Anti-claudin 18.2 antibodies and uses thereof

Abstract

The present disclosure relates to anti-Claudin 18.2 antibodies and uses thereof. In particular, the present disclosure relates to an anti-Claudin 18.2 antibody; murine antibodies, chimeric antibodies, humanized antibodies and antigen-binding fragments thereof comprising the CDRs of the anti-Claudin 18.2 antibody, and uses thereof as a medicament. In particular, the present disclosure relates to the use of an anti-Claudin 18.2 antibody in the manufacture of a medicament for treating a Claudin 18.2 positive disease or disorder.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080019073.9, filed on March 31, 2020, entitled "Anti-Claudin18.2 Antibody and Its Application".

[0002] This application claims priority to Chinese patent application 201910257853.6, filed on April 1, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of antibody drugs. Specifically, this disclosure relates to the Claudin18.2 antibody and its applications. Background Technology

[0004] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans, belonging to the tight junction protein family. Claudin-18 controls molecular flow between cellular layers.

[0006] The Claudin-18 protein structure includes four transmembrane regions and two extracellular loops, with its N-terminus and C-terminus located in the cytoplasm. Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, differing only in the first extracellular loop by eight amino acids. The expression distribution of Claudin 18.1 and Claudin 18.2 differs; Claudin 18.1 is selectively expressed in normal lung cells, while Claudin 18.2 expression is highly restricted in normal cells but frequently ectopically activated and overexpressed in various tumors (such as gastric, lung, and pancreatic cancer). Claudin 18.2 is considered a potential therapeutic target for gastric cancer and other cancer types, and its discovery provides a new treatment option for gastric cancer. Summary of the Invention

[0007] This disclosure provides an anti-Claudin18.2 antibody.

[0008] In some implementations, as described above, the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0009] i) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO: 4; or

[0010] ii) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO: 6. In some embodiments, the anti-Claudin18.2 antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

[0011] iii) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or

[0012] iv) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively.

[0013] Those skilled in the art should understand that item numbers, such as i), ii), a), b), etc., are intended only to make the listed technical solutions or elements clearer and more organized, but do not have any binding force on the technical solutions or elements that follow. The use of the same item number does not imply that the technical solutions or elements that follow are the same.

[0014] In some implementations, the anti-Claudin18.2 antibody, as previously described, is a murine antibody, a chimeric antibody, or a humanized antibody.

[0015] In some implementations, as described above, the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0016] (v) The heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO:3 or 24, and the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO:4 or 21; or

[0017] (vi) The heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable region shown in SEQ ID NO:5 or 31, and the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable region shown in SEQ ID NO:6 or 28.

[0018] In some implementations, as described above, the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0019] (1) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4 or has at least 90% identity with it;

[0020] (2) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:24 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:21 or has at least 90% identity with it;

[0021] (3) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 90% identity with it; or

[0022] (4) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:31 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:28 or has at least 90% identity with it.

[0023] In some embodiments, such as the anti-Claudin18.2 antibody as previously described, wherein the anti-Claudin18.2 antibody is a humanized antibody comprising a frame region derived from a human antibody or a variant thereof, the frame region variant being a reversion mutation having up to 10 amino acids in the light chain frame region and / or heavy chain frame region of the human antibody, respectively.

[0024] In some embodiments, the heavy chain framework region of the human antibody is the same as the framework region of the heavy chain variable region shown in amino acid sequence SEQ ID NO:24, or the light chain variable region of the human antibody is the same as the framework region of the light chain variable region shown in amino acid sequence SEQ ID NO:21; or the heavy chain framework region of the human antibody is the same as the framework region of the heavy chain variable region shown in amino acid sequence SEQ ID NO:31, or the light chain variable region of the human antibody is the same as the framework region of the light chain variable region shown in amino acid sequence SEQ ID NO:28.

[0025] In some implementations, preferably, the frame region variant comprises a mutation selected from (a) or (b) below:

[0026] (a) The light chain variable region contains one or more amino acid reversion mutations selected from 22S, 85I, or 87H, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 48I, 82T, and 69M; or

[0027] (b) The light chain variable region contains one or more amino acid reversion mutations selected from 4L and 22S, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K.

[0028] In some embodiments, such as the anti-Claudin18.2 antibody described above, the frame region variant contains a mutation selected from (a-1) or (b-1) below:

[0029] (a-1) The light chain variable region contains amino acid reversion mutations of 22S, 85I, and 87H, and the heavy chain variable region contains amino acid reversion mutations of 48I and 82T; or

[0030] (b-1) The light chain variable region contains an amino acid reversion mutation selected from 4L.

[0031] In some implementations, as described above, the anti-Claudin18.2 antibody includes:

[0032] (vii) The heavy chain variable region sequence is as shown in SEQ ID NO:3 and the light chain variable region sequence is as shown in SEQ ID NO:4; or

[0033] (viii) The heavy chain variable region sequence as shown in SEQ ID NO: 24, 25, 26 or 27 and the light chain variable region sequence as shown in SEQ ID NO: 21, 22 or 23; or

[0034] (ix) the heavy chain variable region sequence as shown in SEQ ID NO:5 and the light chain variable region sequence as shown in SEQ ID NO:6; or

[0035] (x) The heavy chain variable region sequence is shown as SEQ ID NO: 31, 32, 33 or 34 and the light chain variable region sequence is shown as SEQ ID NO: 28, 29 or 30.

[0036] In some embodiments, as described above, the anti-Claudin18.2 antibody, wherein: the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below:

[0037] (xi) The heavy chain variable region sequence is shown in SEQ ID NO:31 and the light chain variable region sequence is shown in SEQ ID NO:29; or

[0038] (xii) The heavy chain variable region sequence is shown in SEQ ID NO:26 and the light chain variable region sequence is shown in SEQ ID NO:23.

