer immunotherapy. Therefore, developing anti-CLDN18.2 antibodies with higher specificity, lower toxicity, and better clinical efficacy is urgent and necessary, providing cancer patients with more treatment options.

[0005] Summary of the Invention

[0006] The antibody of the present invention specifically recognizes / binds to human CLDN18.2 and can induce the killing of CLDN18.2-expressing cells (e.g., tumor cells) via ADCC and / or CDC, exhibiting superior functional properties compared to known anti-CLDN18.2 antibodies. Therefore, the antibody of the present invention has the potential for the prevention and / or treatment of tumors and possesses significant clinical value.

[0007] The antibody of the present invention

[0008] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0009] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0010] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:75, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to SEQ ID NO:75.

[0011] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:76, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to SEQ ID NO:76, and

[0012] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:77, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to SEQ ID NO:77;

[0013] and / or

[0014] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0015] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:78, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to SEQ ID NO:78.

[0016] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:79, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to SEQ ID NO:79, and

[0017] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:80, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to SEQ ID NO:80.

[0018] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation;

[0019] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:75, VH CDR2 as shown in SEQ ID NO:76, and VH CDR3 as shown in SEQ ID NO:77; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:78 or 96, VL CDR2 as shown in SEQ ID NO:79, and VL CDR3 as shown in SEQ ID NO:80.

[0020] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

[0021] (a) The heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:73; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:74; or

[0022] (b) The heavy chain variable region contains the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:91; and the light chain variable region contains the three CDRs contained in the light chain variable region shown in SEQ ID NO:92.

[0023] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0024] In some preferred embodiments, the antibody or its antigen-binding fragment is 44F7 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0025] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0026] (a) Human CLDN18.2 was bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml or less);

[0027] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0028] (c) Not bound to human CLDN18.1;

[0029] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0030] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0031] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0032] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0033] (1) VH CDR1 as shown in SEQ ID NO:75, VH CDR2 as shown in SEQ ID NO:76, VH CDR3 as shown in SEQ ID NO:77; VL CDR1 as shown in SEQ ID NO:78 or 96, VL CDR2 as shown in SEQ ID NO:79, VL CDR3 as shown in SEQ ID NO:80; or

[0034] (2)(a) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:73; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:74; or (b) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:91; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:92.

[0035] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0036] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0037] (i) The sequence shown in SEQ ID NO:73;

[0038] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:73; or

[0039] (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 with the sequence shown in SEQ ID NO:73;

[0040] And / or,

[0041] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0042] (iv) The sequence shown in SEQ ID NO:74;

[0043] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:74; or

[0044] (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 with the sequence shown in SEQ ID NO:74.

[0045] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0046] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:73 and VL having the sequence shown in SEQ ID NO:74.

[0047] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0048] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0049] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0050] (a) The heavy chain framework region of human immunoglobulin or a variant thereof, said variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to the sequence from which it is derived; and / or

[0051] (b) The light chain framework region of human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to the sequence from which it is derived.

[0052] In some preferred embodiments, the antibody or its antigen-binding fragment comprises a framework region of a human immunoglobulin, such as a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In some preferred embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in the amino acid sequence encoded by a human light chain germline gene.

[0053] In such embodiments, the structural regions (heavy chain and / or light chain structural regions) of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues. In some preferred embodiments, the structural regions (heavy chain and / or light chain structural regions) contain one or more amino acid residues that have been reverted to the corresponding murine residues or conserved amino acid substitutions of the corresponding murine residues (such mutations are called back mutations).

[0054] Therefore, in some preferred embodiments, the antibody or its antigen-binding fragment comprises a framework region of a human immunoglobulin (e.g., a framework region contained in the amino acid sequence encoded by a human germline antibody gene), the framework region optionally comprising one or more reversion mutations from human residues to mouse residues.

[0055] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0056] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0057] (i) The sequence shown in SEQ ID NO:91;

[0058] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 91; or

[0059] (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 with the sequence shown in SEQ ID NO:91;

[0060] And / or,

[0061] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0062] (iv) The sequence shown in SEQ ID NO:92;

[0063] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:92; or

[0064] (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 with the sequence shown in SEQ ID NO:92.

[0065] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0066] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:91 and VL having the sequence shown in SEQ ID NO:92.

[0067] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0068] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0069] (i) The sequence shown in SEQ ID NO:99;

[0070] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 99; or

[0071] (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 with the sequence shown in SEQ ID NO:99;

[0072] And / or,

[0073] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0074] (iv) The sequence shown in SEQ ID NO:100;

[0075] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 100; or

[0076] (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 with the sequence shown in SEQ ID NO:100.

[0077] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0078] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:99 and VL having the sequence shown in SEQ ID NO:100.

[0079] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0080] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0081] (i) The sequence shown in SEQ ID NO:101;

[0082] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 101; or

[0083] (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 with the sequence shown in SEQ ID NO:101;

[0084] And / or,

[0085] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0086] (iv) The sequence shown in SEQ ID NO:102;

[0087] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 102; or

[0088] (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 with the sequence shown in SEQ ID NO:102.

[0089] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0090] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:101 and VL having the sequence shown in SEQ ID NO:102.

[0091] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0092] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0093] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0094] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0095] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0096] and / or

[0097] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0098] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0099] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0100] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0101] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0102] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, and VH CDR3 as shown in SEQ ID NO:5; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, and VL CDR3 as shown in SEQ ID NO:8.

[0103] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:1; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:2.

[0104] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0105] In some preferred embodiments, the antibody or its antigen-binding fragment is 1D10 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0106] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0107] (a) Human CLDN18.2 is bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml, 0.02 μg / ml or less);

[0108] (b) Combined with mouse CLDN18.2 at EC50 of 1 μg / ml or less;

[0109] (c) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0110] (d) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0111] (e) Mediating the internalization of CLDN18.2 into cells (e.g., tumor cells), for example, at an internalization level of at least 10% (e.g., at least 15%, at least 20% or more) as measured by FACS or flow cytometry; said cells express CLDN18.2 on their surface;

[0112] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0113] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0114] (1) VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, VH CDR3 as shown in SEQ ID NO:5; VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, VL CDR3 as shown in SEQ ID NO:8; or

[0115] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:1; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:2.

[0116] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0117] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0118] (i) The sequence shown in SEQ ID NO:1;

[0119] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:1; or

[0120] (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 with the sequence shown in SEQ ID NO:1;

[0121] And / or,

[0122] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0123] (iv) The sequence shown in SEQ ID NO:2;

[0124] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:2; or

[0125] (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 with the sequence shown in SEQ ID NO:2.

[0126] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0127] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:2.

[0128] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0129] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0130] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0131] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0132] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0133] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0134] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0135] and / or

[0136] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0137] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0138] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:15, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0139] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0140] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0141] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:11, VH CDR2 as shown in SEQ ID NO:12, and VH CDR3 as shown in SEQ ID NO:13; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:14, VL CDR2 as shown in SEQ ID NO:15, and VL CDR3 as shown in SEQ ID NO:16.

[0142] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:9; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:10.

[0143] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0144] In some preferred embodiments, the antibody or its antigen-binding fragment is 2F12 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0145] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0146] (a) Human CLDN18.2 was bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml or less);

[0147] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0148] (c) Not bound to human CLDN18.1;

[0149] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0150] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0151] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0152] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0153] (1) VH CDR1 as shown in SEQ ID NO:11, VH CDR2 as shown in SEQ ID NO:12, VH CDR3 as shown in SEQ ID NO:13; VL CDR1 as shown in SEQ ID NO:14, VL CDR2 as shown in SEQ ID NO:15, VL CDR3 as shown in SEQ ID NO:16; or

[0154] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:9; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:10.

[0155] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0156] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0157] (i) The sequence shown in SEQ ID NO:9;

[0158] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:9; or

[0159] (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 with the sequence shown in SEQ ID NO:9;

[0160] And / or,

[0161] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0162] (iv) The sequence shown in SEQ ID NO:10;

[0163] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:10; or

[0164] (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 with the sequence shown in SEQ ID NO:10.

[0165] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0166] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:9 and VL having the sequence shown in SEQ ID NO:10.

[0167] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0168] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0169] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0170] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0171] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0172] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:20, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0173] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0174] and / or

[0175] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0176] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0177] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:23, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0178] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0179] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0180] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:19, VH CDR2 as shown in SEQ ID NO:20, and VH CDR3 as shown in SEQ ID NO:21; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:22, VL CDR2 as shown in SEQ ID NO:23, and VL CDR3 as shown in SEQ ID NO:24.

[0181] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:17; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:18.

[0182] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0183] In some preferred embodiments, the antibody or its antigen-binding fragment is 3F2 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0184] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0185] (a) Human CLDN18.2 was bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml or less);

[0186] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0187] (c) Not bound to human CLDN18.1;

[0188] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0189] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0190] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0191] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0192] (1) VH CDR1 as shown in SEQ ID NO:19, VH CDR2 as shown in SEQ ID NO:20, VH CDR3 as shown in SEQ ID NO:21; VL CDR1 as shown in SEQ ID NO:22, VL CDR2 as shown in SEQ ID NO:23, VL CDR3 as shown in SEQ ID NO:24; or

[0193] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:17; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:18.

[0194] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0195] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0196] (i) The sequence shown in SEQ ID NO:17;

[0197] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:17; or

[0198] (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 with the sequence shown in SEQ ID NO:17;

[0199] And / or,

[0200] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0201] (iv) The sequence shown in SEQ ID NO:18;

[0202] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:18; or

[0203] (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 with the sequence shown in SEQ ID NO:18.

[0204] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0205] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:17 and VL having the sequence shown in SEQ ID NO:18.

[0206] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0207] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0208] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0209] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0210] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:27, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0211] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:28, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0212] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0213] and / or

[0214] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0215] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:30, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0216] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:31, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0217] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0218] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0219] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:27, VH CDR2 as shown in SEQ ID NO:28, and VH CDR3 as shown in SEQ ID NO:29; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:30, VL CDR2 as shown in SEQ ID NO:31, and VL CDR3 as shown in SEQ ID NO:32.

[0220] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:25; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:26.

[0221] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0222] In some preferred embodiments, the antibody or its antigen-binding fragment is 5F9 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0223] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0224] (a) Bind human CLDN18.2 at EC50 of 0.5 μg / ml or less;

[0225] (b) Combined with mouse CLDN18.2 at EC50 of 0.2 μg / ml or less;

[0226] (c) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0227] (d) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0228] (e) Mediating the internalization of CLDN18.2 into cells (e.g., tumor cells), for example, at an internalization level of at least 10% (e.g., at least 15%, at least 20% or more) as measured by FACS or flow cytometry; said cells express CLDN18.2 on their surface;

[0229] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0230] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0231] (1) VH CDR1 as shown in SEQ ID NO:27, VH CDR2 as shown in SEQ ID NO:28, VH CDR3 as shown in SEQ ID NO:29; VL CDR1 as shown in SEQ ID NO:30, VL CDR2 as shown in SEQ ID NO:31, VL CDR3 as shown in SEQ ID NO:32; or

[0232] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:25; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:26.

