RU2822550C2 - Anti-claudin 18.2 antibody and use thereof - Google Patents

Abstract

FIELD: chemistry; biotechnology.

SUBSTANCE: invention relates to biochemistry, in particular to an antibody which specifically binds to claudin 18.2. Also disclosed is a nucleic acid molecule coding said antibody; host cell containing said nucleic acid molecule; conjugate of said antibody; pharmaceutical composition containing said antibody. Disclosed are a method of treating a disease and a method of immunological analysis, in which said antibody is used.

EFFECT: invention provides effective treatment of diseases associated with claudin 18.2.

19 cl, 3 dwg, 16 tbl, 3 ex

Description

This application claims priority to patent application 2019102578536, filed April 1, 2019, which is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to the field of antibody-based drugs. Specifically, the present disclosure relates to antibodies against claudin 18.2 and their uses.

Prior Art

The descriptions herein offer only general information about the present disclosure and do not necessarily constitute prior art.

Claudin-18 (CLDN18) is a protein encoded by the human claudin 18 gene and belongs to the family of tight junction proteins in cells. Claudin-18 may control the flow of molecules between cell layers.

The claudin-18 protein structurally includes four transmembrane regions and two extracellular loops, with the N-terminus and C-terminus located inside the cytoplasm. Claudin-18 has two splice variants, claudin 18.1 and claudin 18.2. The two sequences differ from each other by only eight amino acids in the first extracellular loop. Claudin 18.1 is expressed and distributed in a manner different from claudin 18.2. Claudin 18.1 is selectively expressed in normal lung cells, whereas claudin 18.2 expression is highly restricted in normal cells but is often ectopically activated and overexpressed in a variety of tumors (gastric cancer, lung cancer, pancreatic cancer, etc.). Claudin 18.2 is considered a possible therapeutic target for gastric cancer and other types of cancer. The discovery of this target also offers a new treatment option for gastric cancer.

Summary of the invention

The present disclosure provides an anti-claudin 18.2 antibody.

In some embodiments, an anti-claudin 18.2 antibody, as described above, comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region contains the same sequence of HCDR1, HCDR2 and HCDR3 as the sequences in the heavy chain variable region as shown in SEQ ID NO: 3, and the light chain variable region contains the same sequence of LCDR1, LCDR2 and LCDR3 as the sequences in the light chain variable region as shown in SEQ ID NO: 4; or

ii) the heavy chain variable region contains the same sequence of HCDR1, HCDR2 and HCDR3 as the sequences in the heavy chain variable region as shown in SEQ ID NO: 5, and the light chain variable region contains the same sequence of LCDR1, LCDR2 and LCDR3 as the sequences in the light chain variable region as shown in SEQ ID NO: 6. In some embodiments, the anti-claudin 18.2 antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein:

iii) the heavy chain variable region contains 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 contains LCDR1, LCDR2 and LCDR3 as shown in NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or

iv) the heavy chain variable region contains 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 contains LCDR1, LCDR2 and LCDR3 as shown in NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

Those skilled in the art should understand that item numbers such as i), ii), a), b), etc. are provided only to make the technical solutions or elements listed more clear and easily identifiable, but do not limit in any way the following technical solutions or elements. When the same item number is used, this does not mean that the following technical solutions or elements are the same.

In some embodiments of the anti-claudin 18.2 antibody, as described above, the anti-claudin 18.2 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

In some embodiments, an anti-claudin 18.2 antibody, as described above, comprises a heavy chain variable region and a light chain variable region, wherein:

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

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

In some embodiments, an anti-claudin 18.2 antibody, as described above, comprises a heavy chain variable region and a light chain variable region, wherein:

(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 SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is such as as shown in SEQ ID NO: 4, or has at least 90% identity with SEQ ID NO: 4;

(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 SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is such as as shown in SEQ ID NO: 21, or has at least 90% identity with SEQ ID NO: 21;

(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 SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is such as as shown in SEQ ID NO: 6, or has at least 90% identity with SEQ ID NO: 6; or

(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 SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is such as as shown in SEQ ID NO: 28, or has at least 90% identity with SEQ ID NO: 28.

In some embodiments of the anti-claudin 18.2 antibody, as described above, the anti-claudin 18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody framework region, or a variant framework region, and the variant framework region has a maximum of 10 back mutations on each of the light chain framework regions and/or the heavy chain framework regions of the human antibody.

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

In some embodiments, preferably the framework region variant contains mutation(s) selected from the following (a) or (b):

(a) one or more reverse mutations of amino acids 22S, 85I or 87H contained in the light chain variable region, and/or one or more reverse mutations selected from the group consisting of 48I, 82T and 69M contained in the heavy chain variable region; or

(b) one or more back mutations of amino acids selected from the group consisting of 4L and 22S contained in the light chain variable region, and/or one or more back mutations selected from the group consisting of 38K, 40R, 481, 66K, 67A, 69, 71L and 73K contained in the heavy chain variable region.

In some embodiments of the anti-claudin 18.2 antibody, as described above, the framework region variant contains mutation(s) selected from the following (a-1) or (b-1):

(a-1) back mutations of amino acids 22S, 85I and 87H contained in the light chain variable region, and back mutations of amino acids 48I and 82T contained in the heavy chain variable region; or

(b-1) back mutation of amino acid 4L contained in the light chain variable region.

In some embodiments, anti-claudin 18.2 antibodies, as described above, wherein:

(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

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

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

(x) the heavy chain variable region sequence is as shown in SEQ ID NO: 31, 32, 33 or 34, and the light chain variable region sequence is as shown in SEQ ID NO: 28, 29 or 30;

In some embodiments of the anti-claudin 18.2 antibody as described above, the anti-claudin 18.2 antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, as shown below:

(xi) a heavy chain variable region sequence as shown in SEQ ID NO: 31 and a light chain variable region sequence as shown in SEQ ID NO: 29; or

(xii) a heavy chain variable region sequence as shown in SEQ ID NO: 26 and a light chain variable region sequence as shown in SEQ ID NO: 23.

In some embodiments of the anti-claudin 18.2 antibodies as described above, the light chain variable region and the heavy chain variable region may be a combination of the light and heavy chain variable regions, as shown in the following table:

In some embodiments of the anti-claudin 18.2 antibody as described above, the antibody further comprises antibody constant region(s). In some specific embodiments, the antibody heavy chain constant region is selected from the group consisting of the lgG1, lgG2, lgG3, and lgG4 constant region(s) and variant(s) thereof, and the antibody light chain constant region is selected from the group consisting of kappa region(s), lambda chain of human antibodies and variant(s) thereof. In some specific embodiments, the antibody contains a heavy chain constant region sequence as shown in SEQ ID NO: 7, and a light chain constant region sequence as shown in SEQ ID NO: 8. In some embodiments, the antibody contains a heavy chain having at least 90 %, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity as 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% identical to the amino acid sequence as shown in SEQ ID NO: 36 or 39; or

heavy chain having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 37 or 49, and/or a light chain having at least 90%, 92%, 94%, 95%, 96% 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 38 or 46.

In some embodiments, the anti-claudin 18.2 antibody, as described above, contains:

(c) a heavy chain, as shown in the sequence SEQ ID NO: 35, and/or a light chain, as shown in the sequence SEQ ID NO: 36;

(d) a heavy chain as shown in SEQ ID NO: 42, 43, 44 or 45, and/or a light chain as shown in SEQ ID NO: 39, 40 or 41;

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

(f) a heavy chain as shown in SEQ ID NO: 49, 50, 51 or 52, and/or a light chain as shown in SEQ ID NO: 46, 47 or 48.

In some embodiments of the anti-claudin 18.2 antibody as described above, the antibody competes with an anti-claudin 18.2 antibody or an antigen binding fragment thereof as described above for binding to human claudin 18.2.

In some embodiments, the anti-claudin 18.2 antibody, as described above, contains:

a heavy chain as shown in the amino acid sequence of SEQ ID NO: 44, and a light chain as shown in the sequence of SEQ ID NO: 41; or

a heavy chain as shown in the amino acid sequence of SEQ ID NO: 49, and a light chain as shown in the sequence of SEQ ID NO: 47.

According to yet another aspect of the present disclosure, there is also provided a nucleic acid molecule that encodes an anti-claudin 18.2 antibody as described above.

According to yet another aspect of the present disclosure, an expression vector comprising a nucleic acid molecule as described above is also provided.

According to yet another aspect of the present disclosure, a host cell is also provided that contains a nucleic acid molecule as described above or an expression vector as described above, preferably the cell is a bacterial cell, a fungal cell, an insect animal cell or a mammalian cell.

According to yet another aspect of the present disclosure, there is also provided an antibody-drug conjugate that is formed by conjugating an anti-claudin 18.2 antibody as described above with a cytotoxic drug.

According to yet another aspect of the present disclosure, there is also provided an antibody-drug conjugate comprising or consisting of an anti-claudin 18.2 antibody as described above covalently bound to a cytotoxic drug.

In some embodiments, the present disclosure provides a method for producing an anti-claudin 18.2 antibody as described above.