[0039] In some implementations, such as the anti-Claudin18.2 antibody described above, the light chain variable region and the heavy chain variable region can be a combination of the light and heavy chain variable regions shown in the following table:

[0040] Table 1. Combination of light and heavy chain variable regions of mAb1901 humanized antibody

[0041]

[0042]

[0043] Table 2. Combination of light and heavy chain variable regions of mAb1902 humanized antibody

[0044] Variable region VH11 VH12 VH13 VH14 VL11 VH11VL11 VH12VL11 VH13VL11 VH14VL11 VL12 VH11VL12 VH12VL12 VH13VL12 VH14VL12 VL13 VH11VL13 VH12VL13 VH13VL13 VH14VL13

[0045] In some embodiments, as described above, the anti-Claudin18.2 antibody further comprises an antibody constant region. In some specific embodiments, the heavy chain constant region of the antibody is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 and their variants, and the light chain constant region of the antibody is selected from the constant regions of human antibody κ and λ chains and their variants. In some specific embodiments, the antibody comprises a heavy chain constant region with the sequence shown in SEQ ID NO:7 and a light chain constant region with the sequence shown in SEQ ID NO:8. In some specific embodiments, the antibody comprises: a heavy chain having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:35 or 42, and a light chain having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:36 or 39; or

[0046] Heavy chains having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:37 or 49, and / or light chains having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:38 or 46.

[0047] In some implementations, the anti-Claudin18.2 antibody, as described above, comprises:

[0048] (c) The heavy chain with a sequence as shown in SEQ ID NO:35 and / or the light chain with a sequence as shown in SEQ ID NO:36;

[0049] (d) Heavy chains with sequences as shown in SEQ ID NO:42, 43, 44 or 45 and / or light chains with sequences as shown in SEQ ID NO:39, 40 or 41;

[0050] (e) a heavy chain with a sequence as shown in SEQ ID NO:37 and / or a light chain with a sequence as shown in SEQ ID NO:38; or

[0051] (f) Heavy chains with sequences as shown in SEQ ID NO:49, 50, 51 or 52 and / or light chains with sequences as shown in SEQ ID NO:46, 47 or 48.

[0052] In some embodiments, as described above, the anti-Claudin18.2 antibody competitively binds to human Claudin18.2 with the aforementioned anti-Claudin18.2 antibody or its antigen-binding fragment.

[0053] In some embodiments, the anti-Claudin18.2 antibody, as described above, comprises:

[0054] The heavy chain with the amino acid sequence shown in SEQ ID NO:44, and the light chain with the sequence shown in SEQ ID NO:41; or

[0055] The heavy chain with an amino acid sequence as shown in SEQ ID NO:49, and the light chain with a sequence as shown in SEQ ID NO:47.

[0056] Another aspect of this disclosure provides a nucleic acid molecule that encodes the anti-Claudin18.2 antibody as described above.

[0057] Another aspect of this disclosure provides an expression vector comprising the nucleic acid molecules as described above.

[0058] Another aspect of this disclosure provides a host cell comprising nucleic acid molecules as described above or expression vectors as described above, preferably bacterial cells, fungal cells, insect cells or mammalian cells.

[0059] Another aspect of this disclosure provides an antibody-drug conjugate formed by conjugating an anti-Claudin18.2 antibody with a cytotoxic drug as described above.

[0060] Another aspect of this disclosure provides an antibody-drug conjugate comprising or consisting of the aforementioned anti-Claudin18.2 antibody covalently bound to a cytotoxic drug.

[0061] In some embodiments, this disclosure provides a method for preparing the Claudin18.2 antibody as described above.

[0062] In some embodiments, this disclosure provides a method for preparing the Claudin18.2 antibody-drug conjugate as described above.

[0063] In some embodiments, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-Claudin18.2 antibody as described above, or a nucleic acid molecule as described above, or an antibody-drug conjugate as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0064] In some embodiments, this disclosure provides a method for immunoassay or determination of Claudin18.2, the method comprising contacting a sample to be tested with an anti-Claudin18.2 antibody as described above.

[0065] In some embodiments, this disclosure provides the use of the anti-Claudin18.2 antibody as described above in the preparation of reagents for the immunoassay of human Claudin18.2.

[0066] In some embodiments, this disclosure provides an anti-Claudin18.2 antibody as described above for the immune detection or determination of Claudin18.2.

[0067] In some embodiments, this disclosure provides a kit comprising an anti-Claudin18.2 antibody as described above.

[0068] In some embodiments, this disclosure provides the use of the anti-Claudin18.2 antibody as described above, or the nucleic acid molecule as described above, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above, in the preparation of a medicament for treating cancer or tumors, wherein the cancer or tumor is preferably a Claudin18.2-positive cancer or malignant tumor, more preferably squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, and prostate cancer. The cancers included are: testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma. The lymphomas are selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma. The lung cancers are selected from: non-small cell lung cancer and small cell lung cancer. The leukemias are selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0069] In some embodiments, this disclosure provides a method for treating a disease associated with Claudin18.2, the method comprising administering to a subject a therapeutically effective amount of an anti-Claudin18.2 antibody as described above, or a nucleic acid molecule as described above, or an antibody-drug conjugate as described above, or a pharmaceutical composition as described above, wherein the disease is preferably cancer or tumor; more preferably Claudin18.2-positive cancer or malignant tumor, more preferably selected from: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, and central nervous system cancer. Neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma. More preferably, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0070] In some embodiments, the therapeutically effective amount is a unit dose of the composition containing 0.1 mg to 3000 mg or 1 mg to 1000 mg of the anti-Claudin18.2 antibody or the antibody-drug conjugate as described above.