[0233] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0234] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0235] (i) The sequence shown in SEQ ID NO:25;

[0236] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:25; or

[0237] (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 with the sequence shown in SEQ ID NO:25;

[0238] And / or,

[0239] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0240] (iv) The sequence shown in SEQ ID NO:26;

[0241] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:26; or

[0242] (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 with the sequence shown in SEQ ID NO:26.

[0243] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0244] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:25 and VL having the sequence shown in SEQ ID NO:26.

[0245] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0246] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0247] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0248] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0249] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:35, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0250] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:36, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0251] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:37, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0252] and / or

[0253] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0254] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:38, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0255] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:39, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0256] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:40, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0257] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0258] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:35, VH CDR2 as shown in SEQ ID NO:36, and VH CDR3 as shown in SEQ ID NO:37; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:38, VL CDR2 as shown in SEQ ID NO:39, and VL CDR3 as shown in SEQ ID NO:40.

[0259] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:33; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:34.

[0260] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0261] In some preferred embodiments, the antibody or its antigen-binding fragment is 9F3 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0262] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0263] (a) Bind human CLDN18.2 at EC50 of 0.2 μg / ml or less;

[0264] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0265] (c) Not bound to human CLDN18.1;

[0266] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0267] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0268] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0269] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0270] (1) VH CDR1 as shown in SEQ ID NO:35, VH CDR2 as shown in SEQ ID NO:36, VH CDR3 as shown in SEQ ID NO:37; VL CDR1 as shown in SEQ ID NO:38, VL CDR2 as shown in SEQ ID NO:39, VL CDR3 as shown in SEQ ID NO:40; or

[0271] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:33; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:34.

[0272] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0273] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0274] (i) The sequence shown in SEQ ID NO:33;

[0275] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:33; or

[0276] (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 with the sequence shown in SEQ ID NO:33;

[0277] And / or,

[0278] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0279] (iv) The sequence shown in SEQ ID NO:34;

[0280] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:34; or

[0281] (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 with the sequence shown in SEQ ID NO:34.

[0282] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0283] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:33 and VL having the sequence shown in SEQ ID NO:34.

[0284] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0285] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0286] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0287] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0288] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:43, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0289] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:44, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0290] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0291] and / or

[0292] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0293] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:46, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0294] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:47, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0295] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0296] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0297] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:43, VH CDR2 as shown in SEQ ID NO:44, and VHCDR3 as shown in SEQ ID NO:45; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:46, VL CDR2 as shown in SEQ ID NO:47, and VL CDR3 as shown in SEQ ID NO:48.

[0298] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO: 41; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO: 42.

[0299] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0300] In some preferred embodiments, the antibody or its antigen-binding fragment is 10B11 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0301] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0302] (a) Human CLDN18.2 was bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml or less);

[0303] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0304] (c) Not bound to human CLDN18.1;

[0305] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0306] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0307] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0308] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0309] (1) VH CDR1 as shown in SEQ ID NO:43, VH CDR2 as shown in SEQ ID NO:44, VH CDR3 as shown in SEQ ID NO:45; VL CDR1 as shown in SEQ ID NO:46, VL CDR2 as shown in SEQ ID NO:47, VL CDR3 as shown in SEQ ID NO:48; or

[0310] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:41; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:42.

[0311] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0312] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0313] (i) The sequence shown in SEQ ID NO:41;

[0314] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:41; or

[0315] (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 with the sequence shown in SEQ ID NO:41;

[0316] And / or,

[0317] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0318] (iv) The sequence shown in SEQ ID NO:42;

[0319] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:42; or

[0320] (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 with the sequence shown in SEQ ID NO:42.

[0321] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0322] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:41 and VL having the sequence shown in SEQ ID NO:42.

[0323] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0324] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0325] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0326] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0327] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:51, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0328] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:52, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0329] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:53, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0330] and / or

[0331] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0332] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:54, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0333] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:55, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0334] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:56, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0335] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0336] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:51, VH CDR2 as shown in SEQ ID NO:52, and VH CDR3 as shown in SEQ ID NO:53; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:54, VL CDR2 as shown in SEQ ID NO:55, and VL CDR3 as shown in SEQ ID NO:56.

[0337] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:49; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:50.

[0338] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0339] In some preferred embodiments, the antibody or its antigen-binding fragment is 27B5 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0340] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0341] (a) Bind human CLDN18.2 at EC50 of 0.2 μg / ml or less;

[0342] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0343] (c) Not bound to human CLDN18.1;

[0344] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0345] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0346] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0347] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0348] (1) VH CDR1 as shown in SEQ ID NO:51, VH CDR2 as shown in SEQ ID NO:52, VH CDR3 as shown in SEQ ID NO:53; VL CDR1 as shown in SEQ ID NO:54, VL CDR2 as shown in SEQ ID NO:55, VL CDR3 as shown in SEQ ID NO:56; or

[0349] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:49; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:50.

[0350] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0351] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0352] (i) The sequence shown in SEQ ID NO:49;

[0353] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:49; or

[0354] (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 with the sequence shown in SEQ ID NO:49;

[0355] And / or,

[0356] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0357] (iv) The sequence shown in SEQ ID NO:50;

[0358] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:50; or

[0359] (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 with the sequence shown in SEQ ID NO:50.

[0360] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0361] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:49 and VL having the sequence shown in SEQ ID NO:50.

[0362] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0363] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0364] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0365] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0366] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:59, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0367] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:60, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0368] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:61, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0369] and / or

[0370] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0371] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:62, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0372] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:63, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0373] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0374] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0375] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:59, VH CDR2 as shown in SEQ ID NO:60, and VH CDR3 as shown in SEQ ID NO:61; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:62, VL CDR2 as shown in SEQ ID NO:63, and VL CDR3 as shown in SEQ ID NO:64.

[0376] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:57; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:58.

[0377] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0378] In some preferred embodiments, the antibody or its antigen-binding fragment is 37B1 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0379] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0380] (a) Human CLDN18.2 was bound to EC50 at a concentration of 0.1 μg / ml or less (e.g., 0.05 μg / ml or less);

[0381] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0382] (c) Not bound to human CLDN18.1;

[0383] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0384] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0385] (f) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0386] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0387] (1) VH CDR1 as shown in SEQ ID NO:59, VH CDR2 as shown in SEQ ID NO:60, VH CDR3 as shown in SEQ ID NO:61; VL CDR1 as shown in SEQ ID NO:62, VL CDR2 as shown in SEQ ID NO:63, VL CDR3 as shown in SEQ ID NO:64; or

[0388] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:57; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:58.

[0389] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0390] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0391] (i) The sequence shown in SEQ ID NO:57;

[0392] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:57; or

[0393] (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 with the sequence shown in SEQ ID NO:57;

[0394] And / or,

[0395] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0396] (iv) The sequence shown in SEQ ID NO:58;

[0397] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:58; or

[0398] (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 with the sequence shown in SEQ ID NO:58.

[0399] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0400] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:57 and VL having the sequence shown in SEQ ID NO:58.

[0401] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0402] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0403] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising:

[0404] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0405] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:67, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0406] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:68, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0407] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:69, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0408] and / or

[0409] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0410] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:70, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0411] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:71, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0412] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:72, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0413] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0414] In some preferred embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:67, VH CDR2 as shown in SEQ ID NO:68, and VH CDR3 as shown in SEQ ID NO:69; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:70, VL CDR2 as shown in SEQ ID NO:71, and VL CDR3 as shown in SEQ ID NO:72.

[0415] The present invention also provides an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:65; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:66.

[0416] In some preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0417] In some preferred embodiments, the antibody or its antigen-binding fragment is 44A8 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0418] In some preferred embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0419] (a) Bind human CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0420] (b) Combined with mouse CLDN18.2 at EC50 of 0.1 μg / ml or less;

[0421] (c) Not bound to human CLDN18.1;

[0422] (d) Inducing the killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) through antibody-dependent cell-mediated cytotoxicity (ADCC);

[0423] (e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as tumor cells expressing CLDN18.2) via complement-dependent cytotoxicity (CDC);

[0424] (f) Mediating the internalization of CLDN18.2 into cells (e.g., tumor cells), for example, at an internalization level of at least 10% (e.g., at least 15%, at least 20% or more) as measured by FACS or flow cytometry; said cells express CLDN18.2 on their surface;

[0425] (g) Prevention and / or treatment of tumors (e.g., tumors expressing CLDN18.2) in subjects.

[0426] In some preferred embodiments, the antibody or its antigen-binding fragment comprises:

[0427] (1) VH CDR1 as shown in SEQ ID NO:67, VH CDR2 as shown in SEQ ID NO:68, VH CDR3 as shown in SEQ ID NO:69; VL CDR1 as shown in SEQ ID NO:70, VL CDR2 as shown in SEQ ID NO:71, VL CDR3 as shown in SEQ ID NO:72; or

[0428] (2) The three CDRs contained in the heavy chain variable region shown in SEQ ID NO:65; and the three CDRs contained in the light chain variable region shown in SEQ ID NO:66.

[0429] In one exemplary embodiment, the antibody or its antigen-binding fragment comprises:

[0430] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0431] (i) The sequence shown in SEQ ID NO:65;

[0432] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:65; or

[0433] (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 with the sequence shown in SEQ ID NO:65;

[0434] And / or,

[0435] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0436] (iv) The sequence shown in SEQ ID NO:66;

[0437] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:66; or

[0438] (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 with the sequence shown in SEQ ID NO:66.

[0439] In some preferred embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0440] In some preferred embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:65 and VL having the sequence shown in SEQ ID NO:66.

[0441] In some preferred embodiments, the antibody or its antigen-binding fragment is humanized.

[0442] In some preferred embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. In such embodiments, the heavy chain variable region FR and / or the light chain variable region FR of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region FR and / or the light chain framework region FR may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0443] In some preferred embodiments, the antibody or antigen-binding fragment of the present invention may further comprise a constant region sequence or a variant thereof derived from a mammalian (e.g., mouse or human) immunoglobulin, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence. In some preferred embodiments, the variant has one or more conserved amino acid substitutions compared to its derived sequence.