In some embodiments, the present disclosure provides a method for preparing an anti-claudin 18.2 antibody-drug conjugate as described above.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-claudin 18.2 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.

In some embodiments, the present disclosure provides a method for immunoassaying or detecting claudin 18.2, comprising the step of contacting the above-mentioned anti-claudin 18.2 antibody with a test sample.

In some embodiments, the present disclosure provides the use of an anti-claudin 18.2 antibody, as described above, to produce a reagent for human claudin 18.2 immunoassay.

In some embodiments, the present disclosure provides an anti-claudin 18.2 antibody, as described above, for use in an immunoassay or detection of claudin 18.2.

In some embodiments, the present disclosure provides a kit comprising an anti-claudin 18.2 antibody as described above.

In some embodiments, the present disclosure provides the use of an anti-claudin 18.2 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, for the preparation of a drug for treating a cancer or tumor, wherein the cancer or tumor is preferably a claudin 18.2 positive cancer or malignancy, more preferably a head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central cancer nervous system, neuroendocrine tumor, hypopharyngeal carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular tumor, hepatocellular carcinoma, liver and gallbladder cancer, pancreatic cancer, cancer stomach, gastrointestinal cancer, intestinal cancer, colon cancer, colon and rectal cancer, kidney cancer, clear cell renal carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, cancer skin, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myeloproliferative tumor, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma; the lymphoma is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small cell lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; lung cancer is selected from the group consisting of: non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.

In some embodiments, the present disclosure provides a method of treating a disease associated with claudin 18.2, comprising administering to a subject a therapeutically effective amount of an anti-claudin 18.2 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, a claudin 18.2 positive cancer or malignancy is more preferably selected from the group consisting of the following: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, hypopharyngeal carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular tumor, hepatocellular carcinoma, liver and gallbladder cancer, pancreatic cancer, stomach cancer, cancer gastrointestinal tract, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal 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 tumor, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma. More preferably, the lymphoma is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small cell lymphocytic lymphoma, T-cell rich large B-cell lymphoma / histiocytes and lymphoplasmacytic lymphoma; lung cancer is selected from the group consisting of: non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.

In some embodiments, a therapeutically effective amount refers to 0.1 mg to 3000 mg or 1 mg to 1000 mg of an anti-claudin 18.2 antibody as described above or an antibody-drug conjugate as described above contained in a single dose of the composition.

In some embodiments, the present disclosure provides an anti-claudin 18.2 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, for use in treating a disease, associated with claudin 18.2, where the disease is preferably cancer or tumor; more preferably, a claudin 18.2-positive cancer or malignancy, more preferably selected from the group consisting of the following: squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, hypopharyngeal carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular tumor, hepatocellular carcinoma, liver and gallbladder cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer -intestinal tract, intestinal cancer, colon cancer, colon and rectal cancer, kidney cancer, clear cell renal 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 tumor, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma. More preferably, the lymphoma is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small cell lymphocytic lymphoma, T-cell rich large B-cell lymphoma / histiocytes and lymphoplasmacytic lymphoma; lung cancer is selected from the group consisting of: non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.

In some embodiments, the cancer is gastric cancer, esophageal cancer, lung cancer, or pancreatic cancer.

In some embodiments, the antibody or antibody-drug conjugate may, as described above, play a therapeutic role in cancers with high, medium and low levels of claudin 18.2 expression, as described above.

The anti-claudin 18.2 antibody and antibody-drug conjugate proposed in the present disclosure have excellent affinity for cell surface antigens, favorable endocytic efficiency, strong tumor inhibition efficiency and a wider range of pharmaceutical applications, and are suitable for clinical use as a drug.

Description of graphic materials

Fig. 1: results of FACS analysis (from the English fluorescence-activated cell sorting) of the binding of humanized antibodies to human claudin 18.2 at the cellular level.

Fig. 2: Endocytosis assay of humanized antibodies in NUGC4 cells.

Fig. 3A-Fig. 3C: detection of ADCC (antibody-dependent cell-mediated cytotoxicity) effect of antibodies in NUGC4 cells with different levels of claudin expression 18.2. In FIG. 3A shows the detection of the ADCC effect of antibodies on wild-type NUGC4 cells (with low levels of claudin 18.2 expression); In FIG. 3B shows the detection of the ADCC effect of antibodies on NUGC4 cells with an average claudin expression level of 18.2; In FIG. Figure 3C shows the ADCC effect of antibodies on NUGC4 cells with high levels of claudin 18.2 expression.

Detailed Description of the Invention

Terminology

To make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this disclosure pertains.

The three-letter codes and single-letter codes for amino acids used in the present disclosure are those described in J. Biol. Chem, 243, p3558 (1968).

The term "cytotoxic drug" refers to a substance that inhibits or prevents cell function and/or leads to cell death or destruction. A cytotoxic drug includes compounds that can be used to kill cells, such as toxins, chemotherapeutic agents and others.

The term "toxin" refers to any substance capable of causing deleterious effects on cell growth or proliferation, and such substance may be a low molecular weight toxin and its derivative from bacteria, fungi, plants or animals, including camptothecin derivatives such as exatecan, maytansinoids and their derivatives (CN101573384), such as DM1, DM3, DM4, orlistatin F (AF) and their derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, chain A abrin, modecin, α-sarcin, toxic protein of Aleutites fordii, toxic protein diantin, toxic protein of Phytolaca americana (PAPI, PARI and PAP-S), inhibitor of Momordica charantia, curcin, crotin, inhibitor of Sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

The term "chemotherapeutic drug" is a compound that can be used to treat tumors. Such a definition also includes antihormonal agents capable of modulating, reducing, blocking, or inhibiting hormonal actions that might stimulate tumor growth, and are typically presented as systemic or systemic therapies. They may be hormones themselves. Examples of chemotherapeutic agents include alkylating agents such as thiotepa; cyclosfamide (CYTOXAN™); alkyl sulfonate such as busulfan, improsulfan and pyrosulfan; aziridine such as benaodopa, carboquone, meturedopa and uredopa; aziridine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide; melphalan, novembicin phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as clarithromycin, actinomycin, outramycin, azaserin, bleomycin, cactinomycin, calicheamicin, carabicin, chromomycin, carcinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-oxy-L-norleucine, doxorubicin, epirubicin, ezorubicin, idarubicin , marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin; streptozocin, tubercidin, ubenimex, cinostatin, zorubicin; antimetabolites such as methotrexate, 5-FU; folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; methotrexate analogs such as fludarabine, 6-mercaptoptrerin, thiometopterin, tioguanopterin; pyrimidine analogs such as ancecitabine, azacitidine, 6-azuridine, carmofur, carmofur, cytarabine, dideoxyuridine, doxyluridine, enocytabine, fluorouridine, 5-FU; androgens such as calusterone, drostanolone propionate, epithiostanol, mepithiostane, testolactone; antiadrenergic agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; glucurone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntren; edatraxate; defofamine; colchicine; diaziquinone; elfomitin; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pintostatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; Alternaria tenuis keto acid; triiminequinone; 2,2',2''-trichlorotriethylamine; urethane; vinblastine amide; dacarbazine; mannitol mustard; mitobronitol; dibromoalcohol of euonymus; pipobromance; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepu; taxanes such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinau; etoposide (VP-16); iphosphate; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunorubicin; aminopterin; Xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoic acid; esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition also includes antihormonal agents that can modulate or inhibit the effects of hormones on tumors. For example, antiestrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxyltamoxifene, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

As used herein, the term "antibody" refers to an immunoglobulin, and a complete antibody is a four-peptide chain structure formed by two identical heavy chains linked to two identical light chains by interchain disulfide(s). ) connection(s). Immunoglobulin heavy chain constant regions exhibit different amino acid compositions and arrangements and therefore present different antigenicity. Accordingly, immunoglobulins can be divided into five types, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ, δ, γ, α and ε, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same lg type can be divided into different subtypes; for example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. Light chains can be subdivided into κ or λ chain, taking into account different constant regions. Each of the five types of IgG can have a kappa chain or a lambda chain.

The approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are highly variable, known as variable regions (Fv regions); the remaining amino acid sequences close to the C-terminus are relatively stable, known as constant regions. The variable region includes 3 hypervariable regions (HVR - from the English hypervariable region) and 4 frame regions (FR - from the English framework region) with relatively conservative sequences. These three hypervariable regions, which determine the specificity of the antibody, are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and each heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. These three light chain CDR regions are LCDR1, LCDR2 and LCDR3; and these three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “mouse antibody” refers to monoclonal antibodies against human claudin 18.2 prepared in accordance with knowledge and skill in the art. During production, the test subject is injected with claudin 18.2 or an epitope thereof as an antigen, and a hybridoma expressing an antibody that has the desired sequence or functional characteristics is then isolated. In a preferred embodiment of the present disclosure, the mouse anti-claudin 18.2 antibody or antigen binding fragment thereof further comprises a mouse κ, λ light chain constant region, or a variant thereof, or further comprises a mouse IgG1, IgG2, IgG3 heavy chain constant region, or a variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a mouse antibody together with the constant region of a human antibody, and such antibody can mitigate the immune response caused by the mouse antibody. To create a chimeric antibody, a hybridoma secreting a specific mouse monoclonal antibody is first created, and the variable region gene is cloned from the mouse hybridoma. The constant region gene is then cloned from the human antibody if necessary. A mouse variable region gene is fused to a human constant region gene to form a chimeric gene that can subsequently be inserted into an expression vector. Finally, the chimeric antibody molecule will be expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light capa constant region, a human lambda chain, or a variant thereof. The antibody heavy chain of the chimeric anti-claudin 18.2 antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably contains a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably contains a heavy chain constant region human lgG1, lgG2 or lgG4 or contains a heavy chain constant region of lgG1, lgG2 or lgG4 with amino acid mutation(s) (such as the L234A and/or L235A mutation, and/or the S228P mutation).