[0071] In some embodiments, this disclosure provides an anti-Claudin18.2 antibody, or a nucleic acid molecule, or an antibody-drug conjugate, or a pharmaceutical composition as described above for treating diseases associated with Claudin18.2, wherein the disease is preferably cancer or tumor; more preferably Claudin18.2-positive cancer or malignant tumor, more preferably selected from: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal carcinoma, etc. Laryngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma. More preferably, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0072] In some implementations, the cancer is stomach cancer, esophageal cancer, lung cancer, or pancreatic cancer.

[0073] In some implementations, antibodies and antibody-drug conjugates, as described above, can exert therapeutic effects in cancers as described above that exhibit high, moderate, or low expression of Claudin18.2.

[0074] The Claudin18.2 antibody and antibody-drug conjugate disclosed herein have good affinity for cell surface antigens, good endocytosis efficiency and strong tumor inhibition efficiency, and have a wider drug application window, making them suitable for clinical drug application. Attached Figure Description

[0075] Figure 1 FACS detection results of humanized antibody binding to human Claudin18.2 at the cellular level.

[0076] Figure 2: NuGC4 cell endocytosis experiment with humanized antibodies.

[0077] Figures 3A to 3C Detection of the ADCC effect of antibodies in NUGC4 cells with different Claudin18.2 expression levels. Figure 3A To detect the ADCC effect of the antibody in wild-type NUGC4 cells (low expression of Claudin18.2); Figure 3B To detect the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression; Figure 3C To detect the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression. Detailed Implementation

[0078] the term

[0079] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0080] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0081] The term "cytotoxic drug" refers to substances that inhibit or prevent cell function and / or cause cell death or damage. This includes compounds such as toxins and chemotherapeutic drugs that can kill cells.

[0082] The term "toxin" refers to any substance that can have a harmful effect on cell growth or proliferation. It can be a small molecule toxin from bacteria, fungi, plants, or animals, and its derivatives, including camptothecin derivatives such as ixatecan, maytansine alkaloids and their derivatives (CN101573384) such as DM1, DM3, DM4, orlistatine F (AF) and its derivatives such as MMAF, MMAE, 3024 (WO 2016 / 127790 A1, compound 7), diphtheria toxin, exotoxins, ricin A chain, abrin A chain, modeccin, α-sarcin, tung oil (Aleutites fordii) toxin, carnation (dianthin) toxin, American pokeweed (PAPI, PAPII, and PAP-S) toxin, and bitter melon (Momordica) toxin. The inhibitors include charantia, curcin, crotin, sapaonaria officinalis, gelonin, mitogellin, restrictedocin, phenomycin, enomycin, and trichothecenes.

[0083] The term "chemotherapy drug" refers to compounds that can be used to treat tumors. This definition also includes anti-hormonal agents that modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or whole-body therapy. They themselves can be hormones. Examples of chemotherapy drugs include alkylating agents, such as thiotepa; cyclosphamide; and others. TMAlkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; aziridines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomeleamine; nitrogen mustard. Mustards include chlorambucil, naphthylmustine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and oxynimustine hydrochloride; melphalan, novombhichin, cholesterol phenylacetic acid mustard, prednimustine, trofosfamide, and uracil mustard; nitrosureas include carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics include aclarubicin, actinomycin, autramycin, diazoserine, and bleomycin. Mycins, Actinomycin C, Caliceacin, Carabicin, Chromycin, Carzinophilin, Chromycin, Actinomycin D, Daunorubicin, Detorubicin, 6-Diazon-5-O-L-Leucine, Doxorubicin, Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Potfiromycin, Puromycin, Quelamycin, Rodorubicin, Streptomycin;Streptozocin, tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as dempterin, methotrexate, pteroxate, trimetrexate; pteroxate analogs such as fludarabine, 6-mercaptopterin. Anesthetics such as thiophene and thioguanetine; pyrimidine analogs such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxitluridine, enocitabine, fluorouridine, and 5-FU; androgens such as calusterone and dromostanolong. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; acegluconeol; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntrene; edatraxate; defofamine; colchicine; diaziquone; elfomithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pintostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazine; procarbazine; Razoxane; Sizofiran; Spirogermanium; Alternaria alternifolia ketone acid; Triaminequinone; 2,2',2"-Trichlorrotriethylamine; Urethan; Vincristamide; Dacarbazine; Mannitol mustard; Mitobronitol; Dibromocerinol; Pipobroman; Gacytosine; Ara-C glycoside; Cyclophosphamide; Thiotepa; Taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); esperamicins; capecitabine; and any of the above-mentioned pharmaceutically acceptable salts, acids, or derivatives. This definition also includes anti-hormonal agents that can modulate or inhibit the effects of hormones on tumors, such as anti-estrogenic agents including tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and fareston; and anti-androgenic agents such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

[0084] The "antibody" described in this disclosure refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0085] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0086] The antibodies disclosed herein include murine antibodies, chimeric antibodies, and humanized antibodies.

[0087] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human Claudin18.2 prepared in accordance with the knowledge and skills of the art. Preparation involves using Claudin18.2 or its epitopes as the antigen injection target, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of this disclosure, the mouse anti-Claudin18.2 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or its variants, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or its variants.