[0444] In some preferred embodiments, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0445] The light chain of the antibody or its antigen-binding fragment of the present invention comprises a light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions) compared to the sequence from which it is derived.

[0446] In some preferred embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some preferred embodiments, the heavy chain constant region is a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some preferred embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, preferably, the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

[0447] In some preferred embodiments, the light chain constant region is the κ light chain constant region. In some preferred embodiments, the light chain constant region is the mouse κ light chain constant region. In some preferred embodiments, the light chain constant region is the human κ light chain constant region.

[0448] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises the heavy chain constant region (CH) shown in SEQ ID NO:81; and / or the light chain constant region (CL) shown in SEQ ID NO:82.

[0449] In some preferred embodiments, the antibody of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In some preferred embodiments, the antigen-binding fragment of the present invention is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0450] In this invention, the antibody or antigen-binding fragment thereof may include variants that differ from the antibody or antigen-binding fragment from which they are derived only in the conserved substitution of one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibody or antigen-binding fragment from which they are derived, and substantially retain the aforementioned biological functions of the antibody or antigen-binding fragment from which they are derived.

[0451] Antibody preparation

[0452] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0453] 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)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0454] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some preferred embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0455] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) containing the isolated nucleic acid molecules of the present invention. In some preferred embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc. In some preferred embodiments, the vector is capable of expressing the antibody of the present invention or its antigen-binding fragment in a subject (e.g., a mammal, such as a human).

[0456] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecules of the present invention or the vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG44).

[0457] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the host cell of the present invention 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.

[0458] Derived antibodies

[0459] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to CLDN18.2 (particularly human CLDN18.2). Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0460] Therefore, in some preferred embodiments, the antibody or antigen-binding fragment of the present invention is labeled. In some preferred embodiments, the antibody or antigen-binding fragment of the present invention is labeled with a detectable marker, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable marker described in the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I,35 S, 14 C or 32 P), 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)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., ), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of such markers 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 incorporated herein by reference). Detectable markers as described above can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0461] Bispecific or multispecific molecules

[0462] The antibodies or antigen-binding fragments thereof of the present invention can be used to form bispecific or multispecific molecules. The antibodies or antigen-binding fragments thereof of the present invention can be part of a bispecific or multispecific molecule comprising a second functional module (e.g., a second antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, thereby enabling binding to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be linked to a second antibody or antigen-binding fragment capable of specifically binding to any protein that can be used as a potential target for combination therapy. To generate said bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0463] Therefore, in another aspect, the present invention provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment of the present invention.

[0464] In some preferred embodiments, the bispecific or multispecific molecule specifically binds to CLDN18.2 and additionally specifically binds to one or more other targets.

[0465] In some preferred embodiments, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity against a second target.

[0466] Immunoconjugates

[0467] The antibodies or antigen-binding fragments thereof of the present invention can be conjugated with therapeutic agents to form immunoconjugates. Because immunoconjugates have the ability to selectively deliver one or more therapeutic agents to target tissues (e.g., tumor-associated antigens, such as tumors expressing CLDN18.2), immunoconjugates can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0468] Therefore, in another aspect, the present invention provides an immunoconjugate comprising an antibody of the present invention or an antigen-binding fragment thereof and a therapeutic agent attached to said antibody or antigen-binding fragment thereof.

[0469] In some preferred embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0470] In some preferred embodiments, the therapeutic agent is a cytotoxic agent. In this invention, the cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells).

[0471] In some preferred embodiments, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

[0472] Examples of alkylating agents that can be used in the immunoconjugates of the present invention include, but are not limited to, nitrogen mustards (such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethyleneimines (such as thiotepa), sulfate esters and polyols (such as busulfan, dibromomannitol), nitrosoureas (such as carmustine, lomustine, etc.), and platinum-based antitumor agents (such as cisplatin, oxaliplatin, carboplatin, etc.).

[0473] Examples of mitotic inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, maytansine derivatives (e.g., maytansine, maytanol, C-3 ester of maytanol, etc.), taxane derivatives (e.g., docetaxel, paclitaxel, or nanoparticle paclitaxel, etc.), and vinblastine alkaloids (e.g., vinorelbine sulfate, vincristine, vinblastine, or vinorelbine, etc.).

[0474] Examples of antitumor antibiotics that can be used in the immunoconjugates of the present invention include, but are not limited to, actinomycin, anthracycline antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), calicimycin, and pyromycin.

[0475] Examples of antimetabolites that can be used in the immunoconjugates of the present invention include, but are not limited to, folic acid antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, fluorouridine, cytarabine, capecitabine, gemcitabine), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nerabine, pentostatin).

[0476] Examples of topoisomerase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, camptothecins and their derivatives (e.g., irinotecan, topotecan, etc.), acridine, doxorubicin, epipodophyllotoxin, rose alkaloids, epirubicin, etoposide, propylamine, teniposide, etc.

[0477] Examples of tyrosine kinase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, axitinib, bosutinib, cidinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, letotinib, nilotinib, semasanib, sunitinib, vandetanib, etc.

[0478] Examples of radionuclide agents that can be used in the immunoconjugates of the present invention include, but are not limited to, I. 131 In 111 Y 90 Lu 177 wait.

[0479] In some exemplary embodiments, the therapeutic agent is selected from platinum-based antitumor agents, anthracycline antibiotics, taxanes, nucleoside analogs, camptothecin compounds, and their analogs or homologues, and any combination thereof.

[0480] In some preferred embodiments, the antibody or its antigen-binding fragment of the present invention is optionally conjugated to the therapeutic agent via a linker.

[0481] In this invention, cytotoxic agents can be conjugated to the antibodies or antigen-binding fragments thereof using existing linker techniques in the art. Examples of linker types already used for conjugating cytotoxic agents to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. Linkers that are readily cleaved at low pH within lysosomal compartments or readily cleaved by proteases (e.g., proteases preferentially expressed in tumor tissues, such as cathepsins, such as cathepsins B, C, and D) can be selected.

[0482] Further discussion on the types of cytotoxic agents, linkers, and methods of conjugating therapeutic agents to antibodies can be found in Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, PA et al. (2003) Cancer I mmunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Allen, TM (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53: 247-264.

[0483] Chimeric antigen receptor

[0484] The antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptors (CARs), wherein the CAR comprises an extracellular antigen-binding domain (e.g., scFv) that specifically binds to CLDN18.2, is linked to a transmembrane domain, and is linked to one or more intracellular T cell signaling domains. The intracellular T cell signaling domain may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or a combination thereof. The T cell receptor signaling domain refers to the portion of the CAR containing the intracellular domain of a T cell receptor (e.g., the intracellular portion of the CD3ζ protein). The co-stimulatory signaling domain refers to the portion of the CAR containing the intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an efficient lymphocyte response to an antigen.

[0485] The CAR of the present invention is characterized by its ability to specifically and reactivity target T cells (e.g., tumor cells) expressing CLDN18.2 in a non-MHC-restricted manner. This non-MHC-restricted CLDN18.2 recognition capability endows T cells expressing the CAR of the present invention with the ability to recognize antigens independently of antigen processing.

[0486] Therefore, in another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain of the antibody or antigen-binding fragment of the present invention.

[0487] In some preferred embodiments, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment of the present invention.

[0488] In some preferred embodiments, the antigen-binding domain is scFv.

[0489] In some preferred embodiments, the antigen-binding receptor comprises an antigen-binding fragment of the antibody of the present invention (e.g., scFv).

[0490] In some preferred embodiments, the antigen-binding receptor is expressed by immune effector cells (e.g., T cells).

[0491] In some preferred embodiments, a spacer domain comprising a polypeptide sequence may be present between the antigen-binding domain and the transmembrane domain of the CAR. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some embodiments, the spacer domain may contain an immunoglobulin domain, such as a human immunoglobulin sequence. In some exemplary embodiments, the immunoglobulin domain comprises immunoglobulin CH2 and CH3 domain sequences. In such embodiments, without being bound by any particular theory, it is considered that the CH2 and CH3 domains extend the antigen-binding domain of the CAR from the membrane of the cell expressing the CAR and more accurately mimic the size and domain structure of the native TCR.

[0492] In some preferred embodiments, the transmembrane domain may be derived from a natural or synthetic source. In such embodiments, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains that can be used in the CAR of the present invention may comprise a transmembrane region of at least the α, β, or ζ chain of a T-cell receptor, which may be selected from CD28, CD3ε, CD45, CD4, CD5, CD5, CD64, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine.

[0493] In some exemplary embodiments, the transmembrane domain includes the transmembrane domain of a T cell receptor, such as the CD8 transmembrane domain.

[0494] In some exemplary embodiments, the transmembrane domain includes a transmembrane domain of a T-cell co-stimulatory molecule (e.g., CD137 or CD28).

[0495] In some preferred embodiments, examples of intracellular T-cell domains that can be used in the CAR include cytoplasmic sequences and co-stimulatory molecules of a T-cell receptor (TCR) that cooperate in initiating signal transduction upon antigen-receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0496] In some preferred embodiments, the intracellular region of the CAR may contain a primary cytoplasmic signaling sequence that functions in a stimulatory manner, which may include a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences that may be included in the CAR include those from the proteins CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d.

[0497] In some preferred embodiments, the intracellular region of the CAR may contain an ITAM containing a primary cytoplasmic signaling domain (e.g., CD3ζ) alone or in combination with any other desired cytoplasmic domains available for use in the CAR environment. For example, the cytoplasmic domain of the CAR contains a portion of the CD3ζ chain and an intracellular co-stimulatory signaling domain. The co-stimulatory signaling domain refers to a portion of the intracellular domain of the CAR containing a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0498] In some preferred embodiments, the CAR may include a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or any combination thereof. The order of the one or more T cell signaling domains on the CAR may be changed by those skilled in the art as needed.

[0499] Methods for generating chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for cancer treatment) are known in the art, and detailed descriptions can be found, for example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp. 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000, WO2013 / 126726; and US patent publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety. For example, the nucleic acid molecule encoding the chimeric antigen-binding receptor of the present invention may be contained in an expression vector (e.g., a lentiviral vector) for expression in host cells, such as T cells, to manufacture the CAR. In some exemplary embodiments, a method of using the chimeric antigen receptor includes isolating T cells from a subject, transforming the T cells with an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor, and administering the engineered T cells expressing the chimeric antigen receptor to the subject for treatment, such as for treating a tumor in the subject.

[0500] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the present invention. In some preferred embodiments, the isolated nucleic acid molecule encodes the chimeric antigen receptor of the present invention.

[0501] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising isolated nucleic acid molecules as described above. In some preferred embodiments, the vector of the present invention is, for example, a plasmid.