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody generated by grafting non-human CDR sequences into human antibody variable region frameworks, namely, an antibody produced in different types of human germline antibody framework sequences . Humanized antibodies can overcome heterologous responses caused by chimeric antibodies that carry a large number of heterologous protein components. Such scaffold sequences can be obtained from a publicly available DNA database covering germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid the reduction in activity caused by reduced immunogenicity, framework sequences in the variable region of a human antibody may be subject to minimal back mutations or back mutations while maintaining activity. Humanized antibodies of the present disclosure also refer to humanized antibodies subjected to CDR affinity maturation via yeast display.

In one embodiment of the present disclosure, the antibody or antigen binding fragment thereof further comprises a light chain constant region derived from a human or murine κ, λ chain, or a variant thereof, or further comprises a heavy chain constant region derived from human or murine IgG1, IgG2, IgG3, IgG4, or its variant; preferably contains a heavy chain constant region derived from human lgG1, lgG2 or lgG4, or a variant of lgG1, lgG2 or lgG4 with amino acid mutation(s) (such as the L234A and/or L235A mutation, and/or the S228P mutation).

As described herein, human antibody heavy chain constant region and light chain constant region "variants" refer to heavy and light chain constant region variants disclosed in the prior art that are not altered in the structure and function of the variable areas of the antibody. Exemplary variants include heavy chain constant region variants lgG1, lgG2, lgG3, or lgG4 obtained through site-directed engineering and amino acid substitutions on the heavy chain constant region. Specific substitutions are, for example, the YTE mutation, the L234A and/or L235A mutation, the S228P mutation, and/or mutations resulting in a knob-to-valley structure (which results in the antibody heavy chain having a knob-Fc combination and depression-Fc). It turned out that these mutations impart new properties to the antibody without affecting the function of the variable region of the antibody.

The terms “human antibody (Humab)”, “human-derived antibody”, “full-length human antibody” and “full human antibody” are used interchangeably and could represent human-derived antibodies or genetically modified antibodies. an organism that is “engineered” by any method known in the art to produce specific human antibodies in response to stimulation by an antigen. In some technologies, elements of the human heavy and light chain loci are introduced into cell lines derived from embryonic stem cell lines in which the endogenous heavy and light chain loci are targeted for disruption. Transgenic organisms can synthesize human antibodies specific for human antigens, and these organisms can be used to produce hybridomas that secrete human antibodies. A human antibody may also be one in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Full length human antibodies can also be constructed using gene or chromosomal transfection techniques and phage display technology, or constructed from in vitro activated B cells, all methods known in the art.

The terms “full-length antibody,” “whole antibody,” and “complete antibody” are used interchangeably herein and refer to an antibody in substantially complete form that is distinct from the antigen-binding fragments defined below. The term specifically refers to antibodies that contain constant regions in the light and heavy chains. "Antibody" as used herein includes "full-length antibodies" and antigen-binding fragments thereof.

In some embodiments, a full-length antibody of the present disclosure includes full-length antibodies formed by linking a light chain variable region to a light chain constant region, and linking a heavy chain variable region to a heavy chain constant region, as shown in the table below in a combination of light and heavy chains. Those skilled in the art can select the light chain constant region and the heavy chain constant region from different antibody sources according to the actual need, such as the human antibody-derived light chain constant region and the heavy chain constant region.

The term "antigen binding fragment" or "functional fragment" refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, claudin 18.2). It has been shown that full-length antibody fragments can be used to achieve antigen binding function. Examples of binding fragments included in the term “antigen binding fragment” of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) fragment F(ab') ₂ , a bivalent fragment formed by two Fab fragments connected by a disulfide bridge in the hinge region, (iii) fragment Fd, consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of one arm of the antibody; (v) dsFv, an antigen binding fragment formed by VH and VL through interchain disulfide bonds; and (vi) a diabody, bispecific antibody and multispecific antibody containing fragments such as scFv, dsFv and Fab. In addition, the VL domain and the VH domain of the Fv fragment are linked by a synthetic linker to form a single protein chain, which is a monovalent molecule formed by combining the VL and VH domains (called single chain Fv (scFv); see, for example, Bird et al (1988) Science 242: 423-426; and Huston et al (1988) Proc. Sci USA85:5879-5883). Such single chain antibodies are also intended to be included within the term “antigen binding fragment” of an antibody. Such antibody fragments are prepared using conventional techniques known in the art and are screened for functional fragments using the same method as for the intact antibody. Antigen-binding sites can be obtained through genetic engineering technology or through enzymatic or chemical destruction of intact immunoglobulin. Antibodies can be in the form of different isotypes, for example, IgG antibodies (for example, the IgG1, IgG2, IgG3 or IgG4 subtype), IgG1, IgG2, IgD, IgE or IgM.

A Fab is an antibody fragment with antigen-binding activity produced by treating an IgG antibody molecule with an enzyme that has the same activity as papain.

F(ab') ₂ is an antibody fragment with antigen-binding activity resulting from the cleavage of IgG by an enzyme with the same activity as pepsin.

Fab' is an antibody fragment having antigen-binding activity obtained by cleavage of the above-mentioned F(ab') ₂ .

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

The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) connected to an antibody light chain variable domain (or region; VL) via a linker. Such scFv molecules have the general structure NH ₂ -Vl-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Other linkers that may be used in the present disclosure are described in the following documents, such as, 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.

A diabody is an antibody fragment in which the scFv or Fab is dimerized, and it is an antibody fragment that has bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens may be the same or different.

Bispecific and multispecific antibodies refer to an antibody that can bind to two or more antigens or antigenic determinants.

dsFv is produced by replacing one amino acid residue in each of VH and VL with a cysteine residue, and then joining the polypeptides with a disulfide bond between two cysteine residues. The amino acid residues to be replaced by a cysteine residue can be selected based on prediction of the three-dimensional structure of the antibody according to known methods (eg, Protein Engineering, 7, 697 (1994)).

The term "amino acid difference" or "amino acid mutation" refers to changes or mutations in an amino acid in a variant protein or polypeptide compared to the parent protein or polypeptide, including 1, 2, 3 or more insertions, deletions or substitutions of amino acids, based on the parent protein or polypeptide.

The term “antibody framework” or “FR region” refers to the portion of a variable domain, either VL or VH, that serves as a framework for the antigen binding loops (CDRs) of that variable domain. Essentially, it is a variable domain without a CDR.

The term “complementarity determining region,” “CDR,” or “hypervariable region” refers to one of the six hypervariable regions present in the variable domain of an antibody that primarily facilitate antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) are found in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. CDR amino acid sequence boundaries can be determined using any of a variety of well-known schemes, including the "Kabat" numbering criteria (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering criteria (see Al-Lazikani et al., (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering criteria (Lefranc MP, Immunologist, 7, 132 -136 (1999); Lefranc, MP, etc., Dev. Comp., 27, 55-77 (2003)), etc. For example, in the case of the classical format, following the Kabat criteria, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to Chothia criteria, the amino acid residues of the CDRs in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). Combining both Kabat and Chothia to define CDRs, CDRs consist of amino acid residues 26–35 (HCDR1), 50–65 (HCDR2), and 95–102 (HCDR3) in human VH and amino acid residues 24–34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3) in human VL. According to IMGT criteria, CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). According to IMGT criteria, antibody CDR regions can be identified using the IMGT/DomainGapAlign program.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody binds (eg, a specific site on a claudin 18.2 molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-sequential amino acids in a unique tertiary conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, ed. G.E. Morris, Ed. (1996).

The term “specifically binds to,” “selectively binds to,” “selectively binds to,” or “specifically binds to” refers to the binding of an antibody to a given epitope on an antigen. Typically, an antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, or even less.

The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, an antibody of the present disclosure binds to claudin 18.2 or an epitope thereof with a dissociation constant (KD) of less than about 10 ^-7 M, for example, less than about 10 ^-8 M or 10 ^-10 M, for example, the affinity of the antibody in the present disclosure is cell surface antigen was determined by measuring the KD value by FACS.