[0088] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established. Then, the variable region gene is cloned from the murine hybridoma cells, and the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of this disclosure, the antibody light chain of the chimeric antibody further comprises the light chain constant region of a human κ, λ chain, or a variant thereof. The heavy chain of the Claudin18.2 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4, or using an IgG1, IgG2 or IgG4 variant with amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0089] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a non-human species' CDR sequence into the variable region framework of a human antibody, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the said human antibody variable region framework sequence to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further demonstrated by yeast with affinity maturation mutations of the CDR.

[0090] In one embodiment of this disclosure, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or using an IgG1, IgG2 or IgG4 variant with an amino acid mutation (e.g., L234A and / or L235A mutation, and / or S228P mutation).

[0091] The “variants” of the heavy chain constant region and light chain constant region of human antibodies described in this disclosure refer to prior art variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region; specific substitutions include prior art known YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knock-in-hole structure (giving the antibody heavy chain a knock-Fc and hole-Fc combination), which have been shown to impart novel properties to the antibody without altering the function of the antibody variable region.

[0092] The terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. A human antibody can be derived from a genetically modified organism (GMO) that is "engineered" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, human heavy and light chain locus elements are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted and disrupted. The GMO can synthesize human antibodies specific to human antigens, and can be used to produce hybridomas that secrete human antibodies. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0093] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably herein to refer to an antibody in substantially its complete form, as distinguished from the antigen-binding fragment as defined below. The term specifically refers to antibodies containing constant regions in both the light and heavy chains. This disclosure of “antibody” includes “full-length antibodies” and their antigen-binding fragments.

[0094] In some embodiments, the full-length antibody of this disclosure comprises a full-length antibody formed by linking a light chain variable region to a light chain constant region and a heavy chain variable region to a heavy chain constant region in the light and heavy chain variable region combinations in the table below. Those skilled in the art can select light chain constant regions and heavy chain constant regions from different antibody sources according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.

[0095] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen (e.g., Claudin 18.2). It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, an antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are linked by a synthetic linker, enabling it to produce a single protein chain (referred to as a single-chain Fv (scFv) where the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. The antigen-binding moiety can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. 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.

[0096] Fab is an antibody fragment with antigen-binding activity obtained by treating IgG antibody molecules with an enzyme of the same activity as papain.

[0097] F(ab')2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme of the same activity as pepsin.

[0098] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0099] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment into an expression vector and then introducing the vector into a host.

[0100] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules that contain a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules can have a generic structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other connectors that may be used in this disclosure are described in the following literature, for example, but not limited to: Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; 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.

[0101] A biantibody is an antibody fragment in which scFv or Fab is dimerized, and it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0102] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants.

[0103] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (e.g., Protein Engineering, 7, 697 (1994)).

[0104] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide. This includes the insertion, deletion, or substitution of one, two, three, or more amino acids in the original protein or polypeptide.

[0105] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0106] The terms “complementarity-determining region,” “CDR,” or “hypervariant region” refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) exist in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0107] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody (e.g., a specific site on the Claudin18.2 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0108] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 M or lower affinity (KD) binding.

[0109] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this disclosure have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M is bound to Claudin18.2 or its epitope. For example, in this disclosure, the affinity of the antibody for the cell surface antigen is determined by FACS.

[0110] When the term "competition" is used in the context of competing antigen-binding proteins for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., Claudin18.2 antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIA with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). The assay typically involves using a solid surface or cell to bind purified antigen loaded with either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detection antigen-binding protein. Typically, the detection antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially prevent each other from binding. Further details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0111] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0112] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence when aligning amino acid sequences (introducing gaps where necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0113] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0114] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human Claudin 18.2 or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0115] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells, and NSO cells.

[0116] The antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human Claudin18.2. Positive clones are scaled up in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0117] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0118] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any antibody or antigen-binding fragment of this disclosure, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0119] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will appreciate that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conserved substitutions are described in the table "Exemplary Conserved Amino Acid Substitutions" below.

[0120] Table 3. Exemplary Conserved Substitutions of Amino Acids

[0121]

[0122]

[0123] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this disclosure or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition in a patient.

[0124] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances.

[0125] "Endogenous" refers to substances that are produced in cells, organisms, or the human body, depending on the circumstances.

[0126] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0127] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0128] "Separated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0129] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0130] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0131] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0132] Furthermore, this disclosure includes pharmaceutical agents for treating diseases associated with positive cells of a target antigen (e.g., Claudin18.2), said pharmaceutical agents comprising the anti-Claudin18.2 antibody of this disclosure or an antigen-binding fragment thereof as an active ingredient.

[0133] There are no limitations on the diseases associated with Claudin18.2 in this disclosure, as long as they are diseases related to Claudin18.2. For example, the molecularly induced therapeutic responses of this disclosure include: (1) inhibiting the binding of Claudin18.2 to its receptor / ligand by binding to human Claudin18.2, or (2) killing tumor cells overexpressing Claudin18.2. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of this disclosure are of great use to individuals suffering from tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.

[0134] Furthermore, this disclosure relates to methods for immunodetection or determination of target antigens (e.g., Claudin18.2), reagents for immunodetection or determination of target antigens (e.g., Claudin18.2), methods for immunodetection or determination of cells expressing target antigens (e.g., Claudin18.2), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., Claudin18.2) positive cells, comprising an antibody or antibody fragment of the present disclosure that specifically recognizes the target antigen (e.g., human Claudin18.2) and binds to the amino acid sequence or its three-dimensional structure in the extracellular region as an active ingredient.

[0135] In this disclosure, the method for detecting or determining the amount of a target antigen (e.g., Claudin 18.2) can be any known method. For example, it includes immunoassay or assay methods.