[0502] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecules or vectors as described above. In some preferred embodiments, the host cell is a T cell. In some preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0503] Treatment methods and pharmaceutical compositions

[0504] The antibody or its antigen-binding fragment of the present invention can kill cells by inducing ADCC and / or CDC through binding to CLDN18.2, thereby enabling its use in the prevention and / or treatment of tumors.

[0505] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, or an immunoconjugate of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0506] In some preferred embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents.

[0507] In some preferred embodiments, the additional pharmaceutically active agent is a drug with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive isotopes, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), antiangiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), molecularly targeted drugs (e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, etc.), oncolytic viruses, etc.

[0508] In some preferred embodiments, in the pharmaceutical composition, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent. Therefore, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention can be administered simultaneously, separately, or sequentially with the additional pharmaceutically active agent.

[0509] In some exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0510] In another aspect, the present invention provides a method for reducing the expression level of CLDN18.2 on the cell surface, comprising contacting the cells with an antibody or antigen-binding fragment thereof of the present invention, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition, such that the expression level of CLDN18.2 on the cell surface is reduced; wherein the cells express CLDN18.2 on their surface.

[0511] In some preferred embodiments, the cells are tumor cells expressing CLDN18.2.

[0512] In some preferred embodiments, the method is used to reduce the expression level of CLDN18.2 on the cell surface in vitro for non-diagnostic purposes.

[0513] In another aspect, the use of the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, or pharmaceutical compositions of the present invention in the preparation of medicaments for reducing the expression level of CLDN18.2 on the cell surface is provided.

[0514] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, or pharmaceutical compositions for reducing the expression level of CLDN18.2 on the cell surface.

[0515] In another aspect, the present invention provides a method for inhibiting the growth of tumor cells expressing CLDN18.2 and / or killing said tumor cells, comprising contacting said tumor cells with an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, a pharmaceutical composition, a chimeric antigen receptor, or a host cell expressing said chimeric antigen receptor (e.g., chimeric antigen receptor T cells (CAR-T)).

[0516] The method can be used for therapeutic or non-therapeutic purposes. In some preferred embodiments, the method can be used for non-therapeutic purposes, specifically to inhibit the growth of tumor cells expressing CLDN18.2 and / or kill the tumor cells in vitro.

[0517] In another aspect, the use of the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immune conjugates, pharmaceutical compositions, chimeric antigen receptors or host cells expressing said chimeric antigen receptors (e.g., chimeric antigen receptor T cells (CAR-T)) of the present invention in the preparation of medicaments for inhibiting the growth of tumor cells expressing CLDN18.2 and / or killing said tumor cells is provided.

[0518] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors or host cells expressing said chimeric antigen receptors (e.g., chimeric antigen receptor T cells (CAR-T)) for inhibiting the growth of tumor cells expressing CLDN18.2 and / or killing said tumor cells.

[0519] In another aspect, the present invention provides a method for preventing and / or treating tumors in a subject (e.g., a human), the method comprising administering to a subject in need an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a bispecific or multispecific molecule, an immune conjugate, a pharmaceutical composition, a chimeric antigen receptor, or a host cell expressing said chimeric antigen receptor (e.g., chimeric antigen receptor T cells (CAR-T)).

[0520] In some preferred embodiments, the tumor involves tumor cells expressing CLDN18.2. In some preferred embodiments, CLDN18.2 is expressed on the surface of the tumor cells.

[0521] In some preferred embodiments, the tumor expresses CLDN18.2.

[0522] In some preferred embodiments, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

[0523] In some preferred embodiments, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing said chimeric antigen receptor (e.g., chimeric antigen receptor T cell (CAR-T)) of the present invention is used in combination with an additional drug with antitumor activity. This additional drug with antitumor activity may be administered before, simultaneously with, or after the administration of the antibody or its antigen-binding fragment, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing said chimeric antigen receptor (e.g., CAR-T).

[0524] In some preferred embodiments, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells expressing said chimeric antigen receptors (e.g., CAR-T) of the present invention are administered in combination with additional therapies. Such additional therapies can be any therapy known for use in cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy. Such additional therapies can be administered before, simultaneously with, or after the administration of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells expressing said chimeric antigen receptors (e.g., CAR-T).

[0525] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing said chimeric antigen receptors can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. In addition, sterile injectable 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 carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0526] Furthermore, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immune conjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing said chimeric antigen receptors may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0527] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing said chimeric antigen receptors can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route of administration and / or method will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing said chimeric antigen receptors are administered by intravenous injection or bolus injection.

[0528] The pharmaceutical compositions of the present invention may comprise a “therapeutic effective amount” or a “preventive effective amount” of the antibody or its antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, a pharmaceutical composition, a chimeric antigen receptor, or a host cell (e.g., T cell) expressing said chimeric antigen receptor. A “preventive effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or its antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0529] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0530] In this invention, the subject can be a mammal, such as a human.

[0531] Detection methods and kits

[0532] The antibody or its antigen-binding fragment of the present invention can specifically bind to CLDN18.2, thereby enabling it to be used to detect the presence or level of CLDN18.2 in a sample.

[0533] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof. In some preferred embodiments, the antibody of the present invention or an antigen-binding fragment thereof is labeled with a detectable marker. In one preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable marker.

[0534] In this invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred is that such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), 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)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., The invention includes, but is not limited to, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of these markers 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 incorporated herein by reference). The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, the detectable marker described above can be attached to the antibody or its antigen-binding fragment of the present invention via connectors of different lengths to reduce potential steric hindrance.

[0535] In another aspect, the present invention provides a method for detecting the presence or amount of CLDN18.2 in a sample, comprising the following steps:

[0536] (1) Contact the sample with the antibody or its antigen-binding fragment of the present invention;

[0537] (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.2 or detect the amount of the complex.

[0538] The formation of the complex indicates the presence of CLDN18.2 or cells expressing CLDN18.2.

[0539] In some preferred embodiments, the sample is a cell sample, i.e., a sample containing cells (e.g., tumor cells). In such embodiments, preferably, the complex is formed between the antibody, antigen-binding fragment, or conjugate and CLDN18.2 expressed by the cells in the sample.

[0540] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable marker. In another preferred embodiment, in step (2), a reagent labeled with a detectable marker is used to detect the antibody or antigen-binding fragment of the present invention.

[0541] 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). In some preferred embodiments, the CLDN18.2 is a human CLDN18.2.

[0542] In another aspect, the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or amount of CLDN18.2 in a sample is provided. In some preferred embodiments, the CLDN18.2 is human CLDN18.2.

[0543] In another aspect, the present invention provides a method for detecting whether a tumor can be treated by an anti-tumor therapy targeting CLDN18.2, comprising the following steps:

[0544] (1) Contact the sample containing the tumor cells with the antibody or antigen-binding fragment of the present invention;

[0545] (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.2.

[0546] In some preferred embodiments, the complex is formed between the antibody or its antigen-binding fragment and CLDN18.2 expressed by tumor cells in the sample.

[0547] In some preferred embodiments, the sample is from a subject who has a tumor, is suspected of having a tumor, or is at risk of having a tumor. In some preferred embodiments, the sample is from a tissue or organ in which the cells do not substantially express CLDN18.2 when the tissue or organ is not cancerous. In some preferred embodiments, the tissue is selected from stomach tissue, lung tissue, esophageal tissue, pancreatic tissue, or breast tissue, and the tissue has optionally been diagnosed as being affected by cancer, for example, by visual examination or culture testing of the cells in the tissue or organ. In some preferred embodiments, the tissue is tissue other than stomach tissue. In some preferred embodiments, the tissue is lung tissue, esophageal tissue, pancreatic tissue, or breast tissue. In such embodiments, the presence of CLDN18.2 or cells expressing CLDN18.2, and / or an increase in the amount of CLDN18.2 or cells expressing CLDN18.2 compared to a reference level (e.g., compared to a patient without tumor disease), indicates that the subject is suitable for anti-tumor therapy targeting CLDN18.2.

[0548] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable marker. In another preferred embodiment, in step (2), a reagent labeled with a detectable marker is used to detect the antibody or antigen-binding fragment of the present invention.

[0549] In some preferred embodiments, the CLDN18.2 is a human CLDN18.2.

[0550] In some preferred embodiments, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

[0551] In some preferred embodiments, the tumor is selected from esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

[0552] In another aspect, the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting whether a tumor can be treated by an antitumor therapy targeting CLDN18.2 is provided.

[0553] In some preferred embodiments, the antibody or its antigen-binding fragment carries a detectable label.

[0554] In some preferred embodiments, the CLDN18.2 is a human CLDN18.2.

[0555] In some preferred embodiments, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

[0556] In some preferred embodiments, the tumor is selected from esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

[0557] Terminology Definition

[0558] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0559] As used herein, the term "CLDN18 (Claudin 18)" has the meaning commonly understood by those skilled in the art, referring to the Claudin family of transmembrane proteins within the tight junctions of epithelial and endothelial cells, existing in two splice variants, CLDN18.1 and CLDN18.2. The sequences of CLDN18.1 and CLDN18.2 are well-known in the art and can be found in NCBI database accessions NP_057453.1 and NP_001002026.1, respectively.

[0560] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one 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 antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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. Constant domains do not directly participate in antibody-antigen binding, but exhibit various 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 be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in 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.

[0561] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0562] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat numbering system.

[0563] As used herein, the term "framework region" or "FR" residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0564] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0565] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that 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; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated 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, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0566] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting 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 N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0567] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0568] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0569] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0570] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, 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, edited by Roseburg and Moore, 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 repeating 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 this 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 exist between the VH and VL of the scFv.

[0571] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0572] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0573] As used herein, the term "probody" has the meaning commonly understood by those skilled in the art, referring to a masked antibody that remains inert in healthy tissue but is specifically activated in a disease environment (e.g., via proteolytic cleavage by proteases enriched or specific to the disease environment). Detailed teachings can be found, for example, in Desnoyers et al., Sci. Transl. Med., 5:207ra144, 2013. Similar masking techniques may be used for any antibody or its antigen-binding moiety described herein.

[0574] Each of the above antibody fragments 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.

[0575] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0576] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0577] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0578] 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).

[0579] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to purify the immunospecific antibody. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0580] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion 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 which retains its binding activity to the target antigen in any case (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the variable regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0581] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity).

[0582] In this application, the intended properties of the antibodies of the present invention include: (1) specific recognition / binding to CLDN18.2 (particularly human CLDN18.2); (2) mediating CLDN18.2 internalization; (3) inducing the killing of cells expressing human CLDN18.2 via antibody-dependent cell-mediated cytotoxicity (ADCC); (4) inducing the killing of cells expressing human CLDN18.2 via complement-dependent cytotoxicity (CDC); and (5) the ability to prevent and / or treat tumors. The antibodies of the present invention have one or more of the above-mentioned intended properties.