When the term "competition" is used in the context of antigen-binding proteins that compete for the same epitope, it means that competition occurs between antigen-binding proteins as determined by an assay in which the antigen-binding protein being tested (e.g., an antibody or a functional fragment thereof) , prevents or inhibits (eg, reduces) the specific binding of a reference antigen binding protein (eg, a ligand or a reference antibody) to a common antigen (eg, claudin 18.2 antigen or a fragment thereof). Many types of competitive binding assays are available to determine whether one antigen binding protein competes with another. These tests are, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), competitive “sandwich” analysis (see, for example, Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid-phase direct EIA with biotin-avidin (see, for example, Kirkland et al, 1986, J. Immunol. 137: 3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA based on I ^-125 labeling (see, for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid-phase direct RIA based on biotin-avidin labeling (see, for example, Cheung, et al, 1990, Virology 176: 546-552); and direct labeling-based RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen capable of binding to a solid surface or cell, which is loaded with both an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is determined by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Typically, the antigen binding protein being tested is in excess. Antigen-binding proteins identified by competition analysis (competition with antigen-binding protein) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope that is sufficiently close to the epitope to which a reference antigen-binding protein binds, where the two epitopes interfere spatially with each other, preventing binding. Additional details regarding methods for determining competitive binding are provided herein in the Examples section. Typically, when a competing antigen binding protein is in excess, it will inhibit (e.g., reduce) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70 %, 70-75% or 75% or even more specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97% or even more.

The term "nucleic acid molecule", as used herein, refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that coding sequence.

The term "amino acid sequence identity" refers to the percentage of amino acid residues that are identical between the first and second sequences when the given amino acid sequences are aligned (introducing gaps as necessary) to achieve the maximum percentage of sequence identity, and any conservative substitution is not considered part of the sequence identity. To determine percent amino acid sequence identity, alignment can be achieved in a variety of ways within a given region, for example, using publicly available software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid", which refers to a loop of circular double-stranded DNA into which additional DNA segments can be ligated. In yet another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replicator and mammalian episomal vectors), or could be integrated into the genome of the host cell when introduced into the cell. host and are thus replicated along with the host genome (e.g. non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments thereof are well known in the art, for example, Antibodies: a Laboratory Manual, Cold Spring Harbor, New York, chapters 5-8 and 15. For example, mice can be immunized with human claudin 18.2 or a fragment thereof, and the resulting antibodies can then be annealed, purified and sequenced for amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be prepared by conventional methods. Antibodies or antigen binding fragments thereof of the present disclosure are constructed by including one or more human framework regions onto CDR regions derived from a non-human antibody. Human germline FR sequences can be obtained from ImMunoGeneTics (IMGT) via the website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351, by alignment against human germline antibody variable region gene database sequences. using MOE software.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, 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 NS0 cells.

Antibodies or antigen-binding fragments thereof of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. A vector expressing recombinant immunoglobulin can be stably transfected into host cells. As a more recommended prior art, mammalian expression systems can result in glycosylation of antibodies, especially at highly conserved N-terminal regions in the Fc region. Stable clones were generated by expressing an antibody that specifically binds to human claudin 18.2. Positive clones can be propagated in bioreactors to produce antibodies. The culture medium into which the antibody has been secreted can be purified using conventional techniques. For example, purification can be performed on a Protein A or G Sepharose FF column that has been equilibrated with buffer. Nonspecific binding components are washed away. The bound antibody is eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and then combined. Antibodies can be filtered and concentrated using conventional techniques. Soluble mixtures and multimers can be effectively removed by conventional techniques such as gel filtration or ion exchange. The resulting product is then immediately frozen, for example at -70°C, or can be lyophilized.

The terms “administration,” “dosing,” and “treatment,” when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to bringing an exogenous pharmaceutical, therapeutic, diagnostic agent or composition into contact with the animal, human , subject, cell, tissue, organ or biological fluid. The terms "administration", "dosing" or "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Cell processing includes bringing a reagent into contact with the cell, as well as bringing the reagent into contact with a liquid, which liquid, in turn, is in contact with the cell. The term “administration,” “dosing,” or “treatment” also means in vitro or ex vivo treatment of, for example, a cell, reagent, diagnostic, binding compound, or other cell. The term "treatment", when applied to a human subject, veterinary subject or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

The term “treat” means administering a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments thereof of the present disclosure, orally or externally to a patient having one or more symptoms of a disease for which the therapeutic agent has known therapeutic activity. Typically, the agent is administered in an amount effective to relieve one or more symptoms of a disease in the patient or population being treated by causing regression or inhibiting the progression of such symptom(s) to any clinically measurable extent. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also called a "therapeutically effective amount") may vary depending on various factors such as the pathological condition, the age and body weight of the patient, and the ability of the drug to produce the desired effect. patient's response. Whether a disease symptom has been alleviated can be assessed by any clinical measure commonly used by treating physicians or other qualified health care professionals to assess the severity or progression status of that symptom. While one embodiment of the present disclosure (e.g., a treatment method or product of manufacture) may not be effective in alleviating the target disease symptom(s) in every patient, it should improve the target disease symptom(s) in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student's t test, the chi-square test, the Mann and Whitney U test, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon.

The term "conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), thus that these changes can often occur without changing the biological activity of the protein. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., page 224, 4th edition). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Typical conservative substitutions are outlined below in the table entitled "Typical Amino Acid Conservative Substitutions".

The term “effective amount” or “effective dose” refers to the amount of a drug, compound or pharmaceutical composition required to obtain any one or more beneficial or desired results. For prophylactic applications, beneficial or desirable results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of a disease, including the biological, histological, and behavioral manifestations of a disease state, its complications, and intermediate disease phenotypes throughout the development of the disease state. For therapeutic applications, useful or desirable results include clinical results, such as reducing the incidence of various conditions associated with the target antigen of the present disclosure, or improving one or more symptoms of a condition, reducing the dosage of other agents required to treat a pathological condition, enhancing the effectiveness of another means and/or delays the progression of a pathological condition associated with the target antigen of the present disclosure in patients.

The term "exogenous" refers to substances that are formed outside of organisms, cells, or humans, as the case may be.

The term "endogenous" refers to substances that are produced in organisms, cells, or the human body, as the case may be.

The term "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in both of the two sequences being compared is occupied by the same monomeric subunit—a base or an amino acid—for example, if a position in each of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage identity of two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions being compared, and then multiplied by 100. For example, in an optimal alignment of two sequences, if 6 of 10 positions in the two sequences match or are homologous, then these two sequences have 60% homology; if 95 out of 100 positions in two sequences are the same or homologous, then the two sequences have 95% homology. Typically, when two sequences are aligned, the comparison is made to obtain the maximum percentage of identity. For example, the comparison can be made using the BLAST algorithm, in which the algorithm parameters are selected to provide the maximum match between each sequence over the entire length of each reference sequence. The following references refer to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, S. F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T. L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as those available from NCBI BLAST, are also well known to those skilled in the art.

As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. Thus, the terms “transformant” and “transformed cell” include the primary cells under study and the cultures derived from them, without regard to the number of transfers. It should also be understood that all offspring cannot be exactly identical in DNA content due to targeted or random mutations. Included are progeny with mutations that have the same function or have the same biological activity as shown in the originally transformed cells. Where different designations are intended, this will be clearly understood from the context.

The term “isolated” refers to the case where the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates or other substances such as cell debris and growth media. In general, the term “isolated” is not intended to mean the complete absence of these substances or the absence of water, buffers or salts unless they are in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.

The term "possible" or "perhaps" means that the event or situation that follows may occur, but does not necessarily occur, and the description includes examples in which the event or situation does or does not occur.

The term "pharmaceutical composition" refers to the composition of a mixture of one or more compounds according to the present disclosure or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration into the body, facilitate absorption of the active substance and thereby produce a biological effect.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a composition for the delivery of antibodies or antigen-binding fragments. The carrier may be an antiadhesive agent, a binding agent, a coating agent, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, antibacterial or antifungal agent), a sweetener, an antiabsorption agent, a humectant, an emulsifier, a buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyol (such as glycerin, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oil (such as olive oil), saline, buffer, buffered saline, and an isotonic agent such as for example, sugars, polyol, sorbitol and sodium chloride.

In addition, the present disclosure includes an agent for treating a disease associated with cells positive for a target antigen (such as claudin 18.2), an agent containing an anti-claudin 18.2 antibody or an antigen binding fragment thereof of the present disclosure as an active substance.

The present disclosure is not specifically limited to a disease associated with claudin 18.2, provided that the disease is associated with claudin 18.2. For example, a therapeutic response induced by a molecule of the present disclosure includes: (1) preventing the binding of claudin 18.2 to its receptor/ligand by binding the molecule of the present disclosure to human claudin 18.2, or (2) killing tumor cells overexpressing claudin 18.2. Thus, the molecules of the present disclosure, when contained in preparations and compositions suitable for therapeutic applications, are very useful for such people who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer , colon cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.

In addition, the present disclosure relates to methods for immunodetection or determination of target antigens (e.g., claudin 18.2), reagents for immunodetection or determination of target antigens (e.g., claudin 18.2), methods of immunodetection or determination of cells expressing target antigens (e.g., claudin 18.2), and diagnostic agents for performing diagnosis of diseases associated with cells positive for a target antigen (e.g., claudin 18.2) containing an antibody or antibody fragment of the present disclosure that specifically recognizes the target antigen (e.g., human claudin 18.2) and binds to its extracellular amino acid sequences or tertiary structure as an active substance.