[0136] Immunological detection or assay methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibody or antigen. Examples of immunological detection or assay methods include radiolabeled antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0137] The aforementioned diseases associated with Claudin18.2 positive cells can be diagnosed by detecting or measuring cells expressing Claudin18.2 using antibodies or antibody fragments disclosed herein.

[0138] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0139] In this disclosure, there are no particular limitations on the test sample used to detect or measure the target antigen (e.g., Claudin18.2), as long as it has the potential to contain cells expressing the target antigen (e.g., Claudin18.2), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0140] Depending on the required diagnostic method, diagnostic reagents containing the monoclonal antibody or antibody fragment thereof of this disclosure may also contain reagents for performing an antigen-antibody reaction or for detecting the reaction. Reagents for performing the antigen-antibody reaction include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragment, or conjugates, and substrates corresponding to the labeled antibodies.

[0141] Details of one or more embodiments of the invention have been set forth in the foregoing description. While the invention may be practiced or tested using any methods and materials similar to or the same as those described herein, preferred methods and materials are described below. Other features, objects, and advantages of the invention will become apparent from the description and claims. In the description and claims, the singular form includes plural references unless clearly indicated otherwise in the context. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this invention pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of the invention more fully. These embodiments should not be construed in any way as limiting the scope of the invention, which is defined by the claims.

[0142] Example

[0143] Example 1: Construction of a cell line highly expressing Claudin18.2

[0144] Using Lipofectamine 3000 transfection reagent, the pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into 293T viral packaging cells. The supernatant containing the virus was collected, filtered, and centrifuged at ultra-high speed. The concentrated virus was used to infect the human gastric signet ring cell carcinoma cell line NUGC4. After two to three weeks of selection with puromycin, single-cell sorting was performed using FACS.

[0145] Claudin18.2 expression levels were differentiated based on tumor IHC scores. Cells with Claudin18.2 expression levels comparable to tumors with an IHC score of 3 were considered high-expressing cells, while those with Claudin18.2 expression levels comparable to tumors with an IHC score of 2 were considered moderate-expressing cells. Based on FACS detection of Claudin18.2 expression on the surface of lentivirally infected NUGC4 cells, the NUGC4 / hClaudin18.2 monoclonal cell line with high Claudin18.2 expression was selected. Simultaneously, FACS detection of Claudin18.2 expression on the surface of wild-type NUGC4 cells identified NUGC4 clones with moderate Claudin18.2 expression, while wild-type NUGC4 cells were classified as low-expressing cells.

[0146] The selected monoclonal cell lines were expanded and cryopreserved for future experiments.

[0147] Claudin18.2 sequence Genbank: NP_001002026: (SEQ ID NO:1)

[0148] MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRES

[0149] SGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKAN

[0150] MTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFG

[0151] AALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV. Claudin18.2 DNA sequence: (SEQ ID NO:2)

[0152]

[0153] Example 2: Production of anti-human claudin18.2 monoclonal antibody

[0154] 1 Immunity

[0155] Anti-human Claudin18.2 monoclonal antibodies were generated by immunizing mice.

[0156] SJL white mice were used in the experiment, female, 6 - 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2012 - 0001). Rearing environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 - hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol. The immunization antigen was huClaudin18.2 - HEK293 cells (HEK - 293 stable transfected cell line transfected with human Claudin18.2 plasmid).

[0157] Immunization protocol: Before the first immunization with cells, Gold Adjuvant (Sigma Cat No.T2684) was injected intraperitoneally (IP) into the mice at a dose of 0.1 ml / mouse; half an hour later, each mouse was injected intraperitoneally (IP) with 0.1 ml of cell suspension diluted with normal saline to a concentration of 1×10 8 / ml. After the cells were evenly dispersed, inoculation was carried out at days 0, 14, 28, 42, and 56. Blood was collected on days 21, 35, 49, and 63, and the antibody titer in the mouse serum was determined by ELISA method. After the 4th - 5th immunization, mice with high antibody titer in the serum and a titer tending to plateau were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was carried out by intraperitoneal (IP) injection of 1×10 7 cells.

[0158] 2 Spleen cell fusion

[0159] Spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells ( CRL - 8287 TM ) using PEG - mediated fusion steps to obtain hybridoma cells. The hybridoma cells were resuspended at a density of 0.5 - 1×10 6 / ml with complete medium (IMDM medium containing 20% FBS, 1×HAT, 1×OPI), seeded at 100 μl / well in 96 - well plates, incubated at 37 °C, 5% CO2 for 3 - 4 days, then supplemented with 100 μl / well of HAT complete medium, and continued to be cultured for 3 - 4 days until clones formed. The supernatant was removed, and 200 μl / well of HT complete medium (IMDM medium containing 20% FBS, 1×HT and 1×OPI) was added, and ELISA detection was carried out after culturing at 37 °C, 5% CO2 for 3 days.

[0160] 3 Hybridoma cell screening

[0161] Based on the hybridoma cell growth density, the culture supernatant was analyzed using a combined ELISA method. Cells with strong binding ability to huClaudin18.2-HEK293 cells but not to HEK293 cells were selected and promptly expanded and cryopreserved; after two to three subcloning processes, single-cell clones were obtained.

[0162] Each subcloned cell was subjected to a cell binding assay. Hybridoma clones were obtained through the above experiments, and antibodies were further prepared using serum-free cell culture. The antibodies were then purified according to the purification examples for use in the detection cases.

[0163] Example 3: Humanization of mouse antibodies

[0164] Monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; monoclonal antibody sequences from these cells were cloned, and then humanized, recombinantly expressed, and their activity was evaluated.

[0165] The procedure for cloning sequences from hybridomas is as follows: Hybridoma cells in the logarithmic growth phase are collected, RNA is extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions), and reverse transcription (PrimeScript) is performed. TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NO:3-6.