[0583] The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0584] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of mouse immunoglobulins to the constant region of human immunoglobulins (see, for example, U.S. Patent No. 4,816,567, Cabilly et al.). For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDA 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 preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0585] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent 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 that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies, and then transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon encountering antigen stimulation (see, for example, 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 aforementioned transgenic animals include the HuMAb mouse (Medarex, Inc.) containing a miniloci of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM mouse" carrying a human heavy chain transgene and a human light chain transchromosomal transgene. TM (See patent application WO02 / 43478). Other methods for humanizing antibodies 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).

[0586] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence in the genome of an organism that encodes an immunoglobulin, which has not undergone a maturation process involving genetic rearrangements and mutations that would lead to the expression of a specific immunoglobulin. In this invention, the term "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding the heavy chain of immunoglobulins, including the V (variable), D (diversity), J (joining), and C (constant) genes; similarly, the term "light chain germline gene" refers to a germline antibody gene or gene segment encoding the light chain of immunoglobulins, including the V (variable), J (joining), and C (constant) genes. In this invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene segment is also referred to as a "germline sequence." Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0587] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody binds at a dissociation equilibrium constant of less than about 10-1. -9 M, for example, less than about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or lower affinity (K) D The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0588] As used herein, the term "cytotoxic agent" includes any agent that is harmful to cells (e.g., kills cells), such as chemotherapy drugs, bacterial toxins, plant toxins, or radioactive isotopes.

[0589] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0590] 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.

[0591] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage 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 × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0592] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using 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 or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0593] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this 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.

[0594] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered T-cell receptor having an extracellular antibody-derived targeting domain (e.g., scFv) that binds to one or more intracellular signaling domains of a T-cell receptor. In this invention, the term "chimeric antigen receptor T-cell" refers to a T-cell that expresses a CAR and has antigen specificity determined by the targeting domain of that CAR. Methods for manufacturing CARs (e.g., for cancer treatment) are known in the art and can be found, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000 and WO2013 / 059593; and U.S. Patent Publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety.

[0595] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means 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, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters 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, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). 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 meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium 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. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0596] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0597] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor (e.g., a tumor expressing CLDN18.2) or is at risk of having the aforementioned disease.

[0598] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from 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 purposes 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 characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0599] As used herein, the term "immune effector cells" includes cells of hematopoietic origin that play a role in the immune response, such as lymphocytes, including B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some preferred embodiments, the immune effector cells are T cells.

[0600] As used herein, the term "metastasis" refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastasis is a highly complex process and depends on malignant cells detaching from the primary tumor, invading the extracellular matrix, penetrating the endothelial basement membrane to enter body cavities and blood vessels, and subsequently infiltrating target organs via hematogenous transport. Finally, the growth of the new tumor (i.e., secondary or metastatic tumor) at the target site depends on angiogenesis. Tumor metastasis often occurs even after the primary tumor has been removed, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis," which involves metastasis away from the primary tumor and the local lymph node system. The cells of a secondary or metastatic tumor are similar to those in the original tumor. This means, for example, if ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells (rather than abnormal hepatocytes). The tumor in the liver is then referred to as metastatic ovarian cancer (rather than liver cancer).

[0601] Beneficial effects of the invention

[0602] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0603] The antibodies of this invention specifically recognize / bind to CLDN18.2 and can induce the killing of CLDN18.2-expressing cells (e.g., tumor cells) via ADCC and / or CDC. Therefore, the antibodies of this invention have the potential for the prevention and / or treatment of tumors (particularly tumors expressing CLDN18.2). The humanized antibodies of this invention not only retain the function and properties of the parent antibodies but also have a high degree of humanization, thus allowing safe administration to human subjects without inducing immunogenic reactions. Particularly surprisingly, the antibodies of this invention exhibit significantly enhanced affinity and tumor-killing activity compared to known anti-CLDN18.2 antibodies. Therefore, the antibodies of this invention (particularly the humanized antibodies) have significant clinical value.

[0604] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0605] Figures 1A-1D show the results of the binding activity assays of anti-CLDN18.2 murine antibody with different cell surface CLDN18.2 or CLDN18.1. Figure 1A: HEK293T-hCLDN18.2; Figure 1B: HEK293T-mCLDN18.2; Figure 1C: HEK293T-hCLDN18.1; Figure 1D: HEK293T.

[0606] Figure 2 shows the results of the binding activity assay of the anti-CLDN18.2 chimeric antibody to CLDN18.2 on the cell surface.

[0607] Figures 3A-3C show the ADCC activity assays of the anti-CLDN18.2 chimeric antibody against HEK293T-hCLDN18.2, KATO-III, and NUGC4, respectively. Figure 3A: HEK293T-hCLDN18.2; Figure 3B: KATO-III; Figure 3C: NUGC4.

[0608] Figures 4A-4B show the CDC activity assay results of the anti-CLDN18.2 chimeric antibody against HEK293T-hCLDN18.2 and KATO-III, respectively. Figure 4A: HEK293T-hCLDN18.2; Figure 4B: KATO-III.

[0609] Figure 5 shows the results of the binding activity assay of the anti-CLDN18.2 humanized antibody to CLDN18.2 on the cell surface.

[0610] Figure 6 shows the results of ADCC activity assay for KATO-III by the anti-CLDN18.2 humanized antibody.

[0611] Figure 7 shows the results of the assay of CDC activity of anti-CLDN18.2 humanized antibody against KATO-III.

[0612] Figures 8A-8B show the effects of anti-CLDN18.2 antibody on tumor volume (A) and survival (B) in a mouse tumor model, respectively.

[0613] Sequence information

[0614] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0615] Table 1: Sequence Description

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626] Detailed Implementation

[0627] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0628] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0629] Example 1: Generation of anti-CLDN18.2 murine antibody

[0630] To obtain anti-human CLDN18.2 antibodies, mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., strain code 216) were inoculated using different immunization strategies (Table 2) to induce the production of murine monoclonal antibodies. The antigens include: an expression plasmid expressing the extracellular region 1 (ECL1) of human CLDN18.2 (CLDN18.2-ECL1 DNA; SEQ ID NO:83, vector pcDNA3.1), an expression plasmid expressing the complement C3-encoding sequence of extracellular region 1 of human CLDN18.2 (CLDN18.2-ECL1-C3d DNA; SEQ ID NO:84, vector pcDNA3.1), an expression plasmid expressing the full-length encoding sequence of human CLDN18.2 (hCLDN18.2 DNA; SEQ ID NO:85, vector pcDNA3.1), Chinese hamster ovary cells transfected with human CLDN18.2 (CHO-hCLDN18.2), and kidney embryonic cells transfected with high expression of human CLDN18.2 (HEK293-hCLDN18.2). Adjuvants included: In vivo-jetPEI (Polyplus Transfection, catalog number 201-50G), ODN 1826 VacciGrade (InvivoGen, catalog number vac-1826-1), and complete Freund's adjuvant CFA (InvivoGen, catalog number vac-cfa-60). Routes of administration included intramuscular (im), intraperitoneal (ip), and subcutaneous (sc). Three days after booster immunization, spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 using the polyethylene glycol method to obtain B cell fusions that could both express antibodies and proliferate indefinitely in vitro, and cultured in HAT selective medium. The fused hybridoma cells were seeded in 96-well cell culture plates, and positive clones were selected through primary screening for 2-3 rounds of subcloning.

[0631] Table 2: Immunization regimens for generating monoclonal antibodies

[0632]

[0633] Primary screening: In primary screening, the binding capacity of the supernatant of the grown clones to CLDN18.2 was tested using cells expressing human CLDN18.2. The presence of the reactive antibody in the supernatant was revealed by a DyLight488 goat anti-mouse IgG (Abcam catalog number ab97015) secondary antibody, and the binding capacity was assessed on a full-field cell scanning analyzer (see Example 3).

[0634] Secondary screening: The supernatant of the above-mentioned fusion clones binding to human CLDN18.2 was tested for its binding ability to CLDN18.1 on the cell surface using cells expressing human CLDN18. The presence of reacting antibodies in the supernatant was revealed by using a DyLight488 goat anti-mouse IgG secondary antibody (Abcam catalog number ab97015), and the binding ability was evaluated using a full-field cell scanning analyzer (detailed experimental procedures can be found in Example 3). Ten positive hybridoma monoclonal cell lines were finally obtained, and the following antibodies were isolated and purified from their culture supernatant: 1D10, 2F12, 3F2, 5F9, 9F3, 10B11, 27B5, 37B1, 44A8, and 44F7.

[0635] Example 2: Evaluation of antigen-binding activity of anti-CLDN18.2 murine antibody

[0636] 2.1 Construction of a cell line expressing CLDN18.2

[0637] Lentiviral infection and resistance selection (MOI = 3-10, 5 μg / ml polybrene) were used to overexpress human CLDN18.2 (SEQ ID NO: 86) or CLDN18.1 (SEQ ID NO: 88), or mouse CLDN18.2 (SEQ ID NO: 87), respectively, in HEK293T cells (ATCC), CHOS cells (Invitrogen), and OCUM-1 gastric cancer cells (Nanjing Kebai Biotechnology Co., Ltd.). Lentiviral viruses were provided by Shanghai Jikai Gene Chemical Technology Co., Ltd. Cells were cultured for 2-4 weeks after infection with the appropriate antibiotics for 72 hours, then amplified and cryopreserved for subsequent experiments.

[0638] 2.2 The binding of mouse antibodies to CLDN18.2 and CLDN18.1 on the cell surface was detected using a cell scanning analyzer.

[0639] Using recombinant HEK293T-hCLDN18.2 and OCUM-1-hCLDN18.2 expressing human CLDN18.2 (hCLDN18.2), or HEK293T-mCLDN18.2 expressing mouse CLDN18.2 (mCLDN18.2), and the corresponding negative control cell line HEK293T; or using recombinant HEK293T-hCLDN18.1 and CHOS-hCLDN18.1 expressing human CLDN18.1 (hCLDN18.1), and using DyLight488 goat anti-mouse IgG (Abcam catalog number ab97015) or DyLight488 goat anti-human IgG (Abcam catalog number ab97003) as the secondary antibody, titration binding curves were generated using the following method.