In the present disclosure, the method for detecting or measuring the amount of a target antigen (eg, claudin 18.2) may be any known method. For example, it includes a method of immunoassay or immunodetection.

An immunoassay or immunodetection method is a method of detecting or measuring the amount of an antibody or antigen with a labeled antigen or antibody. Examples of immunoassay or immunodetection methods include radiolabeled antibody (RIA) immunoassay, enzyme-linked immunosorbent assay (EIA or ELISA), fluorescence immunoassay (FIA), immunoluminescent assay, Western blotting, physical chemical method, etc.

The above-mentioned diseases associated with claudin 18.2-positive cells can be diagnosed by detecting or measuring claudin 18.2-expressing cells using antibodies or antibody fragments of the present disclosure.

Cells expressing the polypeptide can be detected by known immunodetection methods, preferably by immunoprecipitation, fluorescent cell staining, immunohistochemical staining, and the like. In addition, a method such as a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present disclosure, samples to be detected or measured for a target antigen (e.g., claudin 18.2) are not particularly limited as long as they may contain cells expressing the target antigen (e.g., claudin 18.2), such as, tissue, cells, blood, plasma, serum, pancreatic secretion, urine, feces, tissue fluid or culture medium.

Depending on the required diagnostic method, a diagnostic agent containing a monoclonal antibody or antibody fragment of the present disclosure could also contain reagents for conducting an antigen-antibody reaction or reagents for detecting this reaction. Reagents for conducting the antigen-antibody reaction include buffers, salts, and the like. Detection reagents include reagents commonly used in immunoassay or immunodetection methods, for example, a labeled secondary antibody that recognizes a monoclonal antibody, antibody fragment, or conjugate thereof, and a substrate corresponding to the label.

Details of one or more embodiments of the present disclosure are set forth in the description above. Preferred methods and materials are described below, although any method or material similar or identical to the method or material described herein may be used in the practice or analysis of the present disclosure. With reference to the specification and claims, other features, objects and advantages of the present disclosure will become apparent. In the specification and claims, the singular form includes the plural aspects unless the context clearly dictates otherwise. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this disclosure pertains. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed as limiting in any way the scope of the present disclosure, and the scope of the present disclosure is determined by the claims.

Examples

Example 1: Construction of a cell line expressing high levels of claudin 18.2

Transfection reagent Lipofectamine 3000 was used to transfect the lentiviral expression vector plasmid pCDH-hClaudin18.2 and the lentiviral system packaging vector pVSV-G, pCMV-dR8.91, into 293T cells packaging the virus; the supernatant of the culture medium containing the virus was collected, filtered and centrifuged at ultra-high speed; concentrated virus was used to infect the human gastric carcinoma signet cell line NUGC4, screened with puromycin for two to three weeks, and then sorted by FACS single-cell sorting.

The expression level of claudin 18.2 was determined in accordance with the tumor IHC score (from the English ImmunoHistoChemistry - immunohistochemistry). Cells with a claudin 18.2 expression level equivalent to the claudin 18.2 expression level of tumors with an IHC score of 3 are considered high expression cells, and cells with a claudin 18.2 expression level equivalent to the claudin 18.2 expression level of tumors with an IHC score of 2 are considered intermediate expression cells . Claudin 18.2 expression on the surface of lentivirus-infected NUGC4 cells was detected by FACS detection, and NUGC4/hClaudin18.2 monoclonal cell lines with the highest levels of claudin 18.2 expression were selected. At the same time, the surface expression of claudin 18.2 on wild-type NUGC4 cells was detected by FACS, and clonal NUGC4 cell lines with an average expression level of claudin 18.2 were selected. Wild-type NUGC4 were cells with low levels of claudin 18.2 expression.

Selected monoclonal cell lines were expanded and cultured, and frozen and stored for subsequent analyses.

Genbank claudin 18.2 sequence: NP_001002026: (SEQ ID NO: 1)

DNA sequence of claudin 18.2: (SEQ ID NO: 2)

Example 2: Production of monoclonal antibody against human claudin 18.2

1. Immunization

Monoclonal antibodies against claudin 18.2 were obtained by immunization of mice

Female albino mice, SJL, aged 6-8 weeks (Beijing Charles River Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001) were used for the experiment. Feeding environment: SPF level (from the English specific pathogen free - free from specific pathogenic microflora). After acquisition, mice were kept in laboratory conditions for 1 week, on a 12/12-hour light/dark cycle, at 20–25°C; humidity 40-60%. The mice, which were adapted to the environment, were then immunized according to the following schedules. The immune system antigen was huClaudin18.2-HEK293 cell (HEK-293 cell line stably transfected with human claudin 18.2 plasmid).

Immunization protocol: Before primary immunization with cells, mice were injected intraperitoneally (i.p.) with TiterMax® Gold adjuvant (Sigma cat. no. T2684), 0.1 ml/mouse; One hour later, each mouse was injected intraperitoneally (i.p.) with cell fluid diluted to a concentration of 1×10 ⁸ /ml with 0.1 ml saline. After the cells were evenly distributed by pipetting, they were inoculated on day 0, day 14, day 28, day 42, and day 56. Blood samples were collected on days 21, 35, 49, and 63; The antibody titer in mouse serum was determined by ELISA (enzyme-linked immunosorbent assay). After 4-5 immunizations, mice with high serum antibody titers that tended to reach a plateau were selected for splenocyte fusion. Three days before fusion with a splenocyte, 1×10 ⁷ cells were injected intraperitoneally (i.p.) for repeated immunization.

2. Fusion with splenocyte

Hybridoma cells were generated by fusing spleen lymphocytes with Sp2/0 myeloma cells (ATCC® CRL-8287 ^™ ) using a PEG (polyethylene glycol)-mediated fusion method. Hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS (Fet al Bovine Serum), 1×NAT and 1×OPI) at a density of 0.5×10 ⁶ - 1×10 ⁶ / ml, inoculated into a 96-well plate in an amount of 100 µl/well, incubated at 37°C and 5% CO ₂ for 3-4 days, 100 µl/well of complete HAT medium was added and maintained for another 3-4 days until formation of clones. The supernatant was removed, 200 μl/well of complete HT medium (IMDM medium containing 20% FBS, 1×HT and 1×OPI) was added, incubated at 37°C, 5% CO ₂ for 3 days and then subjected to ELISA. identification.

3. Screening for hybridoma cells

According to the growth density of the hybridoma cells, the culture supernatant was detected by the binding ELISA method. Cells that have strong binding ability to huClaudin18.2-HEK293 cells, while not binding to HEK293 cells, were selected and then expanded and cryopreserved at their own time; subcloning was performed two to three times until a single cell clone was obtained.

Cells from each subcloning were also analyzed by cell binding assay. Hybridoma clones were obtained by performing screening through the above assay. Antibodies were additionally obtained using a serum-free cell culture method. Antibodies were purified according to the purification example and used in the assay examples.

Example 3: Humanization of Mouse Antibodies

Monoclonal hybridoma cell lines mAb901 and mAb902 with high in vitro activity were selected; monoclonal antibody sequences were cloned and then humanized, recombinantly expressed, and evaluated for activity.

The procedures for cloning sequences from the hybridoma were as follows. Hybridoma cells were harvested at logarithmic growth phase. RNA was isolated using Trizol (Invitrogen, 15596-018) according to the kit instructions and reverse transcribed using PrimeScript™ Reverse Transcriptase (Takara, cat. #2680A). The cDNAs obtained from reverse transcription were amplified by PCR (polymerase chain reaction) using a mouse Ig primer set (Novagen, TB326 Rev. B0503), and the amplified products were sent to the company for sequencing. Amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NO: 3-6;

Mouse heavy chain variable region mAM901 (SEQ ID NO: 3)

Mouse light chain variable region mAM901 (SEQ ID NO: 4)

Mouse heavy chain variable region mAM902 (SEQ ID NO: 5)

Mouse light chain variable region mAM902 (SEQ ID NO: 6)

The above murine heavy and light chain variable regions were respectively linked to the human IgG1 heavy chain constant region and the human kappa light chain constant region as described below, so as to generate chimeric antibodies ch1901 and ch1902.

The constant region was selected from the following sequences:

Human IgG Antibody Heavy Chain Constant Region 1 (SEQ ID NO: 7)

Human kappa light chain constant region: (SEQ ID NO: 8)

Mouse monoclonal antibodies were humanized, as disclosed in many documents in this field. Briefly, the original (mouse antibody) constant domains were replaced with human constant domains, and human germline antibody sequences were selected based on homology between mouse and human antibodies to perform CDR grafting. In the present invention, candidate molecules with favorable activity were selected for humanization, and the results are as follows.

1. Mouse Antibody CDR Regions

The amino acid residues of the VH/VL CDRs in Table 4 were identified and annotated using Kabat numbering criteria.