[0166] mAb1901 mouse heavy chain variable region (SEQ ID NO:3)

[0167] EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS.

[0168] mAb1901 mouse light chain variable region (SEQ ID NO:4)

[0169] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK.

[0170] mAb1902 mouse heavy chain variable region (SEQ ID NO:5)

[0171] EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPN SGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQG TTLTVSS.

[0172] mAb1902 mouse light chain variable region (SEQ ID NO:6)

[0173] DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK.

[0174] The aforementioned murine heavy chain variable region and light chain variable region are linked to the heavy chain constant region and human κ light chain constant region of the human IgG1 antibody, respectively, to form chimeric antibodies ch1901 and ch1902.

[0175] The constant region is selected from the following sequence:

[0176] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO:7)

[0177] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0178] Human κ light chain constant region: (SEQ ID NO:8)

[0179] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0180] Humanization of murine monoclonal antibodies is performed using methods disclosed in many publications in this field. In short, a human constant domain is used to replace the parental (murine antibody) constant domain, and a human germline antibody sequence is selected based on the homology between the murine and human antibodies for CDR transplantation. This invention selects candidate molecules with good activity for humanization, and the results are as follows.

[0181] 1. CDR region of murine antibody

[0182] The amino acid residues of VH / VL CDR in Table 4 were determined and annotated using the Kabat numbering system.

[0183] The CDR sequences of the murine antibodies are shown in Table 4:

[0184] Table 4. CDR sequences of murine antibodies

[0185]

[0186]

[0187] 2. Select FR region sequences from different ancestral groups

[0188] Based on the obtained typical structure of the murine antibody VH / VLCDR, the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a highly homologous human germline template. The human germline light chain framework region was derived from the human κ light chain gene.

[0189] 2.1 Humanization and Reversion Mutation Design of mAb1901

[0190] A suitable human antibody line was selected, and the mouse antibody mAb1901 was humanized. The CDR region of the mouse antibody mAb1901 was transplanted onto a selected humanized template to obtain the humanized variable region. The humanized heavy chain variable region sequence is SEQ ID NO:24, and the light chain variable region sequence is SEQ ID NO:21. This was then recombinated with the IgG constant region to form a complete antibody. Simultaneously, a reverse mutation was performed on the FR region of the V region of the humanized antibody. Exemplary reverse mutation methods and combinations are as follows:

[0191] Table 5. Humanized mAb1901 Antibody and Reversion Mutation*

[0192]

[0193] *The positions of all amino acids in the table are numbered according to the Kabat numbering rule. In the N82T of the heavy chain variable region, 82 is position 82A of the Kabat rule.

[0194] Table 6. Sequences of the light chain variable region and heavy chain variable region of the mAb1901 humanized antibody

[0195]

[0196]

[0197] The corresponding heavy chain variable regions in the table above can be linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of a full-length antibody, and the light chain variable regions can be linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of a full-length antibody. In other embodiments, the heavy chain variable regions and light chain variable regions can also be linked to other heavy chain constant regions and light chain constant regions respectively to form full-length antibodies.

[0198] 2.2 Humanization and Reversion Mutation Design of mAb1902

[0199] A suitable human antibody line was selected, and the mAb1902 murine antibody was humanized. The CDR region of the murine antibody mAb1902 was transplanted onto a selected humanized template to obtain the humanized variable region. The humanized heavy chain variable region sequence is SEQ ID NO:31, and the light chain variable region sequence is SEQ ID NO:28. This was then recombinated with the IgG constant region to form a complete antibody. Simultaneously, a reverse mutation was performed on the FR region of the V region of the humanized antibody. Exemplary reverse mutation methods and combinations are as follows:

[0200] Table 7. Humanized mAb1902 Antibody and its Reverse Mutation Design*

[0201]

[0202] *The positions of all amino acids in the table are numbered according to the Kabat numbering rule.

[0203] Table 8. Sequences of the light chain and heavy chain variable regions of the mAb1902 humanized antibody.

[0204]

[0205]

[0206] The corresponding heavy chain variable regions in the table above are linked with the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable regions are linked with the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody.

[0207] For example, the full-length antibody sequence is as follows:

[0208] Chimeric antibody ch1901:

[0209] ch1901 heavy chain: (SEQ ID NO:35)

[0210] EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0211] ch1901 light chain (SEQ ID NO:36)

[0212] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0213] Chimeric antibody ch1902:

[0214] ch1902 heavy chain (SEQ ID NO:37)

[0215] EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0216] ch1902 light chain (SEQ ID NO:38)

[0217] DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0218] Table 9. mAb1901 humanized antibody

[0219]

[0220] The full-length antibody light and heavy chain sequences are shown below:

[0221] Table 10. mAb1901 humanized antibody light and heavy chain sequences

[0222]

[0223]

[0224] Table 11. Humanized antibodies against mAb1902

[0225]

[0226] The full-length antibody light and heavy chain sequences are shown below:

[0227] Table 12. mAb1902 Humanized antibody light and heavy chain sequences

[0228]

[0229]

[0230] The positive control antibody disclosed in this paper is IMAB-362 (from WO2016166122): IMAB-362 heavy chain (SEQ ID NO: 53).

[0231]

[0232]

[0233] IMAB-362 light chain (SEQ ID NO:54)

[0234]

[0235] The antibodies were cloned, expressed, and purified using conventional gene cloning and recombinant expression methods.