[0640] 10,000 cells were seeded in 100 μL DMEM + 10% FBS per well in a flat-bottomed 96-well plate and incubated overnight to allow cells to adhere or settle to the bottom. The supernatant was removed the next day. A 3-fold serial dilution was performed by diluting 1 / 3 volume (100 μL) in 200 μL DMEM. 100 μL of diluted antibody (using fusion clone supernatant or subclone supernatant for selection) was added to each well of the cell culture plate, and 100 μL of DMEM was added to the corresponding negative control well. The cells were incubated at room temperature for 1 hour. After removing the supernatant, 100 μL of secondary antibody (5 μg / mL, diluted in DMEM) was added to each well, and the cells were incubated at room temperature for 0.5 hours. After staining, the supernatant was removed, the cells were washed once with PBS, and 100 μL of PBS was added to each well before the cells were read.

[0641] Using a full-field cell scanning analyzer (Nexcelom, model...) Image Cytometer was used to measure the experimental plate. During measurement, both the green fluorescence channel and bright field channel corresponding to the second antibody were selected for high-speed scanning imaging of the cells in the wells. The images obtained from the green fluorescence channel were used to count cells binding to the antibody based on parameters set according to the morphology and fluorescence intensity of the fluorescently labeled cells. The images obtained from the bright field channel were used to count adherent cells based on parameters set according to cell morphology. The two sets of data were then divided to obtain the percentage of adherent cells displaying green fluorescence (%fluorescent cells). This percentage was used to determine the binding activity of the anti-CLDN18.2 antibody to cells expressing CLDN18.2. A lower percentage indicates a weaker ability of the tested anti-CLDN18.2 antibody to bind to CLDN18.2 on the cell surface, and vice versa. Data analysis was performed using GraphPad.

[0642] The binding activity assays of anti-CLDN18.2 antibody with HEK293T cells expressing human CLDN18.2, HEK293T cells expressing mouse CLDN18.2, HEK293T cells expressing human CLDN18.1, and control HEK293T are shown in Figures 1A-1D. The horizontal axis represents the logarithm of antibody concentration, and the vertical axis represents the percentage of adherent cells that showed green fluorescence after binding with the CLDN18.2 antibody. The EC50 of the anti-CLDN18.2 antibody's antigen-binding activity was further calculated using curve fitting, and the results are shown in Table 3. The reference antibody was 175D10 (Ganymed Pharmaceuticals AG), disclosed in, for example, CN101312989B and CN103509114B.

[0643] Table 3: Results of the binding activity assay of anti-CLDN18.2 antibody to CLDN18.2

[0644]

[0645] Note: NB indicates that no binding occurred within the measured concentration range.

[0646] The results showed that 1D10, 2F12, 3F2, 5F9, 9F3, 10B11, 27B5, 37B1, 44A8, and 44F7 could all bind to cells expressing human CLDN18.2, and were significantly superior to the reference antibody 175D10. Meanwhile, these antibodies did not bind to the negative control cells (HEK293T) that did not express CLDN18.2.

[0647] The EC50 values ​​of the binding activity of the anti-CLDN18.2 antibody to CLDN18.1 are shown in Table 4. The results showed that 2F12, 3F2, 9F3, 10B11, 27B5, 37B1, 44A8, and 44F7 did not bind to CLDN18.1, demonstrating good binding specificity against CLDN18.2.

[0648] Table 4: Results of binding activity assay of anti-CLDN18.2 antibody to CLDN18.1

[0649]

[0650] Note: NB indicates no binding within the measured concentration range, and blank indicates that this data was not measured for this antibody.

[0651] Example 3: Sequencing of anti-CLDN18.2 murine antibody and preparation of chimeric antibody

[0652] 3.1 Determination of the variable region sequence of anti-human CLDN18.2 murine antibody

[0653] Hybridoma cells were collected by centrifugation, at a rate of 5-10 × 10⁻� 6Add 1 ml TRIzol and 0.2 ml chloroform to each cell, vortex vigorously for 15 seconds, incubate at room temperature for 3 minutes, centrifuge, collect the aqueous phase, add 0.5 ml isopropanol, incubate at room temperature for 10 minutes, collect the precipitate, wash with ethanol, and dry to obtain RNA. Add template RNA and primers to an ice-cold centrifuge tube, ensure correct primer-template pairing, perform reverse transcription, and then perform PCR amplification. After amplification, add 2.5 μl of dNTP / ddNTP mixture to each of four microcentrifuge tubes, incubate at 37°C for 5 minutes, and set aside. Add 1 pmol of PCR amplification product, 10 pmol of sequencing primers, 2 μl of 5× sequencing buffer to an empty microcentrifuge tube, add double-distilled water to a total volume of 10 μl, heat at 96°C for 8 minutes, cool on ice for 1 minute, and centrifuge at 10000g for 10 seconds at 4°C. Add 2 μl of pre-chilled labeling mixture (0.75 μmol / L each of dCTP, dGTP, and dTTP), 5 μCi of α-32P-dATP, 1 μl of 0.1 mol / L DDT, and 2 U of sequencing enzyme. Add water to a final volume of 15 μl, mix well, and incubate on ice for 2 min. Add 3.5 μl of the labeling reaction mixture to four prepared microcentrifuge tubes and incubate at 37°C for 5 min. Add 4 μl of stop solution to each tube. Denature the samples in an 80°C water bath for 5 min. Add 2 μl of the solution to each lane of the sequencing gel, separate the fragments by electrophoresis, and collect the sequence information.

[0654] The VH and VL sequences of the 10 murine antibodies are shown in Table 5 below. Furthermore, the CDR sequences of the 10 murine monoclonal antibodies were determined using the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669) (Table 6).

[0655] Table 5: Amino acid sequences of the variable regions of the light and heavy chains of murine antibodies

[0656]

[0657] Table 6: Sequences of murine antibody CDRs

[0658]

[0659] 3.2 Preparation and Evaluation of Antigen-Binding Activity of Human-Mouse Chimeric Antibodies

[0660] The gene sequences encoding the variable regions of the heavy and light chains of the aforementioned murine antibodies (see SEQ ID NOs: 103-122) were linked to the sequences encoding the constant regions of the heavy and light chains of human antibodies (SEQ ID NO: 81 and SEQ ID NO: 82), respectively, and recombinantly expressed in HEK293 cells (ATCC) to obtain the corresponding chimeric antibodies 1D10-chIgG1, 2F12-chIgG1, 3F2-chIgG1, 5F9-chIgG1, 9F3-chIgG1, 10B11-chIgG1, 27B5-chIgG1, 37B1-chIgG1, 44A8-chIgG1, and 44F7-chIgG1. The binding activity of different concentrations of chimeric antibodies to HEK293T cells (HEK293T-hCLDN18.2) expressing human CLDN18.2 was detected using the method described in Example 2. The results are shown in Figure 2. The horizontal axis represents the logarithm of antibody concentration, and the vertical axis represents the percentage of adherent cells (% fluorescent cells) that showed green fluorescence after binding to the CLDN18.2 antibody. The EC50 of the chimeric antibody's antigen-binding activity was further calculated based on curve fitting, as shown in Table 7. The results showed that the chimeric antibodies 3F2-chIgG1, 5F9-chIgG1, 9F3-chIgG1, 10B11-chIgG1, 27B5-chIgG1, 37B1-chIgG1, 44A8-chIgG1, and 44F7-chIgG1 could all recognize / bind to human CLDN18.2.

[0661] Table 7: Results of the binding activity assay of the chimeric antibody to CLDN18.2

[0662]

[0663] Example 4: Evaluation of ADCC activity induced by anti-CLDN18.2 chimeric antibody

[0664] Target cells were HEK293T-hCLDN18.2 or the human gastric cancer cell lines KATO-III and NUGC4, which naturally express hCLDN18.2. Effector cells were human peripheral blood mononuclear cells (PBMCs) isolated by Ficoll. Target cells were harvested, washed twice with PBS, and stained with the live cell dye Calcein AM (50 μg Calcein AM powder dissolved in 50 μl DMSO, Life Technologies, Cat#C3100MP) to 3 μM at 37°C for 30 minutes in 5% CO2. After staining, the cells were washed twice with PBS, and 5000 target cells were seeded into 100 μL DMEM / well in a flat-bottomed 96-well plate. A 3-fold serial dilution was performed by diluting 1 / 3 volume (100 μL) in 200 μL DMEM. Add 50 μL of diluted antibody to the corresponding well of the cell plate (50 μL of LMEM medium to the non-specific killing control well), and incubate with target cells at 37°C for 30 minutes with 5% CO2. Then, add 50 μL of isolated PBMCs (50,000 cells) to each well as effector cells. Centrifuge at 1000 rpm for 3 minutes to allow cells to settle at the bottom of the plate. Perform cell scanning at different time points (0 h, 2 h, 3 h, 4 h, 6 h) using a full-field cell scanner (Nexcelom, model number...). Image Cytometer was used to measure the experimental plate. During measurement, Calcein AM was selected to simultaneously perform high-speed scanning imaging of the cells in the wells, using both the green fluorescence channel and the bright field channel. The green fluorescence channel image was used to count live cells in the wells based on parameters set according to the morphology and fluorescence intensity of the fluorescently labeled cells. The bright field channel image was used to count the total number of cells in the wells based on parameters set according to cell morphology. The two sets of data were then divided to obtain the percentage of live cells displaying green fluorescence out of the total number of cells. The percentage of non-specific control wells minus the corresponding percentage of the antibody yielded the percentage of cells that underwent specific cell lysis in the presence of the antibody. This value was used to determine the ADCC activity induced by the anti-CLDN18.2 antibody; a lower percentage indicates a weaker ADCC-inducing ability of the tested anti-CLDN18.2 antibody, and vice versa. Data analysis was performed using GraphPad.

[0665] The results of ADCC activity induced by anti-CLDN18.2 antibody are shown in Figures 3A-3C. The horizontal axis represents the logarithm of antibody concentration, and the vertical axis represents the corrected percentage of specific lysis. The EC50 of ADCC induced by the anti-CLDN18.2 chimeric antibody was further calculated using curve fitting, and the results are shown in Table 8. The results show that all detected antibodies can induce PBMC killing of cells expressing human CLDN18.2, and are significantly superior to the reference antibody 175D10.

[0666] Table 8: Results of ADCC activity induced by chimeric antibodies

[0667]

[0668] Note: NA indicates that EC50 is greater than the measured concentration range, and blank indicates that this data was not measured for this antibody.