The CDR sequences of mouse antibodies are shown in Table 4:

2. Sequence selection of human germline FR regions

Based on the obtained typical mouse antibody VH/VL CDR structure, the heavy and light chain variable region sequences were compared to a germline antibody database to obtain a highly homologous human germline template. The human germline light chain framework region was derived from the human kappa light chain gene.

2.1 Humanization and design of mAb1901 reverse mutations

The corresponding human germline antibody was selected to humanize the mouse antibody mAb1901. The CDR regions of the mouse antibody mAb1901 were grafted onto the selected humanization matrix to produce humanized variable regions. The humanized heavy chain variable region sequence as shown in SEQ ID NO: 24 and the light chain variable region sequence as shown in SEQ ID NO: 21 were combined with IgG constant regions to form an intact antibody. At the same time, the FR region in the V region of the humanized antibody was back-mutated, and exemplary back mutations and combinations are as follows:

The corresponding heavy chain variable region listed in the above table can be linked to the heavy chain constant region of human IgG1 as shown in SEQ ID NO: 7 to form the heavy chain of a full-length antibody, and the light chain variable region can be linked to the constant region light chain to human, as shown in SEQ ID NO: 8, to form the light chain of a full-length antibody. In other embodiments, a heavy chain variable region and a light chain variable region may also be separately linked to another heavy chain constant region and a light chain constant region to form a full-length antibody.

2.2 Humanization and design of mAb1902 reverse mutations

The corresponding human germline antibody was selected to humanize the mouse antibody mAb1902. The CDR regions of the mouse antibody mAb1902 were grafted onto the selected humanization matrix to produce humanized variable regions. The sequence of the humanized heavy chain variable region is as shown in SEQ ID NO: 31, and the sequence of the light chain variable region is as shown in SEQ ID NO: 28, and then they were recombined with IgG constant regions to form an intact antibody . At the same time, the FR region in the V region of the humanized antibody was back-mutated, and the methods and combinations of exemplary back mutations are as follows:

The corresponding heavy chain variable region listed in the above table can be linked to the heavy chain constant region of human IgG1 as shown in SEQ ID NO: 7 to form the heavy chain of a full-length antibody, and the light chain variable region can be linked to the constant region light chain to human, as shown in SEQ ID NO: 8, to form the light chain of a full-length antibody.

As an example, the full-length antibody sequence is as follows:

chimeric antibody ch1901:

heavy chain ch1901: (SEQ ID NO: 35)

light chain ch1901 (SEQ ID NO: 36)

chimeric antibody ch1902:

heavy chain ch1902 (SEQ ID NO: 37)

light chain ch1902 (SEQ ID NO: 38)

The light and heavy chain sequences of a full-length antibody are as follows:

The sequences of the light and heavy chains of a full-length antibody are as follows:

The positive control antibody of the present disclosure was IMAB-362 (available from WO2016166122):

Heavy chain IMAB-362 (SEQ ID NO: 53)

Light chain IMAB-362 (SEQ ID NO: 54)

The above antibodies were cloned, expressed and purified by conventional cloning and recombinant gene expression techniques.

In vitro biological assessment

Assay Example 1: Cellular ELISA Binding Assay

Cell-based ELISA was used to detect the binding properties of anti-claudin 18.2 antibodies. NUGC4 cells stably expressing claudin 18.2 were cultured in a 96-well cell plate (Corning, 3599) until cell growth density reached 90%, and 4% paraformaldehyde was added to fix the cells for 1 hour. The plate was washed three times with PBST buffer (PBS (Phosphate buffered saline) containing 0.05% Tween-20, pH 7.4), and then blocked by adding 200 μl/well of 5% full skim milk (Bright skim milk powder) diluted with PBS as a blocking solution and incubated in an incubator at 37°C for 2.5 hours or at 4°C overnight (16-18 hours). After blocking, the blocking solution was discarded and the plate was washed 3 times with PBST buffer, 50 μl/well of assay antibodies diluted in sample diluent (PBS containing 1% skim milk, pH 7.4) was added and placed in an incubator at 37°C for 2 hours. After incubation, the plate was washed 5 times with PBST, and 100 μl/well of secondary goat anti-human antibody labeled HRP (horseradish peroxidase) (Jackson Immuno Research, cat. No. 109-035-003), diluted with diluent, was added sample and incubated at 37°C for 1 hour. After washing the plate with PBST 6 times, 50 μl/well of the chromogenic substrate TMB (tetramethylbenzidine) (KPL, cat. No. 52-00-03) was added 6 times, incubated at room temperature for 10-15 min, and 50 μl was added /well 1M H ₂ SO ₄ to stop the reaction. The absorbance value was read using an MD Versa Max TM microplate reader at 450 nm, and the EC ₅₀ (half maximal effective concentration) value was calculated, indicating the binding of the anti-claudin 18.2 antibody to claudin 18.2 (Results are shown in the table below ).

Assay Example 2: Cellular Antibody Binding Assay

Cell suspension, 1x10 ⁶ /ml, was prepared using NUGC4 cells stably expressing claudin 18.2 and FACS buffer (2% fetal calf serum) (Gibco, 10099141) in PBS (Sigma, P4417-100TAB), pH 7.4) was added to a 96-well round-bottom plate (Corning, 3795) in an amount of 100 µl/well. After centrifugation to remove the supernatant, different concentrations of anti-claudin 18.2 assay antibodies diluted in FACS buffer were added at 50 μl/well and incubated for 1 hour in a refrigerator at 4°C in the dark. After centrifugation and washing with FACS buffer at 300 g 3 times the working concentration of anti-human IgG antibody (H+L) coated with Alexa Fluor 488 (invitrogen, A-11013) was added and incubated in the refrigerator at 4°C in the dark for 40 minutes. After centrifugation and washing with FACS buffer at 300 g 3 times, the geometric mean fluorescence intensity was measured on a BD FACS Cantoll flow cytometer, and the EC50 value was calculated, indicating the binding of anti-claudin 18.2 antibody to NUGC4 cells stably expressing claudin 18.2. The results are shown in Fig. 1.

Assay Example 3: Antibody Endocytosis Assay

Anti-claudin 18.2 assay antibody, prelabeled with DyLight 488 NHS Ester (thermofisher, 46403), was added to NUGC4 cells, 1 x 10 ⁶ /ml, stably expressing claudin 18.2, at a final concentration of 5 μg/ml and placed on ice for incubation for 1 hour in the dark, centrifuged and washed 3 times with pre-cooled FACS buffer (2% fetal calf serum in PBS, pH 7.4), after removing the supernatant, add pre-warmed complete medium and place in a cell incubator at 37°C, 5 % CO ₂ . The cells were removed after 0, 0.5, 1, 2, and 4 hours, respectively, and placed on ice in the dark. After all samples were collected, centrifuged at 300 g at low temperature, elution buffer (0.05 M glycine, 0.1 M sodium chloride, pH 1.7) was added and incubated at room temperature for 7 minutes. Samples were centrifuged and washed with FACS buffer at 300 g once, geometric mean fluorescence intensity was measured on a BD FACS Cantoll flow cytometer, and the endocytosis efficiency of anti-claudin 18.2 antibody against NUGC4 cells stably expressing claudin 18.2 was calculated. The results show (see Figure 2) that the humanized antibodies have favorable endocytosis efficiency.

Assay Example 4: Determination of Antibody Affinity Based on Flow Cytometry

On the day of testing, HEK293/hClaudin18.2 cells were collected in a 96-well U-bottom plate, with 1×10 ⁵ - 2×10 ⁵ cells per well. Anti-claudin 18.2 antibody was added at an initial concentration of 5 μg/ml, 2x gradient dilutions (12 concentration points) and incubated at 4°C for 1 hour. The positive control was IMAB362, and the well without antibody was set as a negative control. The antibody was removed by centrifugation and then 100 μl/well of FITC-conjugated anti-human Fc IgG antibody (200x) was added, incubated at 4°C for 30 minutes in the dark, washed twice with PBS+2% FBS and prepared for detection by flow cytometry. The BD FACS Cantoll was started and preheated, and new analysis was performed using BD FACSDiva software. The negative control sample HEK293/hClaudin18.2 was identified, and the FSC and SSC voltages were adjusted to the appropriate values and maintained. Blank B and standard curve 1 were generated according to the Quantum™ FITC-5 MESF kit instructions, respectively. The FITC voltage was adjusted to the appropriate value and maintained. Samples in a 96-well U-bottom plate were detected at maintained voltage and data recorded. Flowjo software was used to analyze the experimental data to obtain the Geo Mean, and the MESF-Geo Mean standard curve was fitted according to the instructions of the Quantum™ FITC-5 MESF kit. The molar concentrations of anti-claudin 18.2 antibody binding to HEK293/hClaudin18.2 cells and the concentrations of free antibody were calculated according to the fluorescence value of FITC-conjugated anti-human Fc IgG antibody, and the Bmax and dissociation constant KD of the antibody were calculated using a construction-based method Scatchard graphics. The results are shown in Table 15.