[0236] In vitro bioactivity evaluation

[0237] Test Example 1: Cell-level ELISA Combined Experiment

[0238] Cell-based ELISA assays were used to detect the binding properties of Claudin18.2 antibodies. NUGC4 cells stably expressing Claudin18.2 were cultured in 96-well cell culture plates (Corning, 3599). When the cells reached 90% confluence, 4% paraformaldehyde was added to fix the cells for 1 hour. The plates were washed three times with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20), and then 200 μl / well of blocking buffer (5% skim milk, Bright Dairy skim milk powder) diluted in PBS was added. The plates were incubated at 37°C for 2.5 hours or overnight (16-18 hours) at 4°C for blocking. After blocking, the blocking buffer was discarded, and the plates were washed three times with PBST buffer. Then, 50 μl / well of different concentrations of the test antibody diluted with sample dilution buffer (pH 7.4 PBS containing 1% skim milk) was added, and the plates were incubated at 37°C for 2 hours. After incubation, wash the plate 5 times with PBST, add 100 μl / well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted with sample dilution buffer, and incubate at 37°C for 1 hour. After washing the plate 6 times with PBST, add 50 μl / well of TMB chromogenic substrate (KPL, 52-00-03), incubate at room temperature for 10-15 min, and stop the reaction by adding 50 μl / well of 1M H2SO4. Read the absorbance at 450 nm using an MD Versa Max™ microplate reader and calculate the EC50 value of Claudin18.2 antibody binding to Claudin18.2 (results are shown in the table below).

[0239] Table 13. Antibody binding activity

[0240] Antibody IMAB362 ch1901 ch1902 Emax 1.175 1.399 1.272 EC50(nM) 0.108 0.098 0.074

[0241] Table 14-1. Binding activity of humanized mAb1901 antibody

[0242] Antibody Emax EC50(nM) IMAB362 1.115 0.086 h1901-2 1.039 0.076 h1901-3 1.1055 0.22 h1901-4 0.986 0.201 h1901-6 0.937 0.091 h1901-7 0.921 0.166 h1901-8 1.047 0.091 h1901-11 1.44 0.076 h1901-12 1.22 0.116

[0243] Table 14-2. Binding activity of humanized mAb1902 antibody

[0244] Antibody Emax EC50(nM) IMAB362 0.88 0.187 h1902-1 0.87 0.113 h1902-2 0.88 0.107 h1902-3 0.84 0.175 h1902-4 0.82 0.087 h1902-5 0.9 0.098 h1902-6 0.78 0.141 h1902-7 0.75 0.121 h1902-8 0.89 0.132 h1902-9 0.75 0.137 h1902-10 0.89 0.133

[0245] Test Example 2: Antibody Cellular Binding Assay

[0246] NUGC4 cells stably expressing Claudin18.2 were prepared into 1×10⁶ cells using FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)). 6100 μl / well of cell suspension was added to each well of a 96-well round-bottom plate (Corning, 3795). After centrifugation to remove the supernatant, 50 μl / well of different concentrations of Claudin18.2 antibody diluted with FACS buffer was added, and the plates were incubated at 4°C in the dark for 1 hour. After washing three times with FACS buffer at 300g, anti-human IgG (H+L) (invitrogen, A-11013) coated with Alexa Fluor 488 at the working concentration was added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times with FACS buffer at 300g, the geometric mean fluorescence intensity was detected on a BD FACS CantoII flow cytometer, and the EC50 value of Claudin18.2 antibody binding to NUGC4 cells stably expressing Claudin18.2 was calculated. The results are shown in the figure. Figure 1 .

[0247] Test Example 3: Antibody endocytosis experiment

[0248] The Claudin18.2 antibody pre-labeled with DyLight 488NHS Ester (thermofisher, 46403) was added to a final concentration of 5 μg / ml at 1×10⁻⁶ g / ml. 6 NUGC4 cells stably expressing Claudin18.2 were incubated on ice for 1 hour in the dark. After washing three times with pre-chilled FACS buffer (pH 7.4 PBS, 2% fetal bovine serum), the supernatant was removed, and pre-warmed complete culture medium was added. The cells were then incubated at 37°C with 5% CO2. Cells were harvested at 0, 0.5, 1, 2, and 4 hours and stored on ice in the dark. After all samples were collected, the supernatant was removed by centrifugation at 300g. Elution buffer (pH 1.7, 0.05M glycine, 0.1M sodium chloride) was added, and the cells were incubated at room temperature for 7 minutes. After washing once with FACS buffer by centrifugation at 300g, the geometric mean fluorescence intensity was detected using a BD FACSCantoII flow cytometer, and the endocytosis efficiency of the Claudin18.2 antibody on NUGC4 cells stably expressing Claudin18.2 was calculated. Results showed (see...). Figure 2 Humanized antibodies have good endocytosis efficiency.

[0249] Test Example 4: Measurement of Antibody Affinity Based on Flow Cytometry

[0250] On the day of the experiment, HEK293 / hClaudin18.2 cells were collected in 96-well U-bottom plates, 1×10⁶ cells per well. 5 Up to 2×10 5Cells were added. Claudin18.2 antibody was added at an initial concentration of 5 μg / ml, serially diluted 2× (12 concentration points), and incubated at 4°C for 1 hour. IMAB362 was used as a positive control, and a negative control without antibody was also included. After centrifugation to remove the antibody, 100 μl / well of FITC anti-human IgG Fc antibody (200×) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS + 2% FBS before flow cytometry analysis. BD FACS CantoII was started, and after warm-up, BD FACSDiva software was opened to create a new experiment to detect HEK293 / h Claudin18.2 negative control samples. The FSC and SSC voltages were adjusted to appropriate values ​​and saved. (Based on Quantum...) TM Following the FITC-5MESF Kit instructions, blank sample B and standard curve 1 were tested separately. The FITC voltage was adjusted to an appropriate value and saved. Samples in a 96-well plate with a U-shaped bottom were tested at the saved voltage, and the data were recorded. The experimental data were analyzed using Flowjo software to obtain the GeoMean value, based on the Quantum... TM The FITC-5MESF Kit instructions were used to fit the MESF-Geo Mean standard curve. Based on the concentration fluorescence value of the FITC anti-human IgG Fc antibody, the molar concentration of Claudin18.2 antibody binding to HEK293 / hClaudin18.2 cells and the concentration of free antibody were calculated. The Bmax and dissociation constant KD of the antibody were calculated using the Scatchard plot method. The results are shown in Table 15.