[0669] Example 5: Evaluation of the activity of anti-CLDN18.2 chimeric antibody in inducing CDC

[0670] Target cells were HEK293T-hCLDN18.2 or the KATO-III human gastric cancer cell line naturally expressing hCLDN18.2, and effector cells were treated with fresh human serum. Target cells were harvested, washed twice with PBS, and stained with the live cell dye Calcein AM (50 μg Calcein AM powder dissolved in 50 μl DMSO, Life Technologies, Cat#C3100MP) to 3 μM at 37°C for 30 minutes under 5% CO2 conditions. After staining, the cells were washed twice with PBS, and 8000 target cells were seeded into 25 μL DMEM / well in a flat-bottomed 96-well plate. A 3-fold serial dilution was performed by diluting 1 / 3 volume (100 μL) in 200 μL DMEM. Add 25 μL of diluted antibody (25 μL of DMEM medium to the non-specific killing control wells) to each well of the cell plate. Incubate with target cells at 37°C with 5% CO2 for 30 minutes. Then add 50 μL of freshly isolated 20% human serum (diluted with DMEM) to each well as effector cells. Centrifuge at 1000 rpm for 3 minutes to allow cells to settle to the bottom of the plate. Analyze the cells at different time points (0 h, 1 h, 2 h, 3 h, 4 h) using a full-field cell scanning analyzer (Nexcelom, model number...). Image Cytometer was used to measure the experimental plate. During measurement, Calcein AM was selected to simultaneously perform high-speed scanning imaging of the cells in the wells, using both the green fluorescence channel and the bright field channel. The green fluorescence channel image was used to count the viable cells in the wells based on parameters set according to the morphology and fluorescence intensity of the fluorescently labeled cells. The bright field channel image was used to count the total number of cells in the wells based on parameters set according to cell morphology. The two sets of data were then divided to obtain the percentage of viable cells displaying green fluorescence out of the total number of cells. The percentage of non-specific control wells minus the corresponding percentage of the antibody yielded the percentage of cells exhibiting specific cell killing in the presence of the antibody. This value was used to determine the activity of anti-CLDN18.2 antibody in inducing CDC; a lower percentage indicates a weaker ability of the tested anti-CLDN18.2 antibody to induce CDC, and vice versa. Data analysis was performed using GraphPad.

[0671] The results of the assay for CDC activity induced by anti-CLDN18.2 antibody are shown in Figures 4A-4B. The horizontal axis represents the logarithm of antibody concentration, and the vertical axis represents the corrected percentage of specific cell killing. The EC50 of CDC induced by anti-CLDN18.2 antibody was further calculated using curve fitting, and the results are shown in Table 9. The results show that all the antibodies detected can induce the killing effect of complement in human serum on cells expressing human CLDN18.2, and are significantly superior to the reference antibody 175D10.

[0672] Table 9: Results of chimeric antibody-induced CDC activity assay

[0673]

[0674]

[0675] Note: Blank indicates that this data was not measured for this antibody.

[0676] Example 6: Evaluation of the activity of anti-CLDN18.2 antibody in inducing CLDN18.2 internalization

[0677] In this embodiment, the level of CLDN18.2 internalization mediated by anti-CLDN18.2 antibody on the surface of HEK293T-hCLDN18.2 cells was detected by flow cytometry. Two cell lines were incubated with 10 μg / mL chimeric antibody at 37°C for 1 and 4 hours, respectively. After washing several times with PBS containing 2% FBS, 10 μg / mL of a secondary antibody was added and stained at 4°C for 30 minutes. The expression level of CLDN18.2 on the cell surface was then analyzed by flow cytometry.

[0678] MFI 4HMFI of the sample after 4 hours of incubation; MFI 1H For the MFI of a sample after 1 hour of incubation, assuming antibody binding is complete and endocytosis has not yet occurred under these conditions, the MFI is... 背景 For MFI with only a secondary antibody, the antibody-mediated endocytosis percentage of cell surface CLDN18.2 is calculated using the following formula:

[0679] The percentage of internalized CLDN18.2 is calculated as follows: % = 100 - 100 × (MFI) 4H -MFI 1H ) / (MFI 1H -MFI 背景 )

[0680] The results are shown in Table 10. These antibodies mediated varying degrees of endocytosis of CLDN18.2 cells on the surface of HEK293T-hCLDN18.2 cells.

[0681] Table 10: Results of chimeric antibody-induced CLDN18.2 internalization assay

[0682] Antibody endocytosis percentage (%) 1D 10-chIgG 128.75 F9-chIgG 19.327 B5-chIgG 1-2.237 B1-chIgG 1-3.244 F7-chIgG 1-1.544 A8-chIgG 12.5

[0683] Example 7: Humanization and Activity Evaluation of Anti-CLDN18.2 Antibody

[0684] To improve the sequence homology between candidate antibodies and human antibodies and reduce the immunogenicity of antibodies to humans, the murine antibodies provided in the above embodiments can be humanized by designing and preparing them. The murine CDR region can be inserted into a human frame sequence using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent 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). Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody).

[0685] Based on this, the inventors prepared three humanized antibodies to the mouse antibody 44F7, named 7004-09hu09, 7004-09hu10, and 7004-09hu15, respectively, and their amino acid sequences are shown in the table below.

[0686] Table 11: Amino acid sequences of the light and heavy chain variable regions of humanized antibodies

[0687]

[0688] The gene sequences encoding the variable regions of the heavy and light chains of the aforementioned humanized antibody (see SEQ ID NOs: 123-128) were linked to the sequences encoding the constant regions of the heavy chain (SEQ ID NO: 81) and light chain (SEQ ID NO: 82) of the human antibody, respectively, and recombinantly expressed. The binding of the humanized antibody to CLDN18.2 on the surface of HEK293T-hCLDN18.2 cells was detected using a cell scanning analyzer according to the method described in Example 2.2. The results are shown in Figure 5, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the percentage of adherent cells (% fluorescent cells) that showed green fluorescence after binding with the CLDN18.2 antibody. The EC50 of the binding antigen of the anti-CLDN18.2 humanized antibody was further calculated using curve fitting, and the results are shown in Table 12. The results show that all detected humanized antibodies could bind to cells expressing hCLDN18.2, and their affinity was on the same order of magnitude as that of the parental chimeric antibody.

[0689] Table 12: Results of binding activity assay of humanized antibody to CLDN18.2

[0690]

[0691]

[0692] Furthermore, the inventors investigated the ability of the humanized antibody to induce ADCC, and the evaluation method is described in Example 4. KATO-III human gastric cancer cells were used as target cells, and human peripheral blood mononuclear cells isolated by Ficoll were used as effector cells. The results are shown in Figure 6, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the corrected percentage of specific cell lysis. The EC50 of ADCC induced by the anti-CLDN18.2 chimeric antibody was further calculated using curve fitting, and the results are shown in Table 13. The results show that all detected antibodies can induce PBMCs to kill cells expressing human CLDN18.2, and their ADCC-inducing ability is on the same order of magnitude as the parental chimeric antibody and significantly superior to the reference antibody 175D10.

[0693] Table 13: Results of ADCC activity induced by humanized antibodies

[0694]

[0695] The inventors also investigated the ability of the humanized antibody to induce CDC, and the evaluation method is described in Example 5. Human gastric cancer tumor cells KATO-III were used as target cells, and fresh human serum was used as effector cells. The measurement results are shown in Figure 7, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the corrected percentage of specific cell killing. Furthermore, the EC50 of CDC induced by the anti-CLDN18.2 chimeric antibody was calculated using curve fitting, and the results are shown in Table 14. The results show that all detected antibodies can induce the killing effect of complement in human serum on cells expressing human CLDN18.2, and their ability to induce CDC is on the same order of magnitude as the parental chimeric antibody, and significantly superior to the reference antibody 175D10.

[0696] Table 14: Results of CDC activity induced by humanized antibodies

[0697]

[0698] The above results indicate that the humanized antibody of the present invention not only has a high degree of humanization, which can reduce the possibility of immune rejection, but also exhibits antitumor activity comparable to and superior to that of the parental chimeric antibody.

[0699] Example 8: Evaluation of in vivo antitumor activity of anti-CLDN18.2 chimeric antibody

[0700] To investigate the antitumor effect of anti-CLDN18.2 antibody in animals, 1×10⁻⁶ antibodies were subcutaneously injected into nude mice (SCID, Beijing Vital River Laboratory Animal Technology Co., Ltd.). 7 HEK293T-hCLDN18.2 cells were administered intravenously and intraperitoneally twice a week for four weeks, starting from the day of inoculation. Tumor size was observed and measured after drug withdrawal. Figure 8A shows the changes in tumor volume after treatment. The results showed that treatment with the 44F7-chIgG1 antibody not only significantly inhibited tumor growth but also completely eliminated tumors in the treated mice, demonstrating significantly superior antitumor activity compared to the reference antibody 175D10. Furthermore, Figure 8B shows that 44F7-chIgG1 significantly prolonged the overall survival of tumor-bearing mice, exhibiting excellent antitumor activity. Therefore, compared to the reference antibody 175D10, 44F7-chIgG1 also showed a significant advantage in prolonging survival. Such technical results are significant and unexpected.

[0701] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:75, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:76, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:77, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:78, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:79, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:80, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:75, VH CDR2 as shown in SEQ ID NO:76, and VH CDR3 as shown in SEQ ID NO:77; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:78 or 96, VL CDR2 as shown in SEQ ID NO:79, and VL CDR3 as shown in SEQ ID NO:

80.

2. An antibody or its antigen-binding fragment capable of specifically binding to CLDN18.2, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein, (a) The heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:73; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO:74; or (b) The heavy chain variable region contains the three CDRs contained in the heavy chain variable region shown in SEQ ID NO:91; and the light chain variable region contains the three CDRs contained in the light chain variable region shown in SEQ ID NO:92; Preferably, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

3. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, and VH CDR3 as shown in SEQ ID NO:5; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:6, VL CDR2 as shown in SEQ ID NO:7, and VL CDR3 as shown in SEQ ID NO:

8.

4. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:15, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:11, VH CDR2 as shown in SEQ ID NO:12, and VH CDR3 as shown in SEQ ID NO:13; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:14, VL CDR2 as shown in SEQ ID NO:15, and VL CDR3 as shown in SEQ ID NO:

16.

5. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:20, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:23, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:11, VH CDR2 as shown in SEQ ID NO:12, and VH CDR3 as shown in SEQ ID NO:13; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:14, VL CDR2 as shown in SEQ ID NO:15, and VL CDR3 as shown in SEQ ID NO:

16.

6. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:27, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:28, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:30, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:31, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:32, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:27, VH CDR2 as shown in SEQ ID NO:28, and VH CDR3 as shown in SEQ ID NO:29; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:30, VL CDR2 as shown in SEQ ID NO:31, and VL CDR3 as shown in SEQ ID NO:

32.

7. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:35, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:36, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:37, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:38, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:39, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:40, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:35, VH CDR2 as shown in SEQ ID NO:36, and VH CDR3 as shown in SEQ ID NO:37; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:38, VL CDR2 as shown in SEQ ID NO:39, and VL CDR3 as shown in SEQ ID NO:

40.

8. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:43, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:44, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:46, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:47, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:48, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:43, VH CDR2 as shown in SEQ ID NO:44, and VH CDR3 as shown in SEQ ID NO:45; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:46, VL CDR2 as shown in SEQ ID NO:47, and VL CDR3 as shown in SEQ ID NO:

48.

9. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:51, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:52, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:53, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:54, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:55, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:56, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:51, VH CDR2 as shown in SEQ ID NO:52, and VH CDR3 as shown in SEQ ID NO:53; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:54, VL CDR2 as shown in SEQ ID NO:55, and VL CDR3 as shown in SEQ ID NO:

56.

10. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:59, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:60, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:61, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:62, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:63, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:64, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:59, VH CDR2 as shown in SEQ ID NO:60, and VH CDR3 as shown in SEQ ID NO:61; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:62, VL CDR2 as shown in SEQ ID NO:63, and VL CDR3 as shown in SEQ ID NO:

64.

11. An antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, said antibody or antigen-binding fragment thereof comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO:67, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:68, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:69, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; and / or (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:70, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:71, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:72, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:67, VH CDR2 as shown in SEQ ID NO:68, and VH CDR3 as shown in SEQ ID NO:69; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:70, VL CDR2 as shown in SEQ ID NO:71, and VL CDR3 as shown in SEQ ID NO:

72.

12. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:73; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:73; 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 with the sequence shown in SEQ ID NO:73; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:74; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:74; 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 with the sequence shown in SEQ ID NO:74; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:73 and VL having the sequence shown in SEQ ID NO:

74.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody is humanized.

14. The antibody or antigen-binding fragment thereof according to claim 1, 2 or 13, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:91; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 91; 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 with the sequence shown in SEQ ID NO:91; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:92; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:92; 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 with the sequence shown in SEQ ID NO:92; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:91 and VL having the sequence shown in SEQ ID NO:

92.

15. The antibody or antigen-binding fragment thereof according to claim 1, 2 or 13, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:99; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 99; 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 with the sequence shown in SEQ ID NO:99; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:100; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 100; 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 with the sequence shown in SEQ ID NO:100; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:99 and VL having the sequence shown in SEQ ID NO:

100.

16. The antibody or antigen-binding fragment thereof according to claim 1, 2 or 13, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:101; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 101; 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 with the sequence shown in SEQ ID NO:101; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:102; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 102; 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 with the sequence shown in SEQ ID NO:102; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:101 and VL having the sequence shown in SEQ ID NO:

102.

17. The antibody or antigen-binding fragment thereof according to claim 3, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:1; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:1; 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 with the sequence shown in SEQ ID NO:1; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:2; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:2; 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 with the sequence shown in SEQ ID NO:2; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:

2.

18. The antibody or antigen-binding fragment thereof according to claim 4, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:9; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:9; 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 with the sequence shown in SEQ ID NO:9; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:10; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:10; 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 with the sequence shown in SEQ ID NO:10; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:9 and VL having the sequence shown in SEQ ID NO:

10.

19. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:17; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:17; 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 with the sequence shown in SEQ ID NO:17; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:18; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:18; 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 with the sequence shown in SEQ ID NO:18; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:17 and VL having the sequence shown in SEQ ID NO:

18.

20. The antibody or antigen-binding fragment thereof according to claim 6, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:25; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:25; 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 with the sequence shown in SEQ ID NO:25; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:26; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:26; 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 with the sequence shown in SEQ ID NO:26; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:25 and VL having the sequence shown in SEQ ID NO:

26.

21. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:33; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:33; 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 with the sequence shown in SEQ ID NO:33; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:34; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:34; 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 with the sequence shown in SEQ ID NO:34; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:33 and VL having the sequence shown in SEQ ID NO:

34.

22. The antibody or antigen-binding fragment thereof according to claim 8, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:41; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:41; 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 with the sequence shown in SEQ ID NO:41; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:42; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:42; 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 with the sequence shown in SEQ ID NO:42; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:41 and VL having the sequence shown in SEQ ID NO:

42.

23. The antibody or antigen-binding fragment thereof according to claim 9, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:49; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:49; 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 with the sequence shown in SEQ ID NO:49; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:50; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:50; 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 with the sequence shown in SEQ ID NO:50; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:49 and VL having the sequence shown in SEQ ID NO:

50.

24. The antibody or antigen-binding fragment thereof according to claim 10, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:57; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:57; 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 with the sequence shown in SEQ ID NO:57; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:58; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:58; 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 with the sequence shown in SEQ ID NO:58; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:57 and VL having the sequence shown in SEQ ID NO:

58.

25. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:65; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:65; 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 with the sequence shown in SEQ ID NO:65; And / or, (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:66; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:66; 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 with the sequence shown in SEQ ID NO:66; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:65 and VL having the sequence shown in SEQ ID NO:

66.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1-25, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to the sequence from which it is derived; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO:81; Preferably, the light chain constant region is the κ light chain constant region; Preferably, the antibody or its antigen-binding fragment comprises the light chain constant region (CL) shown in SEQ ID NO:

82.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb; and / or the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody, or multispecific antibody.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1-27, wherein, The antibody or its antigen-binding fragment is labeled; preferably, the antibody or its antigen-binding fragment is labeled with a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

29. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or a variable region of the heavy chain and / or a variable region of the light chain thereof.

30. A vector comprising the isolated nucleic acid molecule of claim 29; preferably, the vector is a cloning vector or an expression vector.

31. A host cell comprising the isolated nucleic acid molecule of claim 29 or the vector of claim 30.

32. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-28, comprising culturing a host cell according to claim 31 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.

33. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-28; Preferably, the bispecific or multispecific molecule specifically binds to CLDN18.2 and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity against a second target.

34. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-28 and a therapeutic agent attached to said antibody or antigen-binding fragment thereof; Preferably, the therapeutic agent is selected from cytotoxic agents; Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, and any combination thereof; Preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

35. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-28, a bispecific or multispecific molecule as described in claim 33, or an immunoconjugate as described in claim 34, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents; Preferably, the other pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus. Preferably, the antibody or its antigen-binding fragment, bispecific or multispecific molecule or immunoconjugate is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent.

36. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-28; Preferably, the antibody or its antigen-binding fragment carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. Preferably, the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1-28; Preferably, the second antibody further includes a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

37. A chimeric antigen receptor comprising an antigen-binding domain of an antibody or an antigen-binding fragment thereof as described in any one of claims 1-28; Preferably, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof as described in any one of claims 1-28; Preferably, the antigen-binding domain is scFv; Preferably, the antigen-binding receptor comprises an antigen-binding fragment of the antibody according to any one of claims 1-28; Preferably, the antigen-binding receptor is expressed by immune effector cells (e.g., T cells).

38. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 37.

39. A carrier comprising the isolated nucleic acid molecule of claim 38; preferably, it is used for preparing chimeric antigen receptor T cells.

40. A host cell comprising the isolated nucleic acid molecule of claim 38 or the vector of claim 39; Preferably, the host cell is an immune effector cell (e.g., a T cell or an NK cell); Preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).

41. A method for reducing the expression level of CLDN18.2 on the cell surface, comprising contacting the cells with an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or a bispecific or multispecific molecule as described in claim 33, or an immunoconjugate as described in claim 34, or a pharmaceutical composition as described in claim 35, or a chimeric antigen receptor as described in claim 37, or a host cell as described in claim 40, thereby reducing the expression level of CLDN18.2 on the cell surface; wherein, The cells express CLDN18.2 on their surface; Preferably, the cells are tumor cells expressing CLDN18.

2.

42. A method for inhibiting the growth of tumor cells expressing CLDN18.2 and / or killing said tumor cells, comprising contacting said tumor cells with an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or a bispecific or multispecific molecule as described in claim 33, or an immunoconjugate as described in claim 34, or a pharmaceutical composition as described in claim 35, or a chimeric antigen receptor as described in claim 37, or a host cell as described in claim 40.

43. A method for preventing and / or treating tumors in a subject (e.g., a human), the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-28, or a bispecific or multispecific molecule as described in claim 33, or an immunoconjugate as described in claim 34, or a pharmaceutical composition as described in claim 35, or a chimeric antigen receptor as described in claim 37, or a host cell as described in claim 40; Preferably, the tumor expresses CLDN18.2; Preferably, the tumor involves tumor cells expressing CLDN18.2; preferably, CLDN18.2 is expressed on the surface of the tumor cells; Preferably, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases); Preferably, the subject is a mammal, such as a human; Preferably, the method further includes administering additional drugs with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, radiosensitizers, antiangiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses. Preferably, the method further includes administering additional antitumor therapies, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

44. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-28, or the bispecific or multispecific molecule according to claim 33, or the immunoconjugate according to claim 34, or the pharmaceutical composition according to claim 35, or the chimeric antigen receptor according to claim 37, or the host cell according to claim 40, in the preparation of a medicament for the prevention and / or treatment of tumors in a subject (e.g., a human). Preferably, the drug further comprises additional pharmaceutically active agents; Preferably, the other pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus. Preferably, the tumor expresses CLDN18.2; Preferably, the tumor involves tumor cells expressing CLDN18.2; preferably, CLDN18.2 is expressed on the surface of the tumor cells; Preferably, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases); Preferably, the subject is a mammal, such as a human.

45. A method for detecting the presence or amount of CLDN18.2 (e.g., human CLDN18.2) in a sample, comprising the following steps: (1) Contact the sample with the antibody or antigen-binding fragment thereof as described in any one of claims 1-28; (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.2 or detect the amount of the complex; Preferably, the antibody or its antigen-binding fragment carries a detectable label; Preferably, the CLDN18.2 is a human CLDN18.

2.

46. ​​A method for detecting whether a tumor can be treated with an antitumor therapy targeting CLDN18.2, comprising the following steps: (1) Contact the sample containing the tumor cells with the antibody or antigen-binding fragment thereof as described in any one of claims 1-28; (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.2; Preferably, the antibody or its antigen-binding fragment carries a detectable label; Preferably, the CLDN18.2 is a human CLDN18.2; Preferably, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).

47. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-28 in the preparation of a kit for detecting whether a tumor can be treated by an antitumor therapy targeting CLDN18.2; Preferably, the antibody or its antigen-binding fragment carries a detectable label; Preferably, the CLDN18.2 is a human CLDN18.2; Preferably, the tumor is selected from gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, ear, nose and throat (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastatic cancers (e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases or lymph node metastases).