Assay Example 5: Evaluation of Antibody ADCC Effect

Different NUGC4 cells (high, medium and low claudin 18.2 expression) were digested, centrifuged at 1000 rpm. and resuspended for counting. Cells were resuspended in phenol red free RPMI 1640 (Gibco, cat. no. 11835-030) containing 10% FBS (ultra-low IgG fetal bovine serum, New Zealand, Gibco, 1921005PJ), 3 x 10 ⁵ cells/ml. 25 μl of cells were added to each well of a 96-well plate (Corning, 3903), 7500 cells/well. The antibody was diluted in the above phenol red free medium to obtain a 3x diluted antibody solution, and 25 μl/well of antibody was added to the cell plate and incubated in an incubator at 37°C, 5% CO ₂ for 0.5 hour.

Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm and resuspended for counting. Cells were resuspended in phenol red-free RPMI 1640 containing 10% FBS (ultra-low IgG fetal bovine serum, New Zealand) at 3 x 10 ⁶ cells/ml, and 25 µl of cells were added to an assay plate (7.5 x 10 ⁴ cells/well) and incubated in an incubator at 37°C, 5% CO ₂ for 6 hours.

75 μl/well of Bright-Glo (Promega, E2610) was added to each well of the assay plate, and chemiluminescence was detected by a microplate reader (PerkinElmer, VITOR3).

The results show (see Table 16 and Fig. 3A to Fig. 3C) that both antibody h1901-11 and h1902-5 exhibit very strong ADCC activity in NUGC4 cells expressing claudin 18.2 at low, medium and high levels.

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LIST OF SEQUENCES

<110> JIANGSU HENGRUI MEDICINE CO., LTD.;

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> ANTI-CLAUDIN ANTIBODY 18.2 AND ITS APPLICATION

<130> 702029CPCP

<140> PCT/CN2020/082369

<141> 2020-03-31

<150> 201910257853.6

<151> 2019-04-01

<160> 54

<170> SIPOSequenceListing 1.0

<210> 1

<211> 261

<212> Protein

<213> Homo sapiens

<220>

<221> Peptide

<223> Claudin 18.2 protein

<400> 1

Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 2

<211> 3350

<212> DNA

<213> Homo sapiens

<220>

<221> gene

<223> claudin 18.2

<400> 2

agaattgcgc tgtccacttg tcgtgtggct ctgtgtcgac actgtgcgcc accatggccg 60

tgactgcctg tcagggcttg gggttcgtgg tttcactgat tgggattgcg ggcatcattg 120

ctgccacctg catggaccag tggagcaccc aagacttgta caacaacccc gtaacagctg 180

ttttcaacta ccaggggctg tggcgctcct gtgtccgaga gagctctggc ttcaccgagt 240

gccggggcta cttcaccctg ctggggctgc cagccatgct gcaggcagtg cgagccctga 300

tgatcgtagg catcgtcctg ggtgccattg gcctcctggt atccatcttt gccctgaaat 360

gcatccgcat tggcagcatg gaggactctg ccaaagccaa catgacactg acctccggga 420

tcatgttcat tgtctcaggt ctttgtgcaa ttgctggagt gtctgtgttt gccaacatgc 480

tggtgactaa cttctggatg tccacagcta acatgtacac cggcatgggt gggatggtgc 540

agactgttca gaccaggtac acatttggtg cggctctgtt cgtgggctgg gtcgctggag 600

gcctcacact aattggggt gtgatgatgt gcatcgcctg ccggggcctg gcaccagaag 660

aaaccaacta caaagccgtt tcttatcatg cctcaggcca cagtgttgcc tacaagcctg 720

gaggcttcaa ggccagcact ggctttgggt ccaacaccaa aaacaagaag atatacgatg 780

gaggtgcccg cacagaggac gaggtacaat cttatccttc caagcacgac tatgtgtaat 840

gctctaagac ctctcagcac gggcggaaga aactcccgga gagctcaccc aaaaaacaag 900

gagatcccat ctagatttct tcttgctttt gactcacagc tggaagttag aaaagcctcg 960

atttcatctt tggagaggcc aaatggtctt agcctcagtc tctgtctcta aatattccac 1020

cataaaacag ctgagttatt tatgaattag aggctatagc tcacattttc aatcctctat 1080

ttcttttttt aaatataact ttctactctg atgagagaat gtggttttaa tctctctctc 1140

acattttgat gatttagaca gactccccct cttcctccta gtcaataaac ccattgatga 1200

tctatttccc agcttatccc caagaaaact tttgaaagga aagagtagac ccaaagatgt 1260

tattttctgc tgtttgaatt ttgtctcccc acccccaact tggctagtaa taaacactta 1320

ctgaagaaga agcaataaga gaaagatatt tgtaatctct ccagcccatg atctcggttt 1380

tcttacactg tgatcttaaa agttaccaaa ccaaagtcat tttcagtttg aggcaaccaa 1440

acctttctac tgctgttgac atcttcttat tacagcaaca ccattctagg agtttcctga 1500

gctctccact ggagtcctct ttctgtcgcg ggtcagaaat tgtccctaga tgaatgagaa 1560

aattattttt tttaatttaa gtcctaaata tagttaaaat aaataatgtt ttagtaaaat 1620

gatacactat ctctgtgaaa tagcctcacc cctacatgtg gatagaagga aatgaaaaaa 1680

taattgcttt gacattgtct atatggtact ttgtaaagtc atgcttaagt acaaattcca 1740

tgaaaagctc actgatccta attctttccc tttgaggtct ctatggctct gattgtacat 1800

gatagtaagt gtaagccatg taaaaagtaa ataatgtctg ggcacagtgg ctcacgcctg 1860

taatcctagc actttgggag gctgaggagg aaggatcact tgagcccaga agttcgagac 1920

tagcctgggc aacatggaga agccctgtct ctacaaaata cagagagaaa aaatcagcca 1980

gtcatggtgg cctacacctg tagtcccagc attccgggag gctgaggtgg gaggatcact 2040

tgagcccagg gaggttgggg ctgcagtgag ccatgatcac accactgcac tccagccagg 2100

tgacatagcg agatcctgtc taaaaaaata aaaaataaat aatggaacac agcaagtcct 2160

aggaagtagg ttaaaactaa ttctttaaaa aaaaaaaaaa gttgagcctg aattaaatgt 2220

aatgtttcca agtgacaggt atccacattt gcatggttac aagccactgc cagttagcag 2280

tagcactttc ctggcactgt ggtcggtttt gttttgtttt gctttgttta gagacggggt 2340

ctcactttcc aggctggcct caaactcctg cactcaagca attcttctac cctggcctcc 2400

caagtagctg gaattacagg tgtgcgccat cacaactagc tggtggtcag ttttgttact 2460

ctgagagctg ttcacttctc tgaattcacc tagagtggtt ggaccatcag atgtttgggc 2520

aaaactgaaa gctctttgca accacacacc ttccctgagc ttacatcact gcccttttga 2580

gcagaaagtc taaattcctt ccaagacagt agaattccat cccagtacca aagccagata 2640

ggccccctag gaaactgagg taagagcagt ctctaaaaac tacccacagc agcattggtg 2700

caggggaact tggccattag gttattattt gagaggaaag tcctcacatc aatagtacat 2760

atgaaagtga cctccaaggg gattggtgaa tactcataag gatcttcagg ctgaacagac 2820

tatgtctggg gaaagaacgg attatgcccc attaaataac aagttgtgtt caagagtcag 2880

agcagtgagc tcagaggccc ttctcactga gacagcaaca tttaaaccaa accagaggaa 2940

gtatttgtgg aactcactgc ctcagtttgg gtaaaggatg agcagacaag tcaactaaag 3000

aaaaaagaaa agcaaggagg agggttgagc aatctagagc atggagtttg ttaagtgctc 3060

tctggatttg agttgaagag catccatttg agttgaaggc cacagggcac aatgagctct 3120

cccttctacc accagaaagt ccctggtcag gtctcaggta gtgcggtgtg gctcagctgg 3180

gtttttaatt agcgcattct ctatccaaca tttaattgtt tgaaagcctc catatagtta 3240

gattgtgctt tgtaattttg ttgttgttgc tctatcttat tgtatatgca ttgagtatta 3300

acctgaatgt tttgttactt aaatattaaa aacactgtta tcctacagtt 3350

<210> 3

<211> 120

<212> Protein

<213> Mus musculus

<220>

<221> Domain

<223> Heavy chain variable region of mouse antibody mAb1901

<400> 3

Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Glu Met Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 4

<211> 113

<212> Protein

<213> Mus musculus

<220>

<221> Domain

<223> Mouse antibody light chain variable region mAb1901

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 5

<211> 118

<212> Protein

<213> Mus musculus

<220>

<221> Domain

<223> Heavy chain variable region of mouse antibody mAb1902

<400> 5

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Pro Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 6