[0251] Table 15. Cellular Affinity of Humanized Antibodies

[0252] Antibody IMAB362 h1901-11 h1902-5 KD(nM) 10.2 6.8 1.64

[0253] Test Example 5: Evaluation of Antibody ADCC Effect

[0254] Various NUC4 cells (high, medium, and low expression of Claudin 18.2) were digested, centrifuged at 1000 rpm, resuspended, and counted. Cells were then cultured at 3 × 10⁻⁶ cells / year. 5 Cells were resuspended at a density of 10% FBS (New Zealand ultra-low IgG fetal bovine serum, Gibco, 1921005PJ) in phenol red-free RPMI 1640 (Gibco, 11835-030). 25 μl of cells (7500 cells / well) was added to each well of a 96-well plate (Corning, 3903). Antibody was diluted in the above phenol red-free medium to prepare a 3× antibody dilution, and 25 μl of antibody was added to each well of the cell plate. The plates were incubated at 37°C in a 5% CO2 incubator for 0.5 hours.

[0255] Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, resuspended, and counted. Cells were then cultured at 3 × 10⁻⁶ cells / year. 6 Cells were resuspended at a density of 10% FBS (New Zealand ultra-low IgG fetal bovine serum) in phenol red-free RPMI 1640, and 25 μl of cells (7.5 × 10⁻⁶ cells / ml) were added to each well of the experimental plate. 4 (cells / well). Incubate at 37°C, 5% CO2 for 6 hours.

[0256] Add 75 μl of Bright-Glo (Promega, E2610) to each well of the experimental plate and detect chemiluminescence using a microplate reader (PerkinElmer, VITOR3).

[0257] The results show (see Table 16 and...) Figures 3A-3C In NUGC4 cells with low, medium and high levels of Claudin18.2 expression, antibodies h1901-11 and h1902-5 both showed strong ADCC activity.

[0258] Table 16. ADCC effect of antibody in NUC4 cells with different levels of Claudin18.2 expression

[0259]

Claims

1. An anti-Claudin18.2 antibody comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

2. The anti-Claudin18.2 antibody according to claim 1 is a murine antibody, a chimeric antibody, or a humanized antibody.

3. The anti-Claudin18.2 antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region as shown below: i) The heavy chain variable region sequence is shown in SEQ ID NO:3 and the light chain variable region sequence is shown in SEQ ID NO:4; ii) The heavy chain variable region sequence is shown in SEQ ID NO:25 and the light chain variable region sequence is shown in SEQ ID NO:21; iii) The heavy chain variable region sequence is shown in SEQ ID NO:25 and the light chain variable region sequence is shown in SEQ ID NO:22; iv) The heavy chain variable region sequence is as shown in SEQ ID NO:26 and the light chain variable region sequence is as shown in SEQ ID NO:23; or v) The heavy chain variable region sequence is shown in SEQ ID NO:27 and the light chain variable region sequence is shown in SEQ ID NO:

22.

4. The anti-Claudin18.2 antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region as shown below: The heavy chain variable region sequence is shown in SEQ ID NO:26 and the light chain variable region sequence is shown in SEQ ID NO:

23.

5. The anti-Claudin18.2 antibody according to claim 1, further comprising a heavy chain constant region and a light chain constant region.

6. The anti-Claudin18.2 antibody according to claim 5, wherein the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their variants.

7. The anti-Claudin18.2 antibody according to claim 5, wherein the anti-Claudin18.2 antibody comprises the heavy chain constant region shown in SEQ ID NO:7 and the light chain constant region shown in SEQ ID NO:

8.

8. The anti-Claudin18.2 antibody according to claim 1, comprising: a) The heavy chain shown in SEQ ID NO:35 and the light chain shown in SEQ ID NO:36; b) The heavy chain shown in SEQ ID NO:43 and the light chain shown in SEQ ID NO:39; c) The heavy chain shown in SEQ ID NO:43 and the light chain shown in SEQ ID NO:40; d) The heavy chain shown in SEQ ID NO:44 and the light chain shown in SEQ ID NO:41; or e) The heavy chain shown in SEQ ID NO:45 and the light chain shown in SEQ ID NO:

40.

9. The anti-Claudin18.2 antibody according to claim 1, comprising: the heavy chain shown in SEQ ID NO:44 and the light chain shown in SEQ ID NO:

41.

10. A nucleic acid molecule encoding the anti-Claudin18.2 antibody as described in any one of claims 1 to 9.

11. A host cell comprising the nucleic acid molecule as described in claim 10.

12. A pharmaceutical composition comprising a therapeutically effective amount of the anti-Claudin18.2 antibody according to any one of claims 1 to 9, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

13. A kit comprising the anti-Claudin18.2 antibody according to any one of claims 1 to 9.

14. Use of the anti-Claudin18.2 antibody according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 12, in the preparation of a medicament for treating tumors associated with Claudin18.2, said tumors being selected from: head and neck cancer, esophageal cancer, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

15. The use according to claim 14, wherein the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer.

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