<211> 113

<212> Protein

<213> Mus musculus

<220>

<221> Domain

<223> Mouse antibody mAb1902 light chain variable region

<400> 6

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 7

<211> 330

<212> Protein

<213> Homo sapiens

<220>

<221> Domain

<223> Human IgG1 antibody heavy chain constant region

<400> 7

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 8

<211> 107

<212> Protein

<213> Homo sapiens

<220>

<221> Domain

<223> Human antibody kappa light chain constant region

<400> 8

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 9

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> mAb1901 HCDR1

<400> 9

Asp Tyr Gly Ile His

15

<210> 10

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>HCDR2 mAb1901

<400> 10

Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 11

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>HCDR3 mAb1901

<400> 11

Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr

1 5 10

<210> 12

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR1 mAb1901

<400> 12

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 13

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR2 mAb1901

<400> 13

Gly Ala Ser Thr Arg Ala Ser

15

<210> 14

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR3 mAb1901

<400> 14

Gln Asn Asp Leu Tyr Tyr Pro Leu Thr

15

<210> 15

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>HCDR1 mAb1902

<400> 15

Ser Tyr Trp Met His

15

<210> 16

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>HCDR2 mAb1902

<400> 16

Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly Arg

<210> 17

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>HCDR3 mAb1902

<400> 17

Leu Lys Thr Gly Asn Ser Phe Asp Tyr

15

<210> 18

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR1 mAb1902

<400> 18

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 19

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR2 mAb1902

<400> 19

Trp Ala Ser Thr Arg Glu Ser

15

<210> 20

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>LCDR3 mAb1902

<400> 20

Gln Asn Ala Tyr Thr Tyr Pro Phe Thr

15

<210> 21

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VL1

<400> 21

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 22

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VL2

<400> 22

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 23

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VL3

<400> 23

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 24

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VH1

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 25

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VH2

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 26

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VH3

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 27

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VH4

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 28

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VL11

<400> 28

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 29

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VL12

<400> 29

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 30

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VL13

<400> 30

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 31

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223>VH11

<400> 31

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 32

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VH12

<400> 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 33

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VH13

<400> 33

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 34

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> VH14

<400> 34

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 35

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Heavy chain ch1901

<400> 35

Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Glu Met Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 36

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Light chain ch1901

<400> 36

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 37

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Heavy chain ch1902

<400> 37

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Pro Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 38

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Light chain ch1902

<400> 38

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 39

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L1

<400> 39

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 40

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L2

<400> 40

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 41

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L3

<400> 41

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn

85 90 95

Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 42

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H1

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 43

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H2

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 44

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H3

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 45

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H4

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 46

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L11

<400> 46

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 47

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L12

<400> 47

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 48

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> L13

<400> 48

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 49

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H11

<400> 49

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 50

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H12

<400> 50

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 51

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H13

<400> 51

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 52

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223>H14

<400> 52

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 53

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Heavy chain IMAB-362

<400> 53

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 54

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> Circuit

<223> Light chain IMAB-362

<400> 54

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<---

Claims (44)

1. An antibody that specifically binds to claudin 18.2, containing a heavy chain variable region and a light chain variable region, in which: iii) the heavy chain variable region contains 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 contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or iv) the heavy chain variable region contains 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 contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively. 2. An antibody that specifically binds to claudin 18.2 according to claim 1, which is a murine antibody, a chimeric antibody or a humanized antibody. 3. An antibody that specifically binds to claudin 18.2, according to claim 1 or 2, containing a heavy chain variable region and a light chain variable region, in which: 1) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 4; 2) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 21; 3) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6; or 4) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28. 4. An antibody that specifically binds to claudin 18.2 according to claim 3, which is a humanized antibody containing a framework region derived from a human antibody, or a variant framework region, and the variant framework region has 1-10 back mutation(s) (ii) on the light chain framework region and/or the heavy chain framework region of a human antibody; wherein the framework region variant contains mutation(s) selected from a) or b below: (a) one or more back mutations selected from the group consisting of: 22S, 85I and 87H contained in the light chain variable region, and/or one or more back mutations selected from the group consisting of: 48I, 82T and 69M contained in the variable region of the heavy chain; or (b) one or more back mutations selected from the group consisting of: 4L and 22S contained in the light chain variable region, and/or one or more back mutations selected from the group consisting of: 38K, 40R, 48I, 66K , 67A, 69L, 71L and 73K contained in the heavy chain variable region; more preferably, the framework region variant contains mutation(s) selected from the following a-1) or b-1): a-1) reverse mutations of amino acids 22S, 85I and 87H contained in the variable region of the light chain, and reverse mutations of amino acids 48I and 82T contained in the variable region of the heavy chain; or b-1) reverse mutation 4L contained in the light chain variable region; a back mutation 82T contained in the heavy chain variable region, where "82" indicates position 82A according to Kabat criteria. 5. An antibody that specifically binds to claudin 18.2 according to claim 3, which contains a heavy chain variable region and a light chain variable region as shown below: vii) a heavy chain variable region sequence as shown in SEQ ID NO: 3 and a light chain variable region sequence as shown in SEQID NO: 4; or viii) a heavy chain variable region sequence as shown in SEQ ID NO: 24, 25, 26 or 27, and a light chain variable region sequence as shown in SEQ ID NO: 21, 22 or 23; or ix) a heavy chain variable region sequence as shown in SEQ ID NO: 5 and a light chain variable region sequence as shown in SEQ ID NO: 6; or x) a heavy chain variable region sequence as shown in SEQ ID NO: 31, 32, 33 or 34, and a light chain variable region sequence as shown in SEQ ID NO: 28, 29 or 30. 6. An antibody that specifically binds to claudin 18.2 according to claim 3, wherein the antibody that specifically binds to claudin 18.2 or an antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below: viii) a heavy chain variable region sequence as shown in SEQ ID NO: 31 and a light chain variable region sequence as shown in SEQID NO: 29; or ix) a heavy chain variable region sequence as shown in SEQ ID NO: 26 and a light chain variable region sequence as shown in SEQ ID NO: 23. 7. An antibody that specifically binds to claudin 18.2 according to claim 3, which further comprises a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region is selected from the group consisting of: human lgG1, lgG2, lgG3 and lgG4 constant region(s) and variant(s) thereof, the light chain constant region is selected from the group consisting of: constant region(s) κ and λ chain region(s), human antibodies and variant(s) thereof. 8. The antibody that specifically binds to claudin 18.2 according to claim 3, wherein the antibody that specifically binds to claudin 18.2 comprises a heavy chain constant region as shown in SEQ ID NO: 7 and a light chain constant region as shown in SEQ ID NO: 8. 9. An antibody that specifically binds to claudin 18.2 according to claim 3, wherein the antibody that specifically binds to claudin 18.2 comprises a heavy chain as shown in SEQ ID NO: 35 or 42 and a light chain as shown in SEQ ID NO: 35 or 42 NO: 36 or 39; or a heavy chain as shown in SEQ ID NO: 37 or 49, and a light chain as shown in SEQIDNO: 38 or 46. 10. An antibody that specifically binds to claudin 18.2, according to claim 3, containing: c) a heavy chain as shown in SEQ ID NO: 35, and a light chain as shown in SEQ ID NO: 36; d) a heavy chain as shown in SEQ ID NO: 42, 43, 44 or 45, and a light chain as shown in SEQ ID NO: 39, 40 or 41; e) a heavy chain as shown in SEQ ID NO: 37, and a light chain as shown in SEQ ID NO: 38; or f) a heavy chain as shown in SEQ ID NO: 49, 50, 51 or 52, and a light chain as shown in SEQ ID NO: 46, 47 or 48. 11. An antibody that specifically binds to claudin 18.2, according to claim 3, containing: a heavy chain as shown in SEQ ID NO: 44, and a light chain as shown in SEQ ID NO: 41; or a heavy chain as shown in SEQ ID NO: 49, and a light chain as shown in SEQ ID NO: 47. 12. A nucleic acid molecule encoding an antibody that specifically binds to claudin 18.2, according to any one of paragraphs. 1-11. 13. A host cell for producing an antibody that specifically binds to claudin 18.2, comprising the nucleic acid molecule of claim 12. 14. An antibody-drug conjugate for the treatment of a disease associated with claudin 18.2, which is an antibody-drug conjugate formed by forming an antibody conjugate that specifically binds to claudin 18.2, according to any one of claims. 1-11 with a cytotoxic drug. 15. A pharmaceutical composition for the treatment of a disease associated with claudin 18.2, containing a therapeutically effective amount of an antibody that specifically binds to claudin 18.2, according to any one of paragraphs. 1-11 or the nucleic acid molecule of claim 12 or the antibody-drug conjugate of claim 14 and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 16. A method for immunological analysis or detection of claudin 18.2, including: the step of bringing an antibody that specifically binds to claudin 18.2, according to any one of paragraphs. 1-11 into contact with the sample to be analyzed. 17. A method of treating a disease associated with claudin 18.2, comprising administering to a subject a therapeutically effective amount of an antibody that specifically binds claudin 18.2, according to any one of claims. 1-11 or the nucleic acid molecule of claim 12 or the antibody-drug conjugate of claim 14 or the pharmaceutical composition of claim 15, wherein the disease is a tumor. 18. The method according to claim 17, where the disease is selected from the group consisting of the following: head and neck cancer, esophageal cancer, lung cancer, breast cancer, liver cancer, liver and gallbladder cancer, pancreatic cancer, stomach cancer, cancer colon and rectal and ovarian cancer. 19. The method of claim 18, wherein the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer.