Anti-CLDN18.2 antibody and its diagnostic applications

Abstract

The present disclosure provides herein anti-CLDN18.2 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and diagnostic uses thereof.

Description

[Technical Field]

[0001] Field of Invention

[0001] This disclosure generally relates to novel anti-CLDN18.2 antibodies and their diagnostic use. [Background technology]

[0002] background

[0002] The claudin-18 (CLDN18) molecule (Genbank accession numbers: selective variant 1 (CLDN18A1 or CLDN18.1): NP_057453, NM_016369, and selective variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, Np_001002026) is a transmembrane protein with a molecular weight of approximately 27.9 / 27.72 kD. The CLDN18 protein is present in tight junctions of epithelium and endothelium, organizing a network of interconnections of intramembrane particles between adjacent cells. CLDN18 and occludins are the most prominent transmembrane protein components of tight junctions. Due to their strong intercellular adhesion, these tight junction proteins create a primary barrier to prevent and control extracellular transport of solutes and restrict the lateral diffusion of membrane lipids and proteins to maintain cellular polarity. Therefore, tight junctions play an important role in the structural formation of epithelial tissue.

[0003]

[0003] Over the past decade, targeted therapies and immunotherapies have revolutionized various systemic treatments, but the management of advanced and / or metastatic cancer remains a major challenge. For example, many patients with advanced gastrointestinal cancers such as gastric cancer, pancreatic cancer, cholangiocarcinoma, and lung cancer have not yet achieved significant efficacy with current standard treatments. For most of these patients with advanced cancer, chemotherapy remains the dominant treatment, and the prognosis remains very poor. Given the highly limited expression of CLDN 18.2 in a wide range of tumor types and the frequent occurrence of its ectopic activation, CLDN 18.2 is considered a therapeutic target for the development of drugs for solid tumors expressing CLDN 18.2, including but not limited to GC / GEC, pancreatic cancer, cholangiocarcinoma, and lung cancer.

[0004]

[0004] In order to enable screening and selection of patients expressing CLDN18.2, an IHC detection assay with high specificity and affinity is desired. [Overview of the project]

[0005]

[0005] Throughout this disclosure, the articles “a,” “an,” and “the” are used here to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “antibody” refers to one or more antibodies.

[0006]

[0006] This disclosure provides, in particular, a novel monoclonal anti-CLDN18.2 antibody that specifically recognizes CLDN18.2 without cross-reacting with CLDN18.1. This disclosure further provides the nucleotide sequence encoding the anti-CLDN18.2 antibody and the use of the anti-CLDN18.2 antibody, for example, for diagnostic purposes.

[0007]

[0007] In one embodiment, the present disclosure provides an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody or the antigen-binding fragment exhibits one or more of the following properties: a) No cross-reactivity with human CLDN18.1; b) No cross-reactivity with non-cancerous cells other than gastric epithelial cells, as measured by immunohistochemical assay (IHC); c) No cross-reactivity with non-cancerous human lung tissue as measured by IHC; d) The drug must be able to specifically bind to cells expressing CLDN18.2, and optionally, the cells expressing CLDN18.2 must be pretreated so that CLDN18.2 is denatured or no longer present in its native three-dimensional structure; e) A fusion polypeptide containing the first extracellular loop of human CLDN18.2 was found to have a K10 nM rating by surface plasmon resonance (SPR). d The substance must be able to bind at an EC50 value of 20 ng / ml or less, either by numerical value or by enzyme-linked immunosorbent assay (ELISA); f) No detectable binding to human CLDN 18.2 on the cell surface as measured by flow cytometry (FACS); g) It can specifically bind to an epitope in the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19) by ELISA measurement; and / or h) No cross-reactivity with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples at an antibody concentration of 1 nM by ELISA or 0.5 ug / ml by IHC.

[0008]

[0008] In one embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a) Heavy chain CDR1 containing the amino acid sequence X1X2YX3H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX4GX5X6X7X8YX9EKFKG (SEQ ID NO: 12), and NYX 10 Heavy chain CDR3 containing the amino acid sequence of STFGY (SEQ ID NO: 24); and / or b) Light chain CDR1, KX containing the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2) 11Light chain CDR2 containing the amino acid sequence of SNRFS (SEQ ID NO: 25), and light chain CDR3 containing the amino acid sequence of FQGSHVPFT (SEQ ID NO: 6); Here, X1 is R or T, X2 is N or Y, X3 is F or I, X4 is G or R, X5 is F or G, X6 is D or N, X7 is I or T, X8 is E or V, X9 is S or N, X 10 is G or R, and X 11 It is either V or I.

[0009]

[0009] In certain embodiments, the isolated antibody or its antigen-binding fragment includes: a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5; or b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11.

[0010]

[0010] In certain embodiments, the antibody or antigen-binding fragment provided herein further includes: a) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0011]

[0011] In certain embodiments, the antibody or antigen-binding fragment provided herein includes: a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0012]

[0012] In certain embodiments, the antibody or antigen-binding fragment provided herein includes: a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or b) A heavy chain variable region comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16.

[0013]

[0013] In certain embodiments, the antibodies or antigen-binding fragments provided herein further contain one or more amino acid residue mutations while retaining binding specificity to human CLDN 18.2, and optionally retain binding specificity to a linear epitope containing the amino acid sequence of SEQ ID NO: 19.

[0014]

[0014] In certain embodiments, at least one mutation is a conservative substitution, or all mutations are conservative substitutions.

[0015]

[0015] In certain embodiments, at least one mutation is present in one or more CDR sequences and / or one or more non-CDR sequences in the heavy chain variable region or light chain variable region.

[0016]

[0016] In a particular embodiment, the antibody or its antigen-binding fragment is as follows: a) A heavy chain CDR1 (HCDR1) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 7, and / or b) A heavy chain CDR2 (HCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 3 or SEQ ID NO: 9, and / or c) A heavy chain CDR3 (HCDR3) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 5 or SEQ ID NO: 11, and / or d) A light chain CDR1 (LCDR1) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 2, and / or e) A light chain CDR2 (LCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 4 or SEQ ID NO: 10, and / or f) A light chain CDR3 (LCDR3) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 6. including, and, On the other hand, it retains binding specificity to CLDN18.2 and optionally has a binding affinity at the same level as or higher than that of its parent antibody.

[0017]

[0017] In certain embodiments, the antibody or its antigen-binding fragment includes HCDR1 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 1 or SEQ ID NO: 7, HCDR2 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 3 or SEQ ID NO: 9, HCDR3 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 5 or SEQ ID NO: 11, LCDR1 having 2 or 1 or fewer amino acid mutations in SEQ ID NO: 2, LCDR2 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 4 or SEQ ID NO: 10, and / or LCDR3 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 6, while maintaining binding specificity to CLDN18.2 and optionally having a binding affinity at the same level as or higher than that of its parent antibody.

[0018]

[0018] In certain embodiments, the heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and / or the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 16.

[0019]

[0019] In certain embodiments, the antibody or antigen-binding fragment provided herein further comprises an immunoglobulin constant region, optionally a heavy chain constant region of IgG, and / or a light chain constant region. In certain embodiments, the constant region comprises a mouse constant region, a rabbit constant region, a human constant region, or any other suitable constant region.

[0020]

[0020] In a particular embodiment, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 80% sequence identity thereto, and / or the light chain constant region includes the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80% sequence identity thereto.

[0021]

[0021] In certain embodiments, the antibodies or antigen-binding fragments provided herein are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, labeled antibodies, bivalent antibodies, anti-idiotype antibodies, fusion proteins, dimerized antibodies or polymers, or modified antibodies (e.g., glycosylated antibodies).

[0022]

[0022] In certain embodiments, the antibodies or antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0023]

[0023] In certain embodiments, the antibodies or antigen-binding fragments provided herein are linked to one or more parts. In certain embodiments, the parts include radioisotopes, lanthanides, chemiluminescent labels, chromogenic parts, gold colloid particles, fluorescent labels, enzyme substrate labels, digoxigenin labels, biotin / avidin, haptens, and DNA molecules for detection or particle labeling. In certain embodiments, the parts include biotin or haptens.

[0024]

[0024] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments provided herein for binding to CLDN18.2.

[0025]

[0025] In one aspect, the Disclosure provides isolated polynucleotides encoding an antibody or an antigen-binding fragment thereof, as provided herein. In one aspect, the Disclosure provides vectors comprising isolated polynucleotides, as provided herein. In one aspect, the Disclosure provides host cells comprising vectors, as provided herein.

[0026]

[0026] In yet another aspect, the present disclosure provides a method for expressing an antibody or an antigen-binding fragment thereof provided herein, comprising culturing host cells provided herein under conditions in which a vector provided herein is expressed.

[0027]

[0027] In yet another aspect, the present disclosure provides a method for detecting the presence or expression level of CLDN18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment provided herein under conditions that enable specific binding of an antibody or antigen-binding fragment thereof to human CLDN18.2, and determining the presence or expression level of CLDN18.2 in the sample.

[0028]

[0028] In yet another aspect, the present disclosure provides a method for diagnosing a CLDN18.2-related disease or condition (e.g., cancer) in a subject, the method comprising: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; and b) Determine the presence or expression level of CLDN18.2 in the sample; If CLDN18.2 is found to be present, or if the expression level of CLDN18.2 reaches a threshold level, the subject is diagnosed with a CLDN18.2-related disease or condition (e.g., cancer).

[0029]

[0029] In yet another aspect, the present disclosure provides a method for determining the eligibility of a subject having or at risk of having a CLDN18.2-related disease or condition for treatment with a CLDN18.2-targeting agent, the method comprising: a) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; b) Determine the presence or expression level of CLDN18.2 in the sample; If the presence of CLDN18.2 is detected, or if the expression level of CLDN18.2 reaches a threshold, the subject is determined to be eligible for treatment with a CLDN18.2-targeting agent, or If CLDN18.2 is not detected, or if the expression level of CLDN18.2 is below the threshold, the subject is determined to be ineligible for treatment with a CLDN18.2-targeting agent.

[0030]

[0030] In yet another aspect, the present disclosure provides a method for predicting the therapeutic effect of a CLDN18.2-targeting agent in the treatment of a CLDN18.2-related disease or condition, the method comprising: a) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; b) Determine the presence or expression level of human CLDN18.2 in the sample; c) Predicting the therapeutic effect of CLDN18.2 targeted agents, Here, CLDN18.2-targeting agents are predicted to be effective in the treatment of the target if the presence of CLDN18.2 is found, or if the expression level of CLDN18.2 reaches a threshold level, or Here, CLDN18.2-targeting agents are predicted to be ineffective in the treatment of the target if CLDN18.2 is not found to be present or if the expression level of CLDN18.2 is below the threshold.

[0031]

[0031] In yet another aspect, the present disclosure provides a method for treating a subject who has or is at risk of having a disease or condition related to CLDN 18.2, the method comprising: a) Selecting a suitable patient for treatment, which includes: i) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; ii) To determine the presence or expression level of human CLDN18.2 in the sample; iii) If the presence of CLDN 18.2 is detected, or if the expression level of CLDN 18.2 in the sample reaches a threshold, subjects should be selected as suitable for treatment of CLDN 18.2-related diseases or conditions; b) Administer a therapeutically effective dose of CLDN18.2-targeting agent to the selected subjects.

[0032]

[0032] In yet another aspect, the present disclosure provides a method for treating a subject who has cancer or is at risk of cancer, the method comprising: a) Select the target, including the following: i) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; ii) Determine the presence or expression level of CLDN18.2 in the sample; iii) If CLDN18.2 is not detected, or if the expression level of CLDN18.2 in the sample is below the threshold, the subject should be selected as unsuitable for treatment of a disease or condition related to CLDN18.2; b) Administer standard therapeutic agents other than CLDN18.2 targeting agents to the selected subjects.

[0033]

[0033] In certain embodiments, the sample is a cell sample or a tissue sample. In certain embodiments, the sample is a fixed tissue sample, and optionally a formalin-fixed paraffin-embedded (FFPE) tissue sample. In certain embodiments, CLDN18.2 is cell surface or membrane-bound CLDN18.2.

[0034]

[0034] In certain embodiments, the presence or expression level of CLDN18.2 is determined by immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or immunoblotting.

[0035]

[0035] In certain embodiments, the expression level is quantified based on the percentage of positively stained cells in the sample. In certain embodiments, the expression level is quantified based on the staining intensity of CLDN18.2 in the sample.

[0036]

[0036] In certain embodiments, the disease or condition related to CLDN 18.2 is cancer. In certain embodiments, the sample includes a tumor sample. In certain embodiments, the tumor sample includes tumor tissue or circulating tumor cells. In certain embodiments, the cancer is primary cancer or metastatic cancer.

[0037]

[0037] In certain embodiments, the disease or condition related to CLDN 18.2 is cancer. In certain embodiments, cancer is primary cancer or metastatic cancer. In certain embodiments, cancer is gastric cancer, lung cancer (non-small cell lung cancer or small cell lung cancer), bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumor, cervical cancer, uterine cancer, endometrial cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, bile duct cancer, and / or adenocarcinoma.

[0038]

[0038] In certain embodiments, the cancer is gastric cancer, pancreatic cancer, bile duct cancer, or lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer (NSCLC or SCLC).

[0039]

[0039] In certain embodiments, a drug targeting CLDN18.2 can induce cytotoxicity against cells expressing CLDN18.2. In certain embodiments, the drug targeting CLDN18.2 is a therapeutic anti-CLDN 18.2 antibody or CLDN 18.2 conjugate molecule, a CLDN 18.2-targeted cell therapy, a CLDN18.2-targeted chemical compound, or a CLDN 18.2-targeted therapeutic nucleic acid.

[0040]

[0040] In certain embodiments, a therapeutic anti-CLDN18.2 antibody or a CLDN18.2-binding molecule is conjugated to a cytotoxic agent. In certain embodiments, the therapeutic anti-CLDN18.2 antibody is a bispecific antibody. In certain embodiments, the CLDN 18.2-targeted cell therapy includes CAR-T (chimeric antibody receptor engineered T cells), TCR-T (genetically modified TCR T cells), or CAR-NK (chimeric antibody receptor engineered NK cells) that express a CLDN 18.2-binding chimeric antibody receptor (CAR).

[0041]

[0041] In certain embodiments, the subject is currently receiving or has previously received anti-cancer treatment, or is suffering from a recurrence of cancer.

[0042]

[0042] In yet another aspect, the present disclosure provides a kit comprising an isolated antibody or an antigen-binding fragment thereof provided herein.

[0043]

[0043] In certain embodiments, the kit further includes a reagent set for detecting a complex of an antibody or its antigen-binding fragment bound to CLDN 18.2. In certain embodiments, the reagent set includes an anti-mouse antibody. [Brief explanation of the drawing]

[0044] [Figure 1]

[0044] Figure 1 shows the FACS analysis of anti-CLDN18.2 antibodies 69H2F7E6 and 14G11G2D2 that bind to HEK293-hCLDN18.2 cells and HEK293-hCLDN18.1 cells. Antibodies 18B10D3G9F3 and EPR19202 were used as positive controls. [Figure 2]

[0045] Figure 2 shows immunocytochemistry (ICC) staining of antibodies 69H2 and 14G11 screened on HEK293, HEK293-CLDN18.1, and HEK293-CLDN18.2 cell block sections. Staining with antibody GC182 is used as a control. [Figure 3]

[0046] Figure 3 shows the binding profiles and EC50 values ​​of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182, which bind to the hCLDN 18.2 peptide (amino acid residues 28-37 of hCLDN 18.2). [Figure 4]

[0047] Figure 4 shows the binding profiles and EC50 values ​​of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182, as measured by ELISA, indicating their binding to the recombinant hCLDN18.2 mutant protein (SEQ ID NO: 26), which constitutes the amino acid sequence of the ECL1 loop of hCLDN18.2. [Figure 5]

[0048] Figure 5 shows the binding profiles and EC50 of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182 to recombinant hCLDN18.1 variant protein containing the amino acid sequence of the ECL1 loop of hCLDN18.1 (SEQ ID NO: 27), as measured by ELISA. [Figure 6]

[0049] Figure 6 shows the binding affinity analysis of the anti-CLDN18.2 antibodies, 69H2F7E6, 14G11G2D2, and GC182, with recombinant hCLDN18.2 variant protein using ForteBio. KD, kon, and koff are shown. [Figure 7-1]

[0050] Figures 7A and 7B show immunohistochemical (IHC) analyses of selected antibodies 14G11G2D2, 69H2F7E6, and GC182 on formalin-fixed, paraffin-embedded (FFPE) sections of normal stomach, lung, intestine, kidney, tonsil, thyroid, breast, and skeletal muscle tissue, respectively. Arrows indicate positive staining of GC182 in normal lung tissue. [Figure 7-2]

[0050] Figures 7A and 7B show immunohistochemical (IHC) analysis of selected antibodies 14G11G2D2, 69H2F7E6, and GC182 on formalin-fixed, paraffin-embedded (FFPE) sections of normal stomach, lung, intestine, kidney, tonsil, thyroid, breast, and skeletal muscle tissue, respectively. The arrows indicate positive staining of GC182 in normal lung tissue. [Figure 8]

[0051] Figure 8 shows IHC images of the anti-CLDN18.2 antibodies 14G11G2D2 and EPR19202 in the stomach and skeletal muscle. EPR19202 stained positively in both gastric tissue and skeletal muscle, while 14G11G2D2 stained positively only in gastric tissue. [Figure 9]

[0052] Figure 9 shows representative IHC images of 14G11G2D2 antibody in various tissues of gastric cancer, pancreatic cancer, cholangiocarcinoma, and non-small cell lung cancer (NSCLC), indicating strong (++), moderate (++), weak (+), and negative (-) staining intensity. [Figure 10]

[0053] Figure 10 shows a comparison of IHC staining of gastric sections with the antibody 14G11G2D2- and the antibody conjugate 14G11G2D2-biotin. [Figure 11-1]

[0054] Figure 11 shows the complete arrangement of the present invention. [Figure 11-2]

[0054] Figure 11 shows the complete arrangement of the present invention. [Modes for carrying out the invention]

[0045] Detailed description of the invention

[0055] The following description of the present disclosure is merely intended to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present disclosure, and such equivalent embodiments are understood to be encompassed herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entireties.

[0046] Definitions

[0056] As used herein, the terms "a", "an", "the" and similar terms used in the context of the present invention (particularly in the context of the claims) shall be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0047]

[0057] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. Native intact antibodies are composed of two heavy chains and two light chains. Mammalian heavy chains are classified into α, δ, ε, γ, and μ, and each heavy chain has a variable region (V H ) and first, second, and third constant regions (C H1 , C H2 , C H3 ), and mammalian light chains are classified as λ or κ, each light chain having a variable region (V LAntibodies consist of a variable region and a constant region. Antibodies are "Y" shaped, with the stem of the Y consisting of second and third constant regions of two heavy chains linked via disulfide bonds. Each arm of the Y contains a variable region of a single light chain and a variable region of a single heavy chain linked to the constant region, as well as the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3; heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibody and antigen-binding domains disclosed herein can be defined or identified according to the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196,901 (1987); NRWhitelegg et al, Protein Engineering, v13(12), 819-824 (2000); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EAet al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al, Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)).The three CDRs are sandwiched between supporting extensions called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exert various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). For the purposes of the present invention, this disclosure includes all antibodies and antibody derivatives described herein, which are encompassed by the term “antibody”. As used herein, the term “antibody derivative” refers to any modified form of an antibody, such as a conjugate of an antibody with another drug, an antibody fragment, or a fusion protein comprising an antibody or an antibody fragment.

[0048]

[0058] As used herein, the term “antigen-binding fragment” refers to an antibody fragment formed from an antibody fragment containing one or more CDRs, or any other antibody portion that binds to an antigen but does not constitute an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds-diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multi-specific antibody fragments, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may contain one or more CDRs from a particular antibody.

[0049]

[0059] In relation to antibodies, "Fab" refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) attached to the variable region and first constant region of a single heavy chain by disulfide bonds. Fab can be obtained by papain digestion of the antibody at a residue adjacent to the N-terminus of the disulfide bond between the heavy chains in the hinge region.

[0050]

[0060] "Fab" refers to a Fab fragment that includes part of the hinge region and can be obtained by pepsin digestion of the antibody at residues adjacent to the C-terminus of the disulfide bond between the heavy chains of the hinge region, and thus differs from Fab in a few residues (including one or more cysteines) in the hinge region.

[0051]

[0061] "F(ab')2" refers to a dimer of Fab' that contains two light chains and parts of two heavy chains.

[0052]

[0062] In the context of antibodies, "Fc" refers to the portion of the antibody where the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain are linked via disulfide bonds. IgG and IgM have three heavy chain constant regions in their Fc region (the second, third, and fourth heavy chain constant regions of each chain). Antibodies are obtained by papain digestion. While the Fc portion of antibodies performs various effector functions such as ADCC, ADCP, and CDC, it does not function in antigen binding.

[0053]

[0063] In relation to antibodies, "Fv" refers to the smallest fragment of an antibody that completely contains the antigen-binding site. An Fv fragment is formed by the binding of a variable region of one light chain to a variable region of one heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the binding between the variable regions of a single light chain and a single heavy chain is a disulfide bond.

[0054]

[0064] A "single-chain Fv antibody" or "scFv" refers to an artificial antibody in which the light chain variable region and the heavy chain variable region are linked directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with linkers. In certain embodiments, the "scFv dimer" has V at one site. H V of the other part L In coordination with another V, it forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L V dimerized with the site H -V L A divalent diabody or divalent ScFv(BsFv) containing (linked by a peptide linker). In other embodiments, the "scFv dimer" is V H1 and V L1 V H2 and V L2 V L1 -V H2 (This is also linked by a peptide linker) and related V H1 -V L2 It is a bispecific diabody (which is also linked by a peptide linker), and each coordination pair has a different antigen specificity.

[0055]

[0065] A "single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of scFv attached to the Fc region of the antibody.

[0056]

[0066] "Camelized single-domain antibodies," "heavy chain antibodies," "nanobodies," or "HCAb" are two V HThis refers to antibodies that contain a domain but do not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol.Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No.6,005,079). Heavy chain antibodies were originally obtained from camelids (camels, dromedaries, and llamas). Although they lack a light chain, antibodies from the camelid family possess a true antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domain (VHH domain) of heavy-chain antibodies represents the smallest known antigen-binding unit produced by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0057]

[0067] The "diabody" contains a small antibody fragment with two antigen-binding sites, and this fragment is V in a single polypeptide chain. L V linked to the domain H Domain (V H -V L or V L -V H) include (see, for example, Holliger P. et al, Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). Two domains on the same strand cannot pair due to the linker being too short; therefore, the domains must pair with a complementary domain on another strand, thereby forming two antigen-binding sites. The antigen-binding sites may target different antigens (or epitopes) of the same antigen.

[0058]

[0068] A "domain antibody" refers to an antibody fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In certain embodiments, two or more V H The domains are covalently linked by a peptide linker to form a bivalent or polyvalent domain antibody. The two Vs of a bivalent domain antibody H Domains may target the same antigen or they may target different antigens.

[0059]

[0069] One "(dsFv)2" is a disulfide-stabilized Fv fragment containing three peptide chains, and two V H The site is linked by a peptide linker, and two V L It is bonded to the site with disulfide crosslinks.

[0060]

[0070] A "bispecific DS antibody" is an antibody that targets two different antigens (or epitopes). H1 and V L1 V via a disulfide bond between them L1 -V H2 V is attached to 2 (which is also linked by a peptide linker). H1 -V L2 It can be composed of (linked by a peptide linker).

[0061]

[0071] "Dual-specific dsFv" or "dsFv-dsFv" refers to a disulfide-stabilized Fv fragment that targets two different antigens (or epitopes). It can contain three peptide chains: V H1 -V H2 In this part, the heavy chain is linked by a peptide linker (e.g., a long flexible linker), V L1 and V L2 Each portion is paired via a disulfide crosslink. The heavy and light chains paired by disulfide bonds each have different antigen specificities.

[0062]

[0072] As used herein, the term “humanized” refers to an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, a FR region derived from a human, and optionally a constant region derived from a human.

[0063]

[0073] As used herein, the term “chimera” refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain from one species and the remainder of a heavy chain and / or light chain from a different species. In exemplary cases, a chimeric antibody may consist of a constant region derived from humans and a variable region derived from a non-human species such as a mouse.

[0064]

[0074] As used herein, “anti-CLDN18.2 antibody” or “antibody against CLDN18.2” refers to an antibody that can specifically bind to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity to provide, for example, use for diagnosis and / or therapy.

[0065]

[0075] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0066]

[0076] As used herein, the terms “specific binding,” “specifically binding,” or “binding specificity” refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen.

[0067]

[0077] The "sequence identity percentage (%)" for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after the sequences have been aligned, gaps introduced as necessary, and the greatest possible correspondence has been achieved. Alignment for the purpose of determining the amino acid (or nucleic acid) sequence identity percentage can be achieved using publicly available tools such as BLASTN and BLASTp (available on the website of the National Center for Biotechnology Information (NCBI)). See also Altschul SF et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute; Higgins DGet al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or they may customize the parameters to suit the alignment, for example, by selecting an appropriate algorithm.

[0068]

[0078] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain specific to each amino acid. In this disclosure, amino acid names are represented by standard one- or three-letter codes, the general outline of which is as follows:

[0069] [Table 1]

[0070]

[0079] A "conservative substitution" in amino acid sequences refers to the substitution of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the three-dimensional structure of a protein, and therefore can preserve the biological activity of the protein.

[0071]

[0080] An "isolated" substance is one that has been altered by human intervention from its natural state. When an "isolated" composition or substance exists in nature, it is either altered from its original environment, removed, or both. For example, polynucleotides and polypeptides naturally present in living animals are not "isolated," but the same polynucleotides or polypeptides can be considered "isolated" if they are sufficiently separated from their naturally occurring coexisting substances and exist in a substantially pure state. The terms "isolated nucleic acid" or "polynucleotide" are used interchangeably and refer to a sequence of isolated nucleic acid molecules. In certain embodiments, “isolated antibody or its antigen-binding fragment” means an antibody or antigen-binding fragment having a purity of at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 75%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as determined by electrophoresis (SDS-PAGE, isoelectric focusing, capillary electrophoresis, etc.) or chromatography (ion-exchange chromatography or reverse-phase HPLC, etc.).

[0072]

[0081] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, rodents (e.g., mice, rats and guinea pigs), cats, rabbits, sheep, dogs, cattle, chickens, amphibians and reptiles. In a more preferred embodiment, the subject is human. Unless otherwise noted, the terms "patient," "subject" and "individual" are used interchangeably herein.

[0073]

[0082] As used herein, the term "effector function" refers to the biological activity resulting from the binding of the antibody's Fc region to effectors such as the C1 complex or Fc receptor. Examples include complement-dependent cytotoxicity (CDC) induced by the interaction between C1q on the C1 complex and the antibody, antibody-dependent cytotoxicity (ADCC) induced by the binding of the antibody's Fc region to the Fc receptor on effector cells, and antibody-dependent cell-mediated phagocytosis (ADCP) in which nonspecific cytotoxic cells expressing FcγR recognize the bound antibody on target cells, subsequently causing phagocytosis of the target cells. Effector functions include those that act after antigen binding and those that act independently of antigen binding.

[0074]

[0083] As used herein, “treat,” “administer,” or “remedy” a condition includes preventing or alleviating a condition, slowing the onset or rate of onset of a condition, reducing the risk of developing a condition, preventing or delaying the onset of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, causing complete or partial regression of a condition, curing a condition, or any combination thereof.

[0075]

[0084] "At risk" refers to individuals, i.e., patients, who have been identified as having a higher-than-usual likelihood of developing a disease, particularly cancer, compared to the general population. Furthermore, individuals who have previously had a disease, particularly cancer, or who currently have cancer, are at high risk of developing the disease because they may continue to experience its development. Additionally, individuals who currently have cancer, or who have had cancer in the past, also have a higher risk of cancer metastasis. In the context of this invention, terms such as "protective," "preventive," and "preventive" relate to the prevention or treatment, or both, of the development and / or transmission of a disease in an individual, and more specifically, to minimizing the opportunity for the individual to develop the disease or delaying its development. For example, individuals at risk of tumors as described above are candidates for treatments aimed at preventing tumors. Immunotherapy can be carried out using one of various methods involving drugs that have the function of removing antigen-expressing cells from the patient.

[0076]

[0085] The “standard treatment” therapeutic agents described herein include the administration of standard treatment therapeutic agents to a patient. As used herein, the term “standard treatment” refers to a course of treatment, including drugs or combinations of drugs, radiation therapy (RT), surgery, or other medical interventions, that is recognized by healthcare professionals as appropriate, accepted, and / or widely used for certain patients, diseases, or clinical situations. Standard treatments for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a coalition of 21 leading cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®), which provide detailed and up-to-date information on standard treatments for a wide variety of cancers (see NCCN GUIDELINES®, 2013).

[0077]

[0086] The terms "effective" and "effective" in relation to treatment methods include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in patients. Physiological safety refers to the degree of toxicity or other adverse physiological effects (side effects) at the cellular, organ, and / or biological levels resulting from the administration of a drug.

[0078]

[0087] The “therapeutic effective dose” or “therapeutic effective amount” of a drug or therapeutic agent, such as an antibody, in this disclosure refers to any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or condition, or that promotes the regression of the disease / condition as demonstrated by a reduction in the severity of symptoms of the disease / condition, an increase in the frequency and duration of asymptomatic periods of the disease / condition, or the prevention of impairment or impairment due to the distress of the disease / condition. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as human subjects in clinical trials, animal model systems to predict efficacy in humans, or assays of drug activity in in vitro assays. A therapeutically effective amount of the drug includes a “prophylactic effective amount,” which is any amount of the drug that, when administered alone or in combination with an antineoplastic agent to a subject at risk of developing a CLDN18.2-related disease or condition, such as cancer (e.g., a subject with a pre-malignant condition) or a subject suffering from a relapse of the disease / condition, suppresses the onset or relapse of the disease / condition. In a preferred embodiment, a prophylactic effective amount completely prevents the onset or relapse of a CLDN18.2-related disease or condition (e.g., cancer). To “inhibit” the onset or recurrence of a disease / condition (e.g., cancer) means either to reduce the likelihood of the disease / condition developing or recurring, or to completely prevent the onset or recurrence of the disease / condition.

[0079]

[0088] As used herein, the term “vector” refers to a vehicle into which a genetic element can be operably inserted to produce a protein, RNA, or DNA encoded by that genetic element, or to replicate that genetic element, in order to bring about the expression of that genetic element.

[0080]

[0089] As used herein, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0081]

[0090] The term "CLDN18" refers to claudin 18 and includes any selective variants of CLDN18, such as CLDN18.1 and CLDN18.2. CLDN18.1 and CLDN18.2 differ in their N-terminal regions, including the first transmembrane (TM) region and loop 1, but their primary C-terminal protein sequences are identical.

[0082]

[0091] The term "CLDN18.2" refers to claudin-18 selective variant 2 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include the human CLDN18.2 protein (NCBI Ref Seq No. NP_001002026.1, or Sequence ID No. 20). Exemplary sequences of CLDN18.2 include the Mus musculus (mouse) CLDN18.2 protein (NCBI Ref Seq No. NP_001181852.1) and the Macaca fascicularis (crab-eating monkey) CLDN18.2 protein (NCBI Ref Seq No. XP_015300615.1).

[0083]

[0092] The term "CLDN 18.1" refers to Claudin-18 splice variant 1 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.1 is human CLDN18.1. Exemplary sequences of human CLDN18.1 include human CLDN18.1 protein (NCBI Ref Seq No. NP_057453.1, or SEQ ID NO. 21), Mus musculus (mouse) CLDN18.2 protein (NCBI Ref Seq No. NP_001181851.1), and Macaca fascicularis (crab-eating macaque) CLDN18.2 protein (NCBI Ref Seq No. XP_005545920.1).

[0084]

[0093] The terms "CLDN18.2" and "CLDN18.2" include exemplary sequence variants such as mutants, structural variants, isoforms, allelic variants, species variants, and species homologs, including those occurring naturally.

[0085]

[0094] As used herein, “CLDN18.2-related disease or condition” refers to any disease or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in the expression or activity of CLDN18.2. In some embodiments, a CLDN18.2-related disease is, for example, cancer.

[0086]

[0095] As used herein, “cancer” refers to any medical condition characterized by the proliferation or neoplasm, abnormal growth, invasion or metastasis of malignant cells, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematological malignancies).

[0087]

[0096] As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0088]

[0097] The term "pharmaceutically acceptable" indicates that a specified carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0089]

[0098] As used herein, the terms “metastasis” or “metastatic cancer” refer to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that depends on the detachment of malignant cells from the primary tumor, their invasion into the extracellular matrix, their penetration of the endothelial basement membrane into body cavities and blood vessels, and their subsequent invasion into a target organ after being carried by the blood. Finally, whether a new tumor, i.e., a secondary or metastatic tumor, grows at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the present invention refers to “distant metastasis,” which is a metastasis that is far from the primary tumor and the regional lymph node system. The cells of a secondary or metastatic tumor are similar to the cells of the original tumor. This means, for example, that if gastric cancer metastasizes to the liver, the secondary tumor consists of abnormal gastric cells, not abnormal liver cells. In that case, the tumor in the liver is called metastatic gastric cancer, not liver cancer.

[0090]

[0099] In this specification, when the term "about" is used with respect to a value or parameter, it includes (and describes) embodiments directed toward that value or parameter itself. For example, a statement referring to "about X" includes a statement about "X". A range of numbers includes the numbers that define that range. In general, the term "about" refers to all values ​​of the variable within the indicated value and the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean), whichever is greater, or within 10% of the indicated value. When the term "about" is used in the context of a period (year, month, week, day, etc.), the term "about" refers to that period plus or minus one unit of the next smaller period (e.g., about one year is 11 to 13 months, about six months is 6 months plus or minus one week, about one week is 6 to 8 days, etc.), or within 10% of the indicated value, whichever is greater.

[0091]

[0100] Anti-CLDN18.2 antibody

[0101] This disclosure provides an anti-CLDN18.2 antibody and its antigen-binding fragment.

[0092]

[0102] CLDN18.2 is a selective variant of Claudin-18 (CLDN18). CLDN18 is a member of the tetraspanin family and has four hydrophobic regions. CLDN18 exhibits several different conformations, which may be selectively matched by antibodies (see Sahin U et al. Clinical Cancer Research, 2008, 14(23):7624-7634). In CLDN18-Conformation-1, all four hydrophobic regions function as transmembrane domains (TMs), forming two extracellular loops (loop 1 embraced by hydrophobic regions 1 and 2, and loop 2 surrounded by hydrophobic regions 3 and 4), as described for most CLDN family members. The second conformation (CLDN18-Conformation-2) refers to the extracellular location of the portion between the first and fourth transmembrane domains (loop D3), as described for PMP22, because the second and third hydrophobic domains do not completely traverse the cell membrane. The third three-dimensional structure (CLDN18-Conformation-3) exhibits a large extracellular domain and two internal hydrophobic domains surrounded by the first and fourth hydrophobic regions. Because loop D3 contains a classical N-glycosylation site, the CLDN-18 topological variants, CLDN 18 topology-2 and CLDN 18 topology-3, also contain an additional extracellular N-glycosylation site.

[0093]

[0103] CLDN18 has two selective variants, both present in mice and humans. The selective variants CLDN18.1 and CLDN18.2 differ in the first TM and the 21 amino acids at the N-terminus that make up loop 1, but their C-terminal protein sequences are identical. These two isoforms share 92% amino acid sequence identity, but their expression patterns are mutually exclusive, with CLDN18.1 predominantly expressed in normal lung tissue and CLDN18.2 predominantly expressed in normal gastric tissue (see Niimi T, et al. molecular and cellular biology, 2001, 21(21): 7380-7390). The amino acid sequences of human CLDN18.1 and CLDN18.2 are shown below.

[0094]

[0104] Human claudin18.2 (accession: NP_001002026.1) amino acid sequence (SEQ ID NO: 20): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0105] Human claudin18.1 (accession: NP_057453.1) amino acid sequence (SEQ ID NO: 21): MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNWFMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0106] In normal tissues, CLDN18.2 expression is limited to the basement membrane of mucosal cells, preventing access to therapeutic antibodies. In pathological conditions (such as tumor cells), the polarity of gastric mucosal cells is disrupted, exposing CLDN 18.2 to the cell surface. The CLDN18.2 protein is a pan-cancer target expressed in primary and metastatic lesions of several human cancer types, including gastric, esophageal, pancreatic, and lung tumors, as well as human cancer cell lines (see Sahin Ugur et al. Clin Cancer Res 2008;14(23); Matsuda Y et al. Cancer Science, 2007, 98(7): 1014-1019). Abnormal ectopic expression of CLDN18.2 has been reported in multiple studies in pancreatic, ovarian, biliary tract, and lung adenocarcinomas (e.g., Karanjawala ZE et al, Am J Surg Pathol, 2008 Feb;32(2):188-96; Micke P et al, Int J Cancer.2014 Nov 1;135(9):2206-14; Keira Y et al, Virchows Arch.2015 Mar;466(3):265-77; Coati et al, Br J Cancer.2019 Jul;121(3):257-263; Dottermusch et al, Virchows Arch.2019 Nov;475(5):563-571; Rohde et al, Jpn J Clin Oncol.2019 Sep 1;49(9):870-876; Woll et al, Int J See Cancer. 2014 Feb 1;134(3):731-9; Espinoza et al, Histopathology. 2019 Mar;74(4):597-607; Shinozaki et al, Virchows Arch. 2011 Jul;459(1):73-80).

[0095]

[0107] In one aspect of this disclosure, a monoclonal anti-CLDN18.2 antibody and its antigen-binding fragment are provided herein. The monoclonal anti-CLDN18.2 antibody and antigen-binding fragment provided herein can specifically bind to CLDN18.2 (e.g., human CLDN18.2), a fragment of CLDN18.2, or a fusion polypeptide containing a fragment of CLDN18.2. In certain embodiments, the fragment of CLDN18.2 includes the first extracellular loop of human CLDN18.2, or a sequence within the first extracellular loop of human CLDN18.2. In certain embodiments, the fragment of human CLDN18.2 includes the amino acid sequence described in SEQ ID NO: 26 or SEQ ID NO: 19. In certain embodiments, the fusion polypeptide includes additional amino acid residues bound to the N-terminus and / or C-terminus of the fragment of CLDN18.2. In certain embodiments, the fragment of CLDN18.2 contained in the fusion polypeptide forms a loop.

[0096]

[0108] The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein can specifically bind to CLDN18.2-expressing cells. In certain embodiments, the CLDN18.2-expressing cells are pre-treated cells. As used herein with respect to cells, the term “pre-treated” means that the cells have been treated such that their surface proteins, such as CLDN18.2, are denatured or otherwise no longer in their native three-dimensional structure. For example, CLDN18.2-expressing cells may be pre-treated with one or more chemicals, such as formalin, paraffin, or acetone, or by physical interventions such as freezing or heating. In certain embodiments, the CLDN18.2-expressing cells are formalin-fixed, paraffin-embedded (FFPE) cells.

[0097]

[0109] The binding of the anti-CLDN18.2 antibody and antigen-binding fragment provided herein to the antigen is "half-maximal effective concentration" (EC2). 50 This can be expressed as an EC value, which refers to the concentration of the antibody at which 50% of its maximum effect (e.g., binding) is observed. 50The value can be measured by methods known in the art, such as sandwich assays including ELISA, Western blotting, and other binding assays. EC50 can be measured in a suitable binding assay in which serial dilutions of the antibody are tested for binding to the antigen and the concentration at which 50% of the maximum binding is determined is determined. In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein specifically binds to human CLDN18.2, human CLDN18.2 fragments, fusion polypeptides containing human CLDN18.2 fragments, human CLDN18.2-expressing cells, or pre-treated human CLDN18.2-expressing cells. In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein specifically binds to a fusion polypeptide containing a first extracellular loop of human CLDN18.2 at an EC50 of 50 ng / ml or less (e.g., 40 ng / ml or less, 35 ng / ml or less, 30 ng / ml or less, 20 ng / ml or less, 18 ng / ml or less, 16 ng / ml or less, 15 ng / ml or less, 14 ng / ml or less, 13 ng / ml or less, 12 ng / ml or less) as measured by ELISA.

[0098]

[0110] The binding affinity of the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein to human CLDN18.2 is, for example, the ratio of the dissociation rate to the association rate (k off / k on ) refers to K D It is also possible to characterize it by K. D This can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance (SPR), microscale thermophoresis, and HPLC-MS. In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein is measured by SPR. -6 M or less (for example, 10 -7 M, 10 -7.5 M, 10 -8 M, or 10 -8.5 K (M or below) DIt binds to a fusion polypeptide containing the first extracellular loop of human CLDN18.2. D The lower the value, the higher the affinity.

[0099]

[0111] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment do not cross-react with human CLDN18.1. Anti-CLDN18.2 antibodies that "do not cross-react with" or "do not have cross-reactivity with" human CLDN18.1 are antibodies that do not produce undesirable results, such as false positives, in detection assays (e.g., IHC assays) against human CLDN18.2. In certain embodiments, binding to human CLDN18.1 is undetectable in detection assays (e.g., IHC assays or flow cytometry assays). The level of binding can be determined as the level of antigen-antibody complexes formed at predetermined concentrations of the antibody and antigen. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein bind to human CLDN18.1 at a lower level or affinity (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% lower) than the binding of antibody GC182 to human CLDN18.1.

[0100]

[0112] In certain embodiments, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.3%, or less than 0.1% of human CLDN18.1-expressing cells are positively detected by IHC assay with the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein do not show detectable binding to human CLDN18.1 in IHC assay. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein do not show detectable binding to human CLDN18.1 in non-cancerous human lung tissue samples in IHC assay.

[0101]

[0113] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein do not cross-react with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples at an antibody concentration of 1 nM as measured by ELISA or 0.5 ug / ml as measured by IHC. IHC can be carried out according to the procedures and conditions outlined in Example 8 or 9.

[0114] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein do not cross-react with non-cancerous cells except gastric epithelial cells. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein do not cross-react with non-cancerous human tissues such as lung tissue, intestinal tissue, kidney tissue, tonsil tissue, thyroid tissue, skeletal muscle tissue, or breast tissue. This distinguishes the antibodies provided herein from existing monoclonal anti-CLDN18.2 antibodies. For example, antibody 43-14 A has been shown to bind to human CLDN 18.1 and therefore to cross-react with non-cancerous human lung tissue (see Ventana CLDN 18 (43-14 A) Assay, Ref. 790-7027, 08504148001). As another example, the antibody GC182 has been shown to cross-react with CLDN18.1 and therefore stain non-cancerous human lung tissue (see Example 4 and Figure 2 of this disclosure). As yet another example, the anti-CLDN18.2 antibody EPR19202 (available from Abcam under product name ab222512) has been shown to bind nonspecifically to skeletal muscle tissue and therefore exhibit cross-reactivity with non-cancerous skeletal muscle tissue.

[0102]

[0115] Exemplary anti-CLDN18.2 antibody

[0116] In certain embodiments, the Disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (e.g., human CLDN18.2), comprising the following: a) Heavy chain CDR1 containing the amino acid sequence X1X2YX3H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX4GX5X6X7X8YX9EKFKG (SEQ ID NO: 12), and / or NYX 10 Heavy chain CDR3 containing the amino acid sequence of STFGY (SEQ ID NO: 24), and / or b) Light chain CDR1, KX containing the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2) 11 Light chain CDR2 containing the amino acid sequence of SNRFS (SEQ ID NO: 25), and / or light chain CDR3 containing the amino acid sequence of FQGSHVPFT (SEQ ID NO: 6). Here, X1 is R or T, X2 is N or Y, X3 is F or I, X4 is G or R, X5 is F or G, X6 is D or N, X7 is I or T, X8 is E or V, X9 is S or N, X 10 is G or R, and X 11 It is either V or I.

[0103]

[0117] In certain embodiments, the Disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (e.g., human CLDN18.2), comprising one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6) selected from the set of sequences consisting of SEQ ID NOs. 1-6 or from the set of sequences consisting of SEQ ID NOs. 2, 6, 7 and 9-11.

[0104]

[0118] In certain embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided herein include the heavy chain CDR3 sequence of SEQ ID NO: 5 or 11. Because the heavy chain CDR3 region is centrally located at the antigen-binding site, it is thought to have the most contact with the antigen and to provide the most free energy to the antibody's affinity for the antigen. Furthermore, heavy chain CDR3 is considered to be the most diverse CDR among antigen-binding sites in terms of length, amino acid composition, and three-dimensional structure, due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81 (1983)). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45 (2000)) and desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263:551-67 (1996)).

[0105]

[0119] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein comprises: a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 7, and / or a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 9, and / or a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 11, and / or a light chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 2, and / or a light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 10, and / or a light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 6.

[0106]

[0120] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (e.g., human CLDN18.2), comprising: a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5; or b) A heavy chain CDR1 sequence containing SEQ ID NO: 7, a heavy chain CDR2 sequence containing SEQ ID NO: 9, and a heavy chain CDR3 sequence containing SEQ ID NO: 11.

[0107]

[0121] In certain embodiments, the antibody or antigen-binding fragment provided herein includes: a) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0108]

[0122] In certain embodiments, the antibody or antigen-binding fragment provided herein includes: a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0109]

[0123] While CDRs are known to be involved in antigen binding, it has become clear that not all six CDRs are necessarily essential or immutable. That is, one, two, or three of the CDRs provided above (e.g., one of SEQ ID NOs. 1-6, or SEQ ID NOs. 2, 6, 7, and 9-11) can be substituted, altered, or modified while substantially retaining a specific binding affinity to CLDN18.2. Antibodies having such modified or variant CDRs are also included in this disclosure.

[0110]

[0124] In certain embodiments, the antibody or antigen-binding fragment provided herein includes: a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or b) A heavy chain variable region comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16.

[0111]

[0125] As used herein, the antibody "14G11" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 14.

[0112]

[0126] The antibody "69H2" as used herein refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 15 and the light chain variable region of SEQ ID NO: 16.

[0113]

[0127] Table 1 shows the CDR sequences of the anti-CLDN18.2 antibodies 14G11 and 69H2. The variable region sequences of the heavy and light chains are also shown in Table 2 below.

[0114]

[0128] Table 1. Nucleotide sequences of the CDR region of CLDN18.2 antibody

[0115] [Table 2]

[0116]

[0129] Table 2. Base sequences of the VH / VL region of mouse / recombinant antibody

[0117] [Table 3]

[0118]

[0130] In certain embodiments, the antibodies and antigen-binding fragments provided herein are composed of appropriate framework region (FR) sequences, insofar as the antibodies and antigen-binding fragments can specifically bind to CLDN18.2 (e.g., human CLDN18.2). The CDR sequences provided in Table 1 are derived from mouse antibodies, but these can be grafted onto FR sequences of any suitable species, such as mouse, human, rat, or rabbit, using appropriate methods known in the art, such as recombinant techniques. Since it is well known in the art that the CDR region is flanked by two FR regions in the variable region, the FR sequences can be readily identified by those skilled in the art based on the CDR sequences in Table 1 and the variable region sequences in Table 2.

[0119]

[0131] In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein further comprises an immunoglobulin constant region. The constant region optionally comprises a heavy chain constant region and / or a light chain constant region of IgG. The heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

[0120]

[0132] In certain embodiments, the anti-CLDN18.2 antibody and its fragment provided herein further comprises a constant region of mouse IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein comprises a constant region of a mouse IgG1 isotype. In certain embodiments, the heavy chain constant region of mouse IgG1 has sequence identity with SEQ ID NO: 17 or at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%), and / or the light chain constant region of mouse IgG1 comprises the amino acid sequence of SEQ ID NO: 18 or homologous sequences having sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0121]

[0133] The anti-CLDN18.2 antibodies or their antigen-binding fragments provided herein may be monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, labeled antibodies, bivalent antibodies, anti-idiotype antibodies, fusion proteins, dimerized antibodies or polymers, or modified antibodies (e.g., glycosylated antibodies). Recombinant antibodies are antibodies prepared in vitro using recombinant methods, rather than from animals.

[0122]

[0134] In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein is divalent, tetravalent, hexavalent, or polyvalent. Any molecule that is divalent or higher is considered polyvalent and may include, for example, trivalent, tetravalent, hexavalent, and so on.

[0123]

[0135] In some embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided herein comprises all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-CLDN18.2 antibody and antigen-binding fragment provided herein is a single-domain antibody comprising all or part of the heavy chain variable domain provided herein. Detailed information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0124]

[0136] Antibody variant

[0137] The anti-CLDN18.2 antibodies and their antigen-binding fragments provided herein also include various types of variants of the antibody 14G11 and 69H2.

[0125]

[0138] In certain embodiments, the anti-CLDN18.2 antibodies or their antigen-binding fragments provided herein retain binding specificity to CLDN18.2, particularly human CLDN18.2, or more specifically, epitopes in the amino acid sequence of SEQ ID NO: 19, while retaining binding specificity to one or more non-CDR sequences of the CDR sequences provided in Table 1 above, the heavy chain variable regions or light chain variable regions provided in Table 2 above, and / or constant region sequences (e.g., the Fc region as defined in SEQ ID NO: 17 or 18). These are also referred to as variants of antibodies 14G11 and 69H2, or variants of the antigen-binding fragment. In certain embodiments, the variants retain binding affinity at a level similar to or higher than that of their parent antibodies (e.g., antibodies 14G11 or 69H2). As used herein, “mutation” or “mutated” includes substitutions, insertions, and / or deletions in amino acid sequences or polynucleotide sequences. In certain embodiments, at least one (or all) of the mutations constitute a conservative substitution.

[0126]

[0139] In certain embodiments, the antibody variant includes a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer substitutions in the CDR sequence, FR sequence, or variable region sequence of antibodies 14G11 and 69H2. In certain embodiments, the antibody variant includes one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with those listed in Table 1 (or those listed therein), while retaining binding specificity to CLDN18.2 and optionally having a binding affinity at or above the level of its parent antibody (e.g., antibody 14G11 or 69H2).

[0127]

[0140] In certain embodiments, the antibody variant has a heavy chain variable region sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with that of SEQ ID NO: 13 or 15 (or those thereof), and / or has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with that of SEQ ID NO: 14 or 16 (or those thereof), while retaining binding specificity to CLDN18.2 and optionally having a binding affinity at a level similar to or higher than that of its parent antibody (e.g., antibody 14G11 or 69H2). In some embodiments, a total of 1 to 10 amino acid residues are mutated in a sequence selected from SEQ ID NOs: 13-16. In some embodiments, the mutation occurs in the region outside the CDR (i.e., within the FR). In some embodiments, one or more of the mutations are conservative substitutions. In some embodiments, all of the mutations are conservative substitutions.

[0128]

[0141] In certain embodiments, the Disclosure provides a variant of antibody 14G11 or 69H2, wherein the variant is: a) a heavy chain CDR1 (HCDR1) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 7, and / or b) A heavy chain CDR2 (HCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 3 or SEQ ID NO: 9, and / or c) A heavy chain CDR3 (HCDR3) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 5 or SEQ ID NO: 11, and / or d) A light chain CDR1 (LCDR1) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 2, and / or e) A light chain CDR2 (LCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 4 or SEQ ID NO: 10, and / or f) A light chain CDR3 (LCDR3) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 6. including and On the other hand, it retains binding specificity to CLDN18.2 and optionally has a binding affinity at the same level as or higher than its parent antibody (e.g., antibody 14G11 or 69H2).

[0129]

[0142] In certain embodiments, the antibody variants provided herein include HCDR1 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 1 or SEQ ID NO: 7; HCDR2 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 3 or SEQ ID NO: 9; HCDR3 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 5; LCDR1 having 2 or 1 or fewer amino acid mutations in SEQ ID NO: 11; LCDR2 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 4 or SEQ ID NO: 10; and / or LCDR3 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 6, while retaining binding specificity to CLDN18.2 and optionally having a binding affinity at the same level as or higher than that of its parent antibody (e.g., antibody 14G11 or 69H2). In some embodiments, one or more of the mutations are conservative substitutions. In some embodiments, all of the mutations are conservative substitutions.

[0130]

[0143] In certain embodiments, antibody variants provided herein retain the specific binding specificity of their parent antibody to CLDN18.2 but possess one or more desirable properties conferred by mutation(s). For example, antibody variants may have, to name a few, improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding in a pH-dependent manner, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues). Such variants are also called affinity variants, glycosylation variants, cysteine ​​variants, Fc variants, etc., and are described in more detail below.

[0131]

[0144] a) Affinity variant

[0145] Affinity variants may include modifications or substitutions to one or more CDR sequences provided in Table 1 above, one or more framework (FR) sequences provided herein, or heavy chain or light chain variable region sequences provided in Table 2 above.

[0132]

[0146] Affinity variants may retain the specific binding affinity of the parent antibody to CLDN18.2, or they may have improved CLDN18.2-specific binding affinity compared to the parent antibody. To achieve this objective, various methods known in the art can be used. For example, a library of antibody variants (such as Fab or scFv variants) can be generated, expressed using phage display technology, and then screened for binding affinity to human CLDN18.2. As another example, computer software can be used to virtually simulate the binding of an antibody to human CLDN18.2 and identify amino acid residues on the antibody that form the binding interface. Such residues can be targeted for mutation to avoid a decrease in binding affinity or to provide stronger binding.

[0133]

[0147] b) Glycosylated variants

[0148] The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also include glycosylated variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or its antigen-binding fragment.

[0134]

[0149] An antibody or its antigen-binding fragment may include one or more modifications that introduce or remove a glycosylation site. A glycosylation site is an amino acid residue having a side chain to which a sugar chain (e.g., an oligosaccharide structure) can be attached. Antibody glycosylation is usually either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence, such as asparagine-X-serine or asparagine-X-threonine, where X is any amino acid other than proline. O-linked glycosylation is the attachment of a sugar such as N-acylgalactosamine, galactose, or xylose to a hydroxyamino acid, particularly serine or threonine. Removal of a native glycosylation site can be conveniently achieved, for example, by modifying the amino acid sequence to substitute either the above-mentioned tripeptide sequence (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence. By introducing such tripeptide sequences or serine or threonine residues, new glycosylation sites can be created in a similar manner.

[0135]

[0150] In certain embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided herein include a mutation at N297 (e.g., N297A, N297Q, or N297G) to remove a glycosylation site.

[0136]

[0151] c) Cysteine ​​modified variant

[0152] The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also include cysteine-modified variants, which contain one or more introduced free cysteine ​​amino acid residues.

[0137]

[0153] Free cysteine ​​residues are those that are not part of a disulfide crosslink. Cysteine ​​variants are useful for conjugation at the modified cysteine ​​site with, for example, cytotoxic and / or imaging compounds, labels, or radioisotypes, via maleimide or haloacetyl. Methods for manipulating antibodies or their antigen-binding fragments to introduce free cysteine ​​residues are known in the art; see, for example, WO2006 / 034488.

[0138]

[0154] d) Fc variant

[0001] The anti-CLDN18.2 antibodies and antigen-binding fragments provided herein also include Fc variants, which include one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region, in order to provide alterations in effector functions such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis), and CDC (complement-dependent cell-mediated cytotoxicity). Examples of Fc variants are known in the Art, see, for example, Wang et al., Protein Cell 2018, 9(1):63-73 and Kang et al., Exp&Mol., Med. (2019) 51:138, which are incorporated herein in their entirety.

[0139]

[0155] Antigen-binding fragment

[0156] Also provided herein are anti-CLDN18.2 antigen-binding fragments. In some embodiments, the antibodies and antigen-binding fragments provided herein include all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CLDN18.2 antibodies provided herein, including, for example, exemplary antibodies whose CDR sequences are shown in Table 1, and their different variants (affinity variants, glycosylated variants, Fc variants, cysteine-modified variants, etc.).

[0140]

[0157] In certain embodiments, the anti-CLDN18.2 antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, biceptive dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0141]

[0158] Various techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and formation of an F(ab')2 fragment by chemical bonding of two Fab'-SH fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for antibody fragment production will be apparent to those skilled in the art.

[0142]

[0159] Epitope

[0160] In certain embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein binds to an epitope in the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19).

[0143]

[0161] As used herein, the term “epitope” refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. An epitope may be a structural epitope or a linear epitope. In certain embodiments of this disclosure, the epitope bound by the anti-CLDN18.2 antibody provided herein is linear. Those skilled in the art should be aware that whether an antibody binds to the same or overlapping epitopes as the antibodies of this disclosure (e.g., hybridoma / mouse antibodies 14G11 and 69H2) can be determined without excessive experimentation by checking whether the two antibodies compete for binding to the CLDN18.2 antigen polypeptide.

[0144]

[0162] This disclosure provides a monoclonal antibody that is targeted to a specific epitope located within the N-terminal region of CLDN18.2, which is useful for detecting and identifying cells expressing CLDN18.2 without cross-reactivity with CLDN18.1. According to the sequences of human CLDN18.1 and CLDN18.2, there are eight differences in amino acids located at amino acids 28-70 (i.e., the N-terminal region including the first transmembrane (TM) region and loop l), but the C-terminal sequences of human CLDN18.1 and CLDN18.2 are identical. A linear epitope located at the N-terminus of human CLDN18.2 (amino acids 28-37 of the first extracellular domain, i.e., SEQ ID NO: 19) has been reported by Sahin U et al. (See Sahin Ugur et al, Clin Cancer Res 2008;14(23) December 1, 2008). However, to date, no monoclonal antibodies targeting such peptide fragments have been reported, and to the best of our knowledge, this disclosure provides the first monoclonal antibody that binds to the peptide fragment of SEQ ID NO: 19.

[0145]

[0163] In another aspect, the Disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that competes for binding to CLDN18.2 with antibodies or antigen-binding fragments provided herein, such as 14G11 and 69H2.

[0146]

[0164] The term “competing for binding” as used herein with respect to two antigen-binding proteins (e.g., antibodies) refers to the blocking or reduction of binding of one antigen-binding protein to the other antigen (e.g., human CLDN18.2) to any detectable degree, as determined by a competitive binding assay. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIAs), direct or indirect enzymatic immunoassays (EIAs), and sandwich competitive assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, such assays involve the use of purified antigen bound to a solid surface or cells containing the antigen, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by measuring the amount of the label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. When two antibodies compete for binding to CLDN18.2, they bind to the same or overlapping epitopes, or to adjacent epitopes that are sufficiently close to the epitope bound by the other antibody due to steric hindrance. Typically, when competing antibodies are present in excess, they inhibit (e.g., reduce) the specific binding of the test antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, or 85-90% or more.

[0147]

[0165] Polynucleotides and Recombination Methods

[0166] This disclosure provides an anti-CLDN18.2 antibody and isolated polynucleotides encoding its antigen-binding fragment. As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form and their polymers. Unless otherwise specified, a particular polynucleotide sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly shown. Specifically, degenerate codon substitution can be achieved by generating sequences in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (see Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J Biol Chem. 260:2605-2608 (1985); and Rossolini et al, Mol Cell. Probes 8:91-98 (1994)).

[0167] The DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional methods (for example, methods using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Furthermore, the encoding DNA can also be obtained by synthetic methods.

[0148]

[0168] This disclosure provides vectors (e.g., expression vectors) comprising isolated polynucleotides as provided herein. Vectors can be used to transform, transduce, or transfect host cells to result in the expression of the genetic elements they carry within the host cells. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Vectors may contain various elements for controlling expression, such as promoter sequences, transcription start sequences, enhancer sequences, selectable elements, and reporter genes. Furthermore, vectors may contain origins of replication. Vectors may also contain, but are not limited to, materials that aid in entry into cells, such as viral particles, liposomes, or protein coatings. Vectors may be expression vectors or cloning vectors.

[0149]

[0169] In certain embodiments, the vectors provided herein are expression vectors. In certain embodiments, the expression vectors provided herein comprise a polynucleotide encoding an antibody or antigen-binding fragment provided herein, at least one promoter operably ligated to the polynucleotide sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0150]

[0170] Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages, and M13 phages, pcDNA3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, PCI, pEGFT, pSV2. Examples of plasmids include pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.1, pCMV-SCRIP, pSV2, pSV2, pVITRO, pVIVO, pMAL, pMONO, pSELECT, puno, pDUO, psg5L 2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0171] A vector containing a polynucleotide sequence encoding an antibody or its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expression of DNA in the vector as described herein are the prokaryotes, yeasts, or higher eukaryotes mentioned above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, such as Escherichia, e.g., Escherichia coli, Enterobacter, Ervinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0151]

[0172] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts are also suitable as cloning or expression hosts for anti-CLDN18.2 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, are among the most commonly used lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are generally available and useful herein, including Schizosaccharomyces pombe; for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. Marxianus, which are Kluiveromyces hosts; Yarouia (EP 402,226); Pitia pastris (EP 183,070); Candida; Trichoderma lysia (EP 183,070); 244,234); Neurospora crassae; Schwannomyces such as Schwannomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0152]

[0173] Host cells suitable for the expression of glycosylated antibodies or antigen fragments provided herein are derived from invertebrate cells, multicellular organisms such as plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding to tolerant insect host cells, have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the viruses of this specification according to the present invention, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0153]

[0174] However, interest in vertebrate cells is highest, and the proliferation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34) Examples include buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals NY Acad Sci 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatocellular carcinoma cell lines (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NS0, 293 and their derivatives.

[0154]

[0175] Host cells are transformed with the above-mentioned expression or cloning vector for anti-CLDN18.2 antibody production and cultured in conventional nutrient medium modified as appropriate for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence. In another embodiment, the antibody may be produced by homologous recombination known in the art.

[0155]

[0176] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM), (Sigma) are suitable for culturing host cells. Alternatively, any of the media described in Ham et al., Meth Enz. 58:44 (1979), Barnes et al., Anal Biochem. 102:255 (1980), U.S. Patents 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent 30,985 may be used as culture media for host cells. In any of these culture media, hormones and / or other growth factors (such as insulin, transferrin, and epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), and antibiotics (such as GENTAMYCIN) may be added as needed. TM The cells may be supplemented with drugs, trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used in the host cells selected for expression and would be obvious to those skilled in the art.

[0156]

[0177] When recombinant technology is used, antibodies can be produced by direct secretion into cells, the periplasmic space, or the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992), describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Simply put, the cell paste is thawed over approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cellular residue can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of accidental contamination.

[0157]

[0178] Anti-CLDN18.2 antibodies and their antigen-binding fragments prepared from cells can be purified using methods such as hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0158]

[0179] In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J Immunol Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix on which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as glass with controlled pores or poly(styrene-devinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX is used for purification. TM Resin (JT Baker, Phillipsburg, NJ) is useful. Other methods, depending on the antibody to be recovered, include fractionation using ion exchange columns, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, and heparin-SEPHAROSE. TM Protein purification techniques such as chromatography, anion or cation exchange resins (e.g., polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation are also available.

[0159]

[0180] Following any one or more preliminary purification steps, the mixture containing the antibody of interest and the contaminant may be subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH between approximately 2.5 and 4.5, preferably with a low salt concentration (e.g., approximately 0 to 0.25 M salt).

[0160]

[0181] This disclosure provides a method for expressing an anti-CLDN18.2 antibody or its antigen-binding fragment provided herein, comprising culturing host cells provided herein under conditions for expressing a vector provided herein.

[0161]

[0182] Conjugate

[0183] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment is linked or conjugated to one or more portions. Examples of such portions include, but are not limited to, therapeutic agents (e.g., DNA alkylating agents, topoisomerase inhibitors, tubulin binders, or other anticancer agents), detectable labels, pharmacokinetic modification portions (e.g., polymers such as PEG that extend half-life), or purified portions (e.g., magnetic beads or nanoparticles).

[0162]

[0184] The parts can be attached to the antibody or its antigen-binding fragment directly, via a linker, or via other parts by methods such as covalent bonding, affinity bonding, intercalation, coordinate bonding, complexation, association, blending, or addition. In certain embodiments, the antibody or its antigen-binding fragment is linked to one or more parts via a linker. In certain embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0163]

[0185] In certain embodiments, the antibodies or antigen-binding fragments provided herein may be manipulated to include specific sites outside the epitope-binding portion that can be used for linking to one or more sub-parts. For example, such sites may include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent bonding to the sub-parts.

[0164]

[0186] In some embodiments, an anti-CLDN18.2 antibody or its antigen-binding fragment functions to (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g., FRET (fluorescence resonance energy transfer); (iii) affect mobility, e.g., electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters; or (iv) provide a capture moiety, e.g., affinity, antibody / antigen, or ion complex.

[0165]

[0187] Such parts include, but are not limited to, labels or parts that can be directly detected via imaging, enzymatic reactions, etc. (e.g., radioisotopes, lanthanides, chemiluminescent labels, chromogenic parts, enzyme labels, colloidal gold particles, and fluorescent labels), as well as parts that can be indirectly detected via, for example, molecular interactions. Examples of indirectly detectable labels include biotin / avidin, biotin / streptavidin, digoxigenin labels, haptens, detection DNA molecules, and particle labels (e.g., paramagnetic particles, metallic particle labels, magnetic particle labels, polymer particle labels).

[0166]

[0188] Examples of radioactive isotopes include S, C, I, H, and I. Antibodies can be labeled with radioactive isotopes using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured by scintillation counting using techniques described in (1991), etc. Other radionuclides include: 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 99 Tc, 90 Y, 111 In, 112 In, 32 P,14 C, 15 O, 13 N, 18 F, 86 Y, 88 Y, 90 Y, 51 Cr, 57 Ho, 225 Ra, 60 Co, 59 Fe, 57 Se, 152 Eu, 64 Cu, 67 Cu, 217 Cl, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 212 Pb, 47 Sc, 109 Pd, 234 Th, 40 K, 157 Gd, 55 Mn, 52 Tb and 56 Fe.

[0167]

[0189] Examples of fluorescent or luminescent labels include, but are not limited to, rare earth chelates (europium chelate), fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluoresamine, dansyl, umbelliferone, luciferin, luminal labels, isolaluminal labels, aromatic acridinium ester labels, imidazole labels, acridium salt labels, oxalate ester labels, aequorin labels, 2,3-dihydrophthalenedione, Texas Red, dansyl, lysamine, umbelliferone, phycoglycerin, phycocyanin, or commercially available fluorescent dyes such as SPECTRUM ORANGE® and SPECTRUM GREEN®, and / or one or more derivatives of any of the above. Fluorescent labels can be conjugated to antibodies, for example, using the techniques disclosed in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorometer.

[0168]

[0190] Another type of useful label is enzyme substrate labeling. Various enzyme substrate labels are available (see U.S. Patent No. 4,275,149). Enzymes generally catalyze chemical changes in chromogenic substrates, and these changes can be measured by various techniques. For example, an enzyme may catalyze a change in the color of a substrate, which can be measured by spectrophotometry. Alternatively, an enzyme may alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence have been described above. Chemiluminescent substrates can be electronically excited by a chemical reaction, emitting measurable light (e.g., using a chemiluminometer) or donating energy to a fluorescence acceptor.

[0169]

[0191] Numerous enzyme-substrate combinations are available to those skilled in the art (see U.S. Patents 4,275,149 and 4,318,980), for example: (i) wasabi peroxidase (HRP) with hydrogen peroxide as a substrate, where hydrogen peroxide oxidizes a pigment precursor such as 3,3'-diaminobenzidine (DAB), which produces a brown final product; 3-amino-9-ethylcarbazole (AEC) oxidizes to form a rose-red final product; 4-chloro-L-naphthol (CN) precipitates as a blue final product; and p-phenylenediamine dihydrochloride / pyrocatechol produces a bluish-black product; orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (ii) Alkaline phosphatase (AP) and para-nitrophenyl phosphate, naphthol AS-MX phosphate, Fast Red TR and Fast Blue BB, naphthol AS-BI phosphate, naphthol AS-TR phosphate, 5-bromo-4-chloro-3-indoxulfonate (BCIP), Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) and Iodonitetrazolium Violet (INT); and (iii) β-D-galactosidase (β-D-Gal) and a chromogenic substrate (e.g., p-nitrophenyl-PD-galactosidase) or a fluorescent substrate (e.g., 4-methylumbelliferyl-PD-galactosidase).

[0170]

[0192] Other useful enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalenedione, maleate dehydrogenases, ureases, peroxidases such as horseradish peroxidase (HRPO), glucoamylases, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase, xanthine oxidase), lactoperoxidases, and microperoxidases. The technique of conjugating enzymes to antibodies is described in O'Sullivan et al, *Methods for Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. ed J. Langbne & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

[0171]

[0193] Detection and Usage Method of CLDN18.2

[0194] This disclosure provides a method for detecting the presence or expression level of CLDN18.2 in a sample, comprising contacting the sample with an antibody or its antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2 (e.g., human CLDN18.2), and determining the presence or expression level of CLDN18.2 in the sample.

[0172]

[0195] In another embodiment, a method is provided for diagnosing a CLDN18.2-related disease or condition (e.g., cancer) in a subject, the method comprising: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; and b) Determine the presence or expression level of CLDN18.2 in the sample; Here, if the presence of CLDN18.2 is detected, or if the expression level of CLDN18.2 reaches a threshold level, the subject is diagnosed with a CLDN18.2-related disease or condition (e.g., cancer). In certain embodiments, the sample is not gastric epithelial tissue.

[0173]

[0196] In another embodiment, a method is provided for determining the eligibility of a subject with or at risk of having a CLDN18.2-related disease or condition for treatment with a CLDN18.2-targeting agent, the method comprising: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; and b) Determine the presence or expression level of CLDN18.2 in the sample; If the presence of CLDN18.2 is detected, or if the expression level of CLDN18.2 reaches a threshold, the subject is determined to be eligible for treatment with a CLDN18.2-targeting agent, or If CLDN18.2 is not detected, or if the expression level of CLDN18.2 is below the threshold, the subject is determined to be ineligible for treatment with a CLDN18.2-targeting agent.

[0174]

[0197] In another embodiment, a method is provided for predicting the therapeutic effect of a CLDN18.2-targeting agent in the treatment of a CLDN18.2-related disease or condition in a subject, the method comprising: a) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; b) Determining the presence or expression level of human CLDN18.2 in the sample; and c) Predicting the therapeutic effect of CLDN18.2 targeted agents, Here, CLDN18.2-targeting agents are predicted to be effective in the treatment of the target if the presence of CLDN18.2 is found, or if the expression level of CLDN18.2 reaches a threshold level, or Here, CLDN18.2-targeting agents are predicted to be ineffective in the treatment of the target group if CLDN18.2 is not detected or if the expression level of CLDN18.2 is below the threshold.

[0175]

[0198] In yet another embodiment, a method is provided for treating a subject who has or is at risk of having a CLDN18.2-related disease or condition, the method comprising: a) Selecting a suitable patient for treatment, which includes: i) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; ii) Determining the presence or expression level of human CLDN18.2 in the sample; and iii) If CLDN18.2 is found to be present, or if the expression level of CLDN18.2 in the sample reaches a threshold, select subjects as suitable for treatment with a CLDN18.2-targeting agent; and b) Administer a therapeutically effective dose of CLDN18.2-targeting agent to the selected subjects.

[0176]

[0199] In yet another embodiment, a method for treating a subject who has cancer or is at risk of cancer is provided, the method comprising: a) Select the target, including the following: i) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment provided herein under conditions that enable specific binding of the antibody or its antigen-binding fragment to CLDN18.2; ii) Determining the presence or expression level of CLDN18.2 in the sample; and iii) If CLDN18.2 is not detected, or if the expression level of CLDN18.2 in the sample is below the threshold, the subject should be selected as unsuitable for treatment with a CLDN18.2-targeting agent; and b) Administer standard therapeutic agents other than CLDN18.2 targeting agents to the selected subjects.

[0177]

[0200] According to the present invention, “Sample” may be any sample useful in accordance with the present disclosure, in particular a biological sample such as a tissue sample containing bodily fluids and / or a cell sample, which can be obtained by conventional methods such as tissue biopsy including punch biopsy, and collection of blood, bronchial aspirate, sputum, urine, feces or other bodily fluids. According to the present invention, the term “Sample” also includes a processed sample such as a fraction or isolate of a biological sample, e.g., nucleic acids and peptide / protein isolates.

[0178]

[0201] Any biological sample suspected of containing CLDN18.2 may be detected by the methods provided herein. In some embodiments, a suitable sample may be a cell or tissue sample obtained from the subject to be detected. For example, a sample may include normal and cancerous tissues of the stomach, lungs, breasts, colon, kidneys, bones, brain, muscles, pancreas, bladder, ovaries, and uterus, as well as heart, embryo, or placental tissue. Preferably, the sample includes cells or tissue from the organ being examined, e.g., the organ subject to cancer diagnosis. For example, if the cancer subject to diagnosis is gastric cancer, the sample may include cells or tissue taken from the stomach. In some embodiments, the sample is a tumor sample. In certain embodiments, the tissue sample is a sample having a disease or condition to which CLDN18.2 is associated.

[0179]

[0202] A suitable sample may be a patient-derived physical sample containing or expected to contain tumor or cancer cells. The physical sample may be any tissue sample such as blood, a tissue sample obtained from a primary tumor or tumor metastasis, or any other sample containing tumor or cancer cells. "Primary tumor" refers to a tumor growing at the site of cancer origin. "Metastatic tumor" refers to a secondary tumor that develops at a site different from the site of cancer origin.

[0180]

[0203] The source of the tissue or cell sample may be solid tissue such as fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of the subject's pregnancy or development. In some embodiments, the sample is obtained from an in vitro tissue or cell culture.

[0181]

[0204] Examples of samples used herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, ascites, primary cell cultures or cell lines derived from or exhibiting tumor-like characteristics, and preserved tumor samples such as formalin-fixed paraffin-embedded (FFPE) tumor samples or frozen tumor samples.

[0182]

[0205] In certain embodiments, cell samples are prepared from cell blocks. A cell block is a method of preparing cytological material so that it can be processed, sectioned, stained, and observed as histological sections. In addition to the information obtained from cytological slides, diagnostic information can be provided. In certain embodiments, cell blocks can be prepared from residual fluid, sputum, urinary sediment, gastrointestinal fluid, cell curettage, or fine-needle aspiration. The cells are concentrated or packed by centrifugation or membrane filtration.

[0183]

[0206] Many methods have been developed for preparing cell blocks. Representative methods include the fixation-precipitation method, the bacterial agar method, and the membrane filtration method. In the fixation-precipitation method, the cell precipitate is mixed with a fixative such as Bouin, picric acid, or buffered formalin, and the mixture is centrifuged to pellet the fixed cells. The pellet is collected, placed in a tissue cassette, and then a fixative is added and the cassette is placed in a jar for processing as a tissue sample. The agar method is very similar, but the pellet is cut in half, and the cut side is placed in a drop of melted agar on a glass slide. After the agar hardens, the excess agar is trimmed off. Alternatively, the pellet can be directly suspended in 2% liquid agar at 65°C, and the sample can be centrifuged. The agar cell pellet is solidified at 4°C for 1 hour. The solid agar can be removed from the centrifuge tube and sliced ​​in half. This is then placed in a tissue cassette to complete the tissue processing. Centrifugation can be replaced with membrane filtration. Any of these processes can be used to produce a "cell block sample".

[0184]

[0207] In certain embodiments, cell blocks can be prepared using special resins including Lowicryl resin, LR White, LR Gold, Unicryl, and MonoStep. These resins have low viscosity and can be polymerized at low temperatures and under ultraviolet light. Embedding is performed by gradually cooling the sample during dehydration, transferring the sample to the resin, and finally polymerizing the block at low temperatures and under appropriate ultraviolet wavelengths.

[0185]

[0208] Cell block sections can be stained with hematoxylin-eosin, Hoechst stain, or DAPI for cytomorphological examination, while additional sections are used for CLDN18.2 testing by exposing them to anti-CLDN18.2 antibody (i.e., primary antibody) for a sufficient time and under appropriate conditions to allow the antibody to bind to the CLDN18.2 protein in the cell block sections. Unbound primary antibody or excess primary antibody may be removed by washing.

[0186]

[0209] In some embodiments, the sample may include, for example, preservatives, anticoagulants, buffers, nutrients, antibiotics, etc. In certain embodiments, the sample may be exposed to and / or contain one or more fixatives. Exemplary fixatives suitable for the methods provided herein include formalin, glutaraldehyde, osmium tetroxide, acetic acid, ethanol, acetone, picric acid, chloroform, potassium dichromate, and mercury chloride and / or stabilization by microwave heating or refrigeration.

[0187]

[0210] In some embodiments, the sample includes a fixed tissue sample. In some embodiments, the fixed tissue sample is formalin-fixed paraffin-embedded (FFPE) tissue. FFPE tissue sections may be about 3–4 millimeters, preferably 4–40 micrometers, and these are mounted on microscope slides and dried. Examples of paraffin include, but are not limited to, paraplast, proloid, and tissue may. In the case of fixed tissue samples such as FFPE tissue samples, the sample may be deparaffinized before contact with the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein.

[0188]

[0211] In some embodiments, deparaffinized samples may be further processed to enable antigen retrieval. Antigen retrieval refers to any technique that reverses epitope masking and restores epitope-antibody binding. While fixation is essential for preserving tissue morphology, this process can sometimes adversely affect antibody binding and detection. Immobilization can alter the biochemical properties of proteins, masking the target epitope and preventing it from binding to the antibody. Epitope masking can be caused by crosslinking of amino acids within the epitope, crosslinking of unrelated peptides in or near the epitope, changes in the conformation of the epitope, or changes in the electrostatic charge of the antigen. The need for antigen retrieval depends on, but is not limited to, several variables, including the target antigen, the antibody used, the type of tissue, and the method and duration of fixation. Antigen detection techniques generally include protease-induced epitope detection (using enzymes such as PIER, proteinase K, trypsin, and / or pepsin) and heat-induced epitope detection (using HIER, microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths).

[0189]

[0212] In certain embodiments, the presence or expression level of cell surface or membrane-bound CLDN18.2 is detected or determined by methods provided herein. The phrase “cell surface or membrane-bound CLDN18.2” means that CLDN18.2 is associated with and located on the cell membrane of a cell, and at least a portion of CLDN18.2 is exposed to the extracellular space of the cell and accessible from outside the cell, for example, by extracellular antibodies. In certain embodiments, the portion of CLDN18.2 exposed to the extracellular space contains at least 4, at least 8, at least 10, at least 12, or at least 20 amino acid residues. In normal tissues other than gastric epithelial cells, CLDN18.2 is thought to reside within tight junctions of the epithelium or endothelium and is not accessible from outside the cell by antibodies, and is therefore not considered to be cell surface or membrane-bound CLDN18.2.

[0190]

[0213] The presence or expression level of CLDN18.2 protein in a sample can be determined based on the presence or level of a complex of the CLDN18.2 antigen bound to an antibody or its antigen-binding fragment disclosed herein. Any suitable method can be used to detect the antibody-antigen complex, for example, immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), immunoblotting (e.g., Western blotting), flow cytometry (FACS). TM These include enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunoassays (EIA), and radioimmunoassays (RIA). For an overview of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds., 7 th See (ed. 1991). Furthermore, immunoassays can be performed using one of several configurations extensively reviewed in Enzyme Immunoassay (Maggio, ed., 1980) and Harlow & Lane, supra. For general immunoassays, see Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7 th See also ed 1991.

[0191]

[0214] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein may be detectably labeled (e.g., primary antibodies) or unlabeled but detectably labeled secondary molecules (e.g., detectably labeled secondary antibodies).

[0192]

[0215] In certain embodiments, the presence or expression level of the CLDN18.2 protein in a sample is determined according to IHC or ICC. IHC refers to the step of detecting an antigen (e.g., a protein) in cells of a tissue section, for example, cells of the tissue described herein. Immunohistochemical staining is widely used for the diagnosis of abnormal cells, such as those found in carcinomas. In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment disclosed herein can be used as the primary antibody in IHC or ICC. Generally, IHC or ICC can be performed for the direct or indirect detection of the CLDN18.2 protein (e.g., human CLDN18.2 protein) in a sample, and the expression of CLDN18.2 can be evaluated using appropriate imaging equipment.

[0193]

[0216] To enable the direct detection of an antigen (e.g., CLDN18.2), an anti-CLDN18.2 antibody or its antigen-binding fragment disclosed herein, linked to a detectable label, can be used, thereby enabling direct visualization without requiring further antibody interactions.

[0194]

[0217] For indirect detection of an antigen (e.g., CLDN18.2), the anti-CLDN18.2 antibody or antigen-binding fragment thereof disclosed herein, which is unlabeled but can react with a second detectably labeled molecule (e.g., a detectably labeled secondary antibody), can be used. For example, the anti-CLDN18.2 antibody or antigen-binding fragment thereof disclosed herein is unlabeled, and may be further contacted with a secondary antibody (e.g., an anti-idiotypic antibody) bound to a detectable label, so that the antigen can be detected indirectly. As another example, the anti-CLDN18.2 antibody provided herein can be conjugated to biotin, which can react with detectably labeled avidin, or vice versa. Since biotin selectively binds to avidin, it enables indirect detection of an antibody-antigen complex. In yet another example, the anti-CLDN18.2 antibody provided herein can be bound to a small hapten, which can react with an anti-hapten antibody linked to the detectable label described herein. Exemplary types of labels are described above herein, and any suitable detectable label can be used.

[0195]

[0218] ICC or IHC assays can be performed using an automated pathology system, which may comprise automatic staining (conventional staining, histochemical techniques, immunostaining), automated in situ hybridization systems, automated slide preparation (coverslipping, slide drying), and integrated slide and cassette labeling, as described in Roja et al., Review of imaging solutions for integrated, quantitative immunohistochemistry in Pathology daily practice, Folia Histochemica et Cytobiologica, Vol.47, No.3, 349-354, 2009.

[0196]

[0219] An exemplary IHC assay is a commercially available Dako EnVision intended for use with a Dako Autostainer instrument (Dako, an Agilent Technologies Company, Glostrup, Denmark) TM FLEX detection system may be employed. These reagents can be used directly in other autostainers or for manual staining without using an autostainer.

[0197]

[0220] After completion of the staining process, a sample (e.g., a slide) is analyzed for CLDN18.2 staining by a human, for example a pathologist, or by a computer programmed to distinguish between specific and non-specific staining results. The analysis may be performed directly by viewing the sample under a microscope at low, medium (10-20×), and high (40-60×) magnifications, or may be performed by viewing high-resolution images of the slide captured at low, medium, and high magnifications.

[0198]

[0221] The presence of CLDN18.2 expression in a sample can be confirmed by the presence of positively stained cells, for example cells having at least partial membrane staining of any intensity for anti-CLDN18.2 antibody staining. In certain embodiments, a normal sample can be used as a control sample, and the presence of CLDN18.2 expression in a test sample can be determined relative to the control sample. The term "normal", as used in the terms "normal sample" or "normal tissue", refers to a sample or tissue from a healthy or non-cancerous subject, or a sample or tissue from healthy or non-cancerous tissue. Preferably, the test sample and the control sample are comparable in terms of sample type, for example both are fixed tissue samples. A test sample may be determined to be positive for CLDN18.2 expression if the test sample shows an increase in staining intensity or in the number of positively stained cells compared to the control sample.

[0199]

[0222] In certain embodiments, the expression level of CLDN18.2 can be quantified using any suitable method known in the art, for example, by determining the relative proportion of positively stained cells and the staining intensity on the cell membrane.

[0200]

[0223] In certain embodiments, CLDN18.2 expression levels are quantified based on the percentage of positively stained cells (i.e., CLDN18.2-positive cells) in the sample. CLDN18.2-positive cells are those that have at least partial membrane staining of any intensity for anti-CLDN18.2 antibody staining. For example, to assess CLDN18.2 expression in a sample, an observer can examine the number of membrane CLDN18.2+ cells in one or more selected fields under a microscope and calculate or estimate the percentage of cells positive for CLDN18.2. If the sample is highly heterogeneous, the sample can be divided into zones, each zone can be scored separately, and then combined into a set of percentage values.

[0201]

[0224] In certain embodiments, expression levels are quantified based on the staining intensity for CLDN18.2 (e.g., membrane-bound CLDN18.2) in the sample. For example, an intensity score such as the 4-point HSCORE can be calculated based on the staining intensity ranging from 0 (no staining), 1+ (weak staining), 2+ (clear staining), 3+ (strong staining), and 4+ (very strong / saturated signal), and multiplied by the percentage (0-100%) of cells staining at each intensity (see McCarty, KSJr, et al, Cancer Res. 46(suppl 8):4244s-4248s(1986) for details). As another example, the Alfred score can be calculated based on a total score (TS, range 0-8) by adding together the percentage score (PS) and the intensity score (IS). PS is the percentage of positive tumor cells from 0 to 5 (0 = no positive cells, 1 = 1 / 100 cells positive, 2 = 1 / 10 cells positive, 3 = 1 / 3 cells positive, 4 = 2 / 3 cells positive, 5 = all tumor cells positive). IS refers to the average staining intensity of positive tumor cells from 0 to 3 (0 = negative, 1 = weak staining, 2 = intermediate staining, 3 = strong staining) (for details, see Alfred DC et al. Mod Pathol. 11:155-168 (1998)).

[0202]

[0225] In certain embodiments, samples are evaluated by two independently operating observers, and the percentages or scores are then combined. In certain other embodiments, the identification of positive and negative cells is performed or scored using appropriate software.

[0203]

[0226] In certain embodiments, the level of CLDN18.2 can be determined, for example, by normalizing to a control value or a standard curve. The control value can be determined in advance from a negative control sample or a blank control sample, or simultaneously.

[0204]

[0227] In certain embodiments, for diagnostic or clinical use, the expression level of CLDN18.2 in a test sample (e.g., exposed on the cell surface) is compared to a threshold.

[0205]

[0228] A "threshold level" or "threshold value" for CLDN18.2 expression refers to an expression level that can distinguish between positive and negative cell surface CLDN18.2 expression, or an expression level that can rule out a CLDN18.2-related condition, such as cancer, or the development of cancer or the risk of developing cancer in a subject, or a threshold level that can monitor the treatment response in a subject receiving cancer treatment. In certain embodiments, the threshold is determined relative to a control expression level.

[0206]

[0229] For example, the threshold can be the level at which a sample is scored as having positive expression of CLDN18.2, and therefore the level at which it is eligible for treatment with a CLDN18.2-targeting agent. If the level of CLDN18.2 in the sample reaches or exceeds the threshold, it may indicate the presence of a CLDN18.2-related disease or condition and / or a potential response to a CLDN18.2-targeting agent.

[0207]

[0230] The threshold can be determined by a person skilled in the art, taking into account various factors, such as the type of sample, the detection method, the disease or condition to be diagnosed, and / or the CLDN18.2 targeting agent to be used.

[0208]

[0231] In certain embodiments, the presence of CLDN18.2 or CLDN18.2-expressing cells and / or an increased amount of CLDN18.2 compared to, for example, a subject without CLDN18.2-related disease (e.g., a non-cancer subject) compared to a threshold level indicates the presence or risk (i.e., potential development) of CLDN18.2-related disease or condition (e.g., cancer) in the patient.

[0209]

[0232] In certain embodiments, the threshold is the percentage (%) of CLDN18.2-positive cells having at least partial membrane staining of any intensity. In some embodiments, the threshold levels for the percentage (%) of CLDN18.2-positive cells are 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0210]

[0233] In certain embodiments, the sample is from a subject having or at risk of having a CLDN18.2-related disease or condition. As used herein, the term “CLDN18.2-related disease or condition” refers to a disease or condition characterized by increased expression of CLDN18.2 in the cells of diseased tissue or organ compared to the condition in healthy or non-cancerous tissue or organ other than the stomach. The increase may be, for example, by at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, or more. In certain embodiments, the expression of CLDN18.2 in the cells of diseased tissue or organ is above the detection limit and / or high enough to allow binding by a CLDN18.2-specific antibody added to the cells. In some embodiments, the increased expression of CLDN18.2 is observed only in diseased tissue, and the expression of CLDN18.2 in normal tissue is undetectable.

[0211]

[0234] In some embodiments, CLDN18.2-related diseases or conditions are characterized by increased expression of CLDN18.2 on the cell surface or in membrane-bound form.

[0212]

[0235] In some embodiments, the CLDN18.2-related disease or condition is cancer. In some embodiments, cancer cells express or abnormally express CLDN18.2, while corresponding normal cells either do not express CLDN18.2 or express it at low levels. As a tight junction protein, CLDN18.2 is considered a good therapeutic target for CLDN18.2-related diseases such as tumors and is thought to be useful in selecting patients for treatment with CLDN18.2-targeted agents. Unlike normal epithelial tissue (excluding gastric epithelial cells) where CLDN binds to form classical tight junctions, CLDN expressed in tumor cells often does not form such classical tight junctions. As a result, tumor cells are thought to be more likely to expose free CLDN that can be subjected to extracellular antibody binding and immunotherapy.

[0213]

[0236] CLDN18.2 is used to treat gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC, squamous / non-squamous), small cell lung cancer (SCLC)), bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer (including basal breast cancer, tubular breast cancer, and lobular breast cancer), liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumors, cervical cancer, uterine cancer, endometrial cancer, liver cancer, gallbladder cancer, colon cancer, and colorectal cancer. It is a valuable target for the prevention and / or treatment of primary tumors such as cancer, rectal cancer, anal cancer, esophageal cancer, digestive tract cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, cholangiocarcinoma, and / or adenocarcinoma, and / or their metastases, particularly gastric cancer metastases such as Krukenberg tumor, peritoneal metastases, and lymph node metastases.

[0214]

[0237] The sample is preferably cancer cells or tissue, and is particularly selected from the group consisting of neoplastic gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer cells or tissue.

[0215]

[0238] In certain embodiments, cancers include renal cell carcinoma, gastric cancer, mesothelioma, melanoma, cervical cancer, thymic cancer, myeloma, mycoses fungoids, Merkel cell carcinoma, hepatocellular carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancies, basal cell carcinoma, sweat gland carcinoma, thyroid medullary carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, and medullary cell carcinoma. Bronchial cancer, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / polycytic B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and negative PTLD, diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal cancer, HHV8-associated primary pleural effusion lymphoma, non-Hodgkin lymphoma, multiple myeloma, Waldenstorm macroglobulinemia, heavy chain syndrome, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma. This includes, but is not limited to, spinal axial tumors, brainstem gliomas, astrocytomas, medulloblastomas, cranial gliomas, ependymomas, pineal glandomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, melanomas, neuroblastomas, and retinoblastomas.

[0216]

[0239] In certain embodiments, the cancer is a cancer that expresses CLDN18.2. The presence or expression level of CLDN18.2 in a sample indicates whether cancer cells express CLDN18.2 and, therefore, whether the cancer is likely to respond to treatment with a CLDN18.2-targeting agent.

[0217]

[0240] As used herein, the "CLDN18.2 targeting agent" refers to one or more agents that target CLDN18.2 protein or nucleic acid (DNA or mRNA) in cells, tissues or in vivo. The CLDN18.2 targeting agent can reduce / eliminate the expression of CLDN18.2 gene product, inhibit / disrupt the signal transduction of CLDN18.2, or induce cytotoxicity against cells that abnormally express CLDN18.2.

[0218]

[0241] In certain embodiments, the CLDN18.2 targeting agent comprises a therapeutic anti-CLDN18.2 antibody, a CLDN18.2 binding molecule, a CLDN18.2-targeted cell therapy, a CLDN18.2-targeted chemical compound, or a CLDN18.2-targeted therapeutic nucleic acid. In certain embodiments, the CLDN18.2-targeted agent is capable of inducing cytotoxicity against cells expressing CLDN18.2.

[0219]

[0242] In certain embodiments, the CLDN18.2 targeting agent comprises a therapeutic anti-CLDN18.2 antibody or a CLDN18.2-binding molecule. In certain embodiments, the therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule is capable of inducing ADCC, CDC or ADCP against CLDN18.2-expressing cells. Alternatively, the therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule can, for example, bind to a cytotoxic agent to form an antibody-drug conjugate (ADC). In certain embodiments, the cytotoxic agent can be any agent that is harmful to cells, or can damage or kill cells. In certain embodiments, the cytotoxic agent is optionally a toxin, a chemotherapeutic agent (such as a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a growth inhibitor, or other anticancer agent), or a radioisotope. In other embodiments, the therapeutic anti-CLDN18.2 antibody can be a bispecific antibody that binds to different antigens or different epitopes on the CLDN18.2 protein.

[0220]

[0243] In certain embodiments, the CLDN18.2 targeting agent includes CLDN18.2-targeted cell therapy. In certain embodiments, the CLDN18.2-targeted cell therapy includes CAR-T (Chimeric Antibody Receptor Engineered T Cell), TCR-T (Gene Modified TCR T Cell), or CAR-NK (Chimeric Antibody Receptor Engineered NK Cell) expressing a CLDN18.2-conjugated chimeric antibody receptor (CAR). A chimeric antigen receptor (CAR) is an artificial chimeric receptor that combines the antigen-binding domain of an antibody with one or more signaling domains for T cell activation. Immune cells such as T cells and nature killer (NK) cells can be genetically engineered to express CARs or recombinant TCRs (see D. Li et al, Sig Transduct Target Ther 4, 35 (2019), S. Kloess et al, Transfus Med Hemother; 46:4-13 (2019), Wang W. et al, Cancer Letters, 472:175-180 (2020) for details). T cells that express CARs are called CAR-T cells. CARs mediate antigen-specific cellular immune activity in T cells, enabling CAR-T cells to eliminate cells expressing target antigens (e.g., tumor cells). In one embodiment, the binding of CAR-T cells provided herein to CLDN18.2 expressed on cells such as cancer cells results in proliferation and / or activation of the CAR-T cells, which can then release cytotoxic factors such as perforin, granzyme, and granulosin, thereby initiating cytolysis and / or apoptosis of cancer cells.

[0221]

[0244] In certain embodiments, therapeutic nucleic acids targeting CLDN18.2 may be short interfering nucleic acids (siNAs), short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), and short-haired RNAs (shRNAs) that can mediate RNA interference (RNAi) to the CLDN18.2 gene sequence or another gene sequence within a CLDN18.2-expressing cell.

[0222]

[0245] In certain embodiments, the CLDN18.2 targeting agent promotes cancer regression in the subject. In preferred embodiments, a therapeutically effective dose of the CLDN18.2 targeting agent promotes cancer regression to the point of cancer disappearance. "Promoting cancer regression" means that, by administering an effective dose of the agent alone or in combination with an antineoplastic agent, a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, or a reduction in or prevention of disease-related disability.

[0223]

[0246] In certain embodiments, the subject is currently receiving or has previously received anticancer treatment, or is suffering from a recurrence of cancer. Anticancer treatments include, but are not limited to, chemotherapy agents, anticancer drugs, radiotherapy, immunotherapy, angiogenesis inhibitors, targeted therapy, cell therapy, gene therapy, hormone therapy, palliative care, surgery for cancer treatment (e.g., tumor resection), or one or more antiemetics for complications arising from chemotherapy.

[0224]

[0247] Relapse or recurrence refers to the recurrence of a disease previously suffered from. For example, if a patient develops cancer and, after successful treatment, subsequently develops the disease again, this newly developed disease can be considered a relapse or recurrence. However, according to this disclosure, a relapse or recurrence of cancer may occur at the site of the original cancer, but it does not necessarily have to. Therefore, for example, if a patient suffers from a stomach tumor and is successfully treated, a relapse or recurrence may refer to the development of a tumor in the stomach, or a tumor in a different site. Furthermore, tumor relapse or recurrence includes situations where a tumor develops not only at the site of the original tumor, but also in a different site. Preferably, the original tumor treated by the patient is a primary tumor, and the tumor in a different site is a secondary or metastatic tumor.

[0225]

[0248] kit

[0249] In certain embodiments, the Disclosure provides a kit comprising an isolated antibody or its antigen-binding fragment provided herein, and in certain embodiments, the kit disclosed herein is a diagnostic kit. The kit is useful in detecting the presence or amount of CLDN18.2 in a biological sample, or may be useful in a diagnostic method provided herein.

[0226]

[0250] In certain embodiments, the kit includes an antibody or its antigen-binding fragment provided herein, optionally labeled for detection. In certain embodiments, the antibody or its antigen-binding fragment is conjugated with an indirectly detectable portion.

[0227]

[0251] In certain embodiments, the kit further includes a set of reagents for detecting a complex of an antibody or its antigen-binding fragment conjugated to CLDN18.2, which is useful in a variety of detection assays, including, for example, immunoassays such as IHC, ICC, or ELISA (sandwich or competitive format).

[0228]

[0252] In yet another embodiment of the present invention, reagents useful for performing immunohistochemistry on FFPE tumor tissue sections are provided in a kit along with instructions for performing an IHC assay.

[0229]

[0253] Any indirectly detectable label or portion disclosed herein may be used. In certain embodiments, the indirectly detectable portion comprises biotin. In such embodiments, the reagent set comprises detectably labeled avidin or steptabidine.

[0230]

[0254] In certain embodiments, a detectable label is conjugated to an antibody or its antigen-binding fragment provided herein. In certain embodiments, the kit comprises an isolated antibody or its antigen-binding fragment provided herein and a secondary antibody to which the detectable label is conjugated. In some embodiments, the secondary antibody comprises an antibody that specifically binds to a primary antibody (e.g., an antibody or its antigen-binding fragment provided herein). In some embodiments, the secondary antibody may be an anti-mouse antibody, an anti-rabbit antibody, or an anti-human antibody.

[0231]

[0255] Detectable labels may require combination with one or more components before use, such as buffers, antibody-enzyme conjugates, enzyme substrates, etc., and such reagents may be included in the kit. For example, if the detection portion contains an enzyme, the kit will include the substrates and cofactors required by the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other reagents may be included, such as blocking reagents to reduce nonspecific binding to the solid phase surface, washing reagents, and enzyme substrates. The relative amounts of the various reagents can be widely varied to provide the concentration of reagents in solution that substantially optimizes the sensitivity of the assay. In particular, reagents are usually supplied as lyophilized dry powders and may include excipients that provide a reagent solution with the appropriate concentration upon dissolution. Instructions (as inserts or labels) showing guidelines for the preparation of the detection system and / or assay system may also be included in the kit.

[0232]

[0256] The components of the kit may be pre-attached to a solid support or coated onto the surface of the solid support when using the kit. The solid-phase surface may be in the form of a material suitable for immobilizing proteins, peptides, and polypeptides, such as a tube, beads, microtiter plate, or microsphere.

[0233]

[0257] Containers for use in such kits typically comprise at least one vial, test tube, flask, bottle, syringe, or other suitable container in which one or more detection compositions may be placed and preferably dispensed. Kits disclosed herein will also typically comprise means for sealing and housing vials for commercial sale, e.g., injection- or blow-molded plastic containers in which the desired vials may be held. If radioactive labeling, chromogenicity, fluorogenicity, or other types of detectable labeling or detection means are included in the kit, the labeling agent may be provided in the same container as the detection composition itself, or instead may be placed in a second, different container means in which this second composition can be placed and appropriately dispensed. Alternatively, the detection reagent may be prepared in a single container means, and in most cases, the kit will also typically comprise means for sealing and housing vials for commercial sale and / or convenient packaging and delivery.

[0234]

[0258] Apparatus or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such apparatus may include a fluid handling system optionally comprising a chamber or tube into which a sample can be introduced, a valve or pump for guiding the flow of the sample through the apparatus, optionally a filter for separating plasma or serum from blood, a mixing chamber for adding a capture agent or detection reagent, and optionally a detection device for detecting the amount of a detectable label bound to a capture agent immunocomplex. The flow of the sample can be passive (e.g., by capillary force, hydrostatic pressure, or other force that does not require further operation of the apparatus once the sample is applied), active (e.g., by the application of force generated via a mechanical pump, electroosmotic pump, centrifugal force, or pneumatic pressure increase), or a combination of active and passive forces. [Examples]

[0235]

[0259] While this disclosure has been specifically shown and described with reference to certain embodiments (some of which are preferred embodiments), it should be understood by those skilled in the art that various modifications in form and detail can be made without departing from the spirit and scope of this disclosure as disclosed herein.

[0236]

[0260] Example 1: Preparation of immunotherapy antigens

[0261] 1. Design of antigen peptides

[0262] To limit the antibody epitope to CLDN18.2 rather than CLDN18.1, MabSpace Biosciences (Suzhou) Co., Limited designed a CLDN18.2-specific peptide (Genbank accession number: NP_001002026). The coding sequence of this peptide (SEQ ID NO: 19: DQWSTQDLYN) was located at amino acid residues Asp28~Asn37 (D28~N37) of CLDN18.2.

[0237]

[0263] 2. Peptide synthesis

[0264] The antigen peptide was synthesized at SANGON BIOTECH (Shanghai) and used for animal immunization.

[0238]

[0265] Example 2: Antibody Production

[0266] 1. Fusion of immunity and hybridoma

[0267] Different strains of mice aged 6-8 weeks were immunized with 40 μg / mouse peptide (KLH-bound) using CFA as an adjuvant. Each mouse received 11 boosts (multiple site subcutaneous injections) three times a week for 4 weeks, followed by two more boosts at one-week intervals, three days after the primary immunization. Serum titer was measured by adding 100 μl / well of serially diluted mouse serum to a plate coated with the antigen peptide, incubating at 4°C for 30 minutes, washing three times with washing solution, adding 100 μl / well of goat anti-mIgG-HRP, and incubating again at 4°C. After washing the plate three times with washing solution, 100 μl / well of goat anti-mIgG-HRP was added and incubated again at 4°C. After washing with washing solution, TMB was added and reacted with HRP on the plate. Subsequently, the OD450nm of the plate was read with a microplate reader, and titer was analyzed using Graphpad Prism 6 software. We selected mice with higher titers and performed the following fusion procedure.

[0239]

[0268] 2.Fusion

[0269] Four days before fusion, each mouse was intraperitoneally boosted with 20 μg of peptide. On the day of fusion, the spleen was aseptically removed and treated with a single-cell suspension. Red blood cells were lysed to obtain splenic cells. Viable logarithmically proliferating myeloma cells (SP2 / 0) and mouse splenic cells were mixed in a 1:1 ratio in fusion medium and electrofused for 1 minute. The cells were resuspended and cultured in a 96-well culture plate at 37°C in a 5% CO2 incubator. After 7 days of culture, the medium was replaced with fresh medium. Screening of the hybridoma supernatant was started 2-3 days after the fresh medium change.

[0240]

[0270] Example 3: Binding screening, subcloning of positive hybridoma clones, and small-scale antibody production.

[0271] 1. Binding screening using ELISA assay

[0272] Hybridoma supernatant was collected for antigen binding screening. 100 μl / well of hybridoma supernatant was added to a plate coated with antigen peptide and incubated at 4°C for 1 hour. After washing the plate three times with washing solution, 100 μl / well of goat anti-mIgG-HRP was added and incubated again at 4°C. After washing with washing solution, TMB was added and reacted with HRP on the plate. Subsequently, the OD490 nm of the plate was read using a microplate reader. ELISA-positive clones were selected and developed. Two days later, the binding of hybridoma supernatant to the selected clones was similarly tested, and positive clones were proceeded to subcloning.

[0241]

[0273] 2. Subcloning of positive hybridoma clones

[0274] Cells were selected from wells containing positive hybridomas with the desired binding profile and subjected to limited dilution in a 96-well plate. The diluted cells were grown for 7 days. Once a sufficient cell volume was reached, the supernatant was collected from each well and re-screened using the same ELISA binding assay as described above.

[0242]

[0275] From each 96-well plate, the clone with the highest cell binding activity was developed in a secondary limited dilution into a 96-well plate containing 200 μl of hybridoma growth medium per well. After 7 days, the supernatant of the cells in the 96-well plate was analyzed using the same ELISA binding assay. Subcloning was performed at least twice until 90 / 96 wells showed a positive binding signal. Two subclones with the highest binding activity from each clone (i.e., 69H2 and 14G11) were identified, expanded, and cultured for purified antibody production. Isotypes were determined by standard methods.

[0243]

[0276] 3. Small-scale antibody production

[0277] Hybridoma cells were inoculated and cultured for 14 days. Monoclonal antibodies (mAbs) were purified from the hybridoma cell culture medium by protein A affinity chromatography (protein A high performance (Bio-Rad)).

[0244]

[0278] After chromatographic purification, these mAbs were combined with PBS by dialysis, followed by a filtration step.

[0245]

[0279] Example 4: Antibody immunocytochemistry screening of cell blocks

[0280] 1. Preparation of cell block sections

[0281] HEK293-human CLDN18.2 cells (hereinafter, HEK293-CLDN18.2) and HEK293-human CLDN18.1 cells (hereinafter, HEK293-CLDN18.1) were constructed by MabSpace Biosciences (Suzhou) Co. Limited. Briefly, HEK293 cells (Shanghai Institute of Biological Sciences, Cat#GNhu43) were transfected with pcDNA3.1 / hCLDN18.2 or pcDNA3.1 / hCLDN18.1 plasmids, selected with G418, and stable expression cell lines HEK293-CLDN18.2 or HEK293-CLDN18.1 were obtained. The expression of CLDN18.1 on HEK293-CLDN18.1 and the expression of CLDN18.2 on HEK293-CLDN18.2 were tested and confirmed by FACS using positive control antibodies. As shown in Figure 1, HEK293-CLDN18.1 cells showed a positive binding signal to the antibody EPR19203 (available from Abcam under product name ab222513) that binds to CLDN18.1, while HEK293-CLDN18.2 cells showed a positive binding signal to the antibody 18B10 (see PCT application PCT / CN2019 / 101563) that specifically binds to human CLDN18.2.

[0282] HEK293 cells with high expression levels of human CLDN18.2 (HEK293-CLDN18.2) or CLDN18.1 (HEK293-CLDN18.1) were collected and fixed in 4% neutral buffered paraformaldehyde (PFA) at room temperature for 30 minutes. After centrifugation, the cells were resuspended in PBS and then dispersed in 200 μl of molten agar at 57°C, and immediately solidified at 4°C. The agar-cell mix was dehydrated with gradient alcohol and clarified with xylene. After immersion in paraffin wax at 60°C, the agar-cell mix was embedded in paraffin wax using standard procedure, sections were prepared to a thickness of 3 μm, and immediately mounted on positively charged slides.

[0246]

[0283] 2. Immunocytochemical screening of antibodies using cell block sections

[0284] To screen for CLDN18.2-specific and highly sensitive antibodies, immunocytochemistry (ICC) was performed on paraffin-embedded (FFPE) HEK293, HEK293-CLDN18.2, and HEK293-CLDN18.1 cell block sections. After deparaffinization and rehydration, all sections were processed using EnVision. TM Antigen retrieval was performed by boiling at 97-99°C for 25 minutes in FLEX Target Retrieval Solution (Dako, K8002), followed by rapid cooling and EnVision TM The antibodies were blocked with FLEX Peroxidase-Blocking Reagent (Dako, K8002) and incubated with appropriately diluted antibodies. Antibody binding was performed using EnVision. TM Visualize with FLEX+ and Mouse (LINKER), then use EnVision. TM FLEX / hRP and EnVision TM FLEX Substrate Working Solution (Dako, K8002) was used. Sections were counterstained with hematoxylin and mounted using permanent mount medium. No significant differences were observed in the staining patterns, and the staining intensity varied from weak to strong between antibodies.

[0247]

[0285] As shown in Table 3 and Figure 2, clones of antibodies 69H2 and 14G11 stained strongly and specifically on the HEK293-CLDN18.2 surface at 1 nM, but were negative for HEK293 and HEK293-CLDN18.1. To further investigate sensitivity, they were titrated to 0.5 nM. 69H2F7E6, 69H2D3B1, and 69H2E1D3 are all clones of antibody 69H2, and share the same heavy and light chain sequences as 69H2. 14G11G2D2, 14G11A4E1, and 14G11F5E1 are all clones of antibody 14G11, and share the same heavy and light chain sequences as 14G11.

[0248]

[0286] As a control, staining with the anti-CLDN18.2 antibody GC182 was used, which positively stained both HEK293-CLDN18.2 and HEK293-CLDN18.1 cells. Antibody GC182 has the heavy chain variable region sequence of SEQ ID NO: 22 and the light chain variable region sequence of SEQ ID NO: 23, and was produced by Mabspace Bioscience according to the sequence disclosed in WO2013167259. The results in Table 3 indicate that antibody GC182 may cross-react with CLDN18.1 and is therefore not specific to CLDN18.2.

[0249]

[0287] GC182 heavy chain variable region sequence (SEQ ID NO: 22) QIQLVQSGPELKKFGETVKISCKASGYTFTDYSIHWVKQAPGKGLKWMGWINTETGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRTGFDYWGQGTTLTVSS

[0288] GC182 light chain variable region sequence (SEQ ID NO: 23) DIVMTQAAFSIPVTLGTSASISCRSSKNLLHSDGITYLYWYLQRPGQSPQLLIYRVSNLASGVPNRFSGSESGTDFTLRISRVEAEDVGVYYCVQVLELPFTFGGGGTKLEIK

[0289] Table 3 Antibody screening of cell block sections by immunocytochemical staining

[0250] [Table 4-1]

[0251] [Table 4-2]

[0252]

[0290] Example 5: Production of selected antibodies by cloning and recombination

[0291] Recombinant antibody production

[0292] The light and heavy chain variable region sequences of mouse anti-human CLDN18.2 antibodies 14G11 and 69H2 were obtained from candidate hybridoma cell lines by polymerase chain reaction (PCR) amplification. After sequencing analysis and confirmation, the variable region genes, including the sequence of the light chain variable region (VL) fused to the mouse κ constant region and the sequence of the heavy chain variable region (VH) fused to the mouse IgG1 constant region, were cloned into the recombinant expression vector pcDNA3.1(+) for antibody production and purification.

[0253]

[0293] The heavy and light chains of the 14G11 antibody and the 69H2 antibody were ligated to the constant region of the mouse IgG1 heavy chain and the constant region of the kappa light chain, respectively, as shown below:

[0294] Mouse IgG1 heavy chain constant region (SEQ ID NO: 17): AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0295] Mouse Kappa light chain constant region (SEQ ID NO: 18) RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0296] Recombinant antibody expression and purification

[0297] ExpiCHO cells were transfected with equal amounts of DNA from heavy-chain and light-chain vectors using the ExpiCHO transfection kit. The transfected cells were cultured in a shake flask at 125 rpm, 37°C, and 8% CO2. Cell cultures were collected on day 10, and the collected antibodies were purified by affinity chromatography. The purity levels of the obtained antibodies were analyzed using SDS-PAGE and size exclusion chromatography (TSK gel G3000SWXL, TOSOH).

[0254]

[0298] Example 6: Evaluation of binding selectivity between hCLDN18.2 and hCLDN18.1 by ELISA and FACS assays.

[0299] Specific binding of hCLDN18.2 peptide (aa28-37) by ELISA.

[0300] A 96-well plate was coated with 1 μg / ml peptide (100 μL / well), blocked with blocking buffer, and then 1 μg / ml peptide was added. Subsequently, serially diluted antibodies (150 to 0.0732 ng / ml) in blocking buffer were added, and the plate was incubated at room temperature for 1.5 hours. After washing the plate six times with washing solution, 100 μl / well of Goat pAb to Ms IgG (HRP) was added, and the plate was incubated at room temperature for 1.5 hours. After washing with washing solution, TMB was added and reacted with HRP on the plate. The OD450 nm of the plate was then read using SpectraMax i3x (Molecular Devices). It was found that 14G11G2D2 and 69H2F7E6 specifically bound to the peptide with high affinity, while GC182 could not recognize the epitopes in the linear peptide (Figure 3).

[0255]

[0301] ELISA method analysis of binding selectivity between recombinant anti-Claduin18.2 antibody and recombinant hCLDN18.2 and hCLDN18.1 proteins.

[0302] This assay used the human recombinant CLDN18.2(N-6His) variant (available from Novoprotein under catalog number NC101) and the human recombinant CLDN18.1(N-8His) variant (available from Novoprotein under catalog number CR54). The human recombinant CLDN18.2(N-6His) variant is a fusion polypeptide containing the extracellular domain of human CLDN18.2 (residues Ala24-Ala81, SEQ ID NO: 26), which is flanked by sequences that allow the extracellular domain of human CLDN18.2 to fold or associate to form a loop. Similarly, the human recombinant CLDN18.1(N-8His) variant is a fusion polypeptide containing the extracellular domain of human CLDN18.1 (residue Asp28-Leu76, SEQ ID NO: 27), which is flanked by sequences that allow the extracellular domain of human CLDN18.1 to fold or associate to form a loop.

[0256]

[0303] 1 μg / ml of human recombinant CLDN18.2 (N-6His) variant or human recombinant CLDN18.1 (N-8His) variant (100 μL / well) was coated onto a highly bounded, transparent polystyrene 96-well plate and blocked with blocking buffer. Antibodies (100-0.0244 ng / ml) serially diluted with blocking buffer were added and incubated at room temperature for 1.5 hours. The plates were washed six times with washing solution (PBS + 0.1% Tween), and 100 μl / well of Goat pAb (HRP) against Ms IgG was added and incubated at room temperature for 1.5 hours. After washing with washing solution, TMB was added and reacted with HRP on the plate. Subsequently, the OD450nm of the plate was read using SpectraMax i3x (Molecular Devices). Both 14G11G2D2 and 69H2F7E6 bound to human recombinant CLDN18.2(N-6His) with high affinity, and their EC50 values ​​were 12.75 ng / ml and 13.87 ng / ml, respectively (Figure 4). Neither 14G11G2D2 nor 69H2F7E6 showed specific binding to human recombinant CLDN18.1(N-8His) protein (Figure 5). As a control, GC182 did not show specific binding to human recombinant CLDN18.2(N-6His) or human recombinant CLDN18.1(N-8His) (Figures 4, 5). This indicates that 14G11G2D2 and 69H2F7E6 bind to an epitope within the extracellular domain of CLDN18.2, while GC182 does not recognize this epitope.

[0304] FACS analysis of HEK293-hCLDN18.2 and HEK293-hCLDN18.1 cell lines

[0305] Logarithmic phase HEK293-hCLDN18.2 or HEK293-hCLDN18.1 cells were harvested, washed, and resuspended. Antibody (20 ug / mL) diluted in blocking buffer was added, and the cells were incubated at 4°C for 1 hour. The cells were then washed twice with blocking buffer, and a secondary antibody (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, ThermoFisher) in blocking buffer was added. After incubation at 4°C for 1 hour, the cells were washed twice with blocking buffer and resuspended in blocking buffer. The cells were then transferred to FACS tubes, and antibody binding to the cells was detected using flow cytometry (BD Accuri C6). Antibodies 18B10 and EPR19203 were used as positive controls for analysis in HEK-hCLDN18.2 and HEK-hCLDN18.1 cells, respectively. As shown in Figure 1, neither antibody 14G11G2D2 nor 69H2F7E6 can bind human Claudin 18.2 or Claudin 18.1 expressed on the cell surface in their native three-dimensional structure.

[0257]

[0306] Example 7: Measurement of the binding affinity of anti-CLUDIN18.2 to recombinant hCLDN18.2 protein by ForteBio.

[0307] 100 nM human recombinant CLDN18.2(N-6His) (Novoprotein, NC101) protein was loaded onto a pre-wet Ni-NTA biosensor (PALL, 18-5101) in 1× Kinetics Buffer ((PBS + 0.1% BSA + 0.02% Tween20)) for 200 seconds. After equilibration with 1× Kinetics Buffer as a baseline (60 seconds), the sensor was immersed in serially diluted antibodies (50 nM and 25 nM) in 1× Kinetics Buffer, incubated for 200 seconds for association (kon), and then dissociated (koff) in 1× Kinetics Buffer for 200 seconds. The biosensor was regenerated in a regeneration solution for 5 seconds, followed by neutralization in a neutralization solution for 5 seconds, and this procedure was repeated three times. All procedures were performed at 30°C using Octet RED 96 (PALL). The binding affinity KD was calculated and analyzed using the ratio of koff to kon (Figure 6). The KD value for 69H2F7E6 was 1.48 nM, and the KD value for 14G11G2D2 was 0.213 nM.

[0258]

[0308] Example 8: Verification of antibody specificity using normal tissue

[0309] CLDN18.2 is a highly selective gastric antigen whose expression is limited to short-lived differentiated epithelial cells of the gastric mucosa, in contrast to CLDN18.1, whose expression is limited to the lungs. Based on this, the selected antibodies were analyzed in various relevant normal tissues to confirm that they bind to CLDN18.2 but not to CLDN18.1. Immunohistochemistry (IHC) was performed on 4% neutral buffered formalin-fixed, paraffin-embedded (FFPE) sections of normal intestine, kidney, tonsil, thyroid, skeletal muscle, stomach, lung, and mammary gland. After deparaffinization and rehydration, all sections were processed in EnVision. TM Antigen retrieval was performed by boiling at 97-99°C for 25 minutes in FLEX Target Retrieval Solution (Dako, K8002), followed by rapid cooling and EnVision TMThe antibodies were blocked with FLEX Peroxidase-Blocking Reagent (Dako, K8002) and incubated with appropriately diluted antibodies. Antibody binding was performed using EnVision. TM Visualize with FLEX+ and Mouse (LINKER), then use EnVision. TM FLEX / hRP and EnVision TM FLEX Substrate Working Solution (Dako, K8002) was used. Sections were counterstained with hematoxylin and mounted using permanent mount medium.

[0259]

[0310] Clones 14G11G2D2 and 69H2F7E6 were selected, and their specificity for FFPE normal tissue was further analyzed. Both 69H2F7E6 and 14G11G2D2 showed good results in terms of staining intensity, pattern, and selectivity. Both 69H2F7E6 and 14G11G2D2 showed strong staining intensity in FFPE gastric sections, but showed negligible staining in lung, intestinal, kidney, skeletal muscle, tonsil, thyroid, and breast sections (see Table 4, Figures 7A and 7B). All stained cells were epithelial cells; other cell types such as lymphocytes, blood vessels, fibroblasts, and smooth muscle cells were not stained. On the other hand, the anti-CLDN18.2 antibody GC182 showed strong and moderate staining intensity in FFPE gastric and lung sections, respectively, and was confirmed to bind to both CLDN18.2 and CLDN18.1. Furthermore, 14G11G2D2 showed better results than 69H2F7E6 and was selected as a candidate for further IHC testing. The results of the IHC staining analysis are shown in Table 4, Figure 7A, and Figure 7B.

[0260]

[0311] Furthermore, the specificity and sensitivity of the selected antibodies were compared with the existing anti-CLDN18.2 antibody EPR19202 (Abcam, ab222512) in normal tissue. IHC was performed as described above. As shown in Figure 8, antibody EPR19202 at a concentration of 0.374 ug / ml showed weaker staining intensity on gastric tissue than antibody 14G11G2D2 at a lower concentration, i.e., 0.15 ug / ml, indicating that EPR19202 is less sensitive than 14G11G2D2. In particular, antibody EPR19202 showed nonspecific staining in skeletal muscle (Figure 8), while antibody 14G11G2D2 maintained its specificity for CLDN18.2 and did not show cross-reactivity with normal tissue where CLDN18.2 is absent.

[0261] [Table 5-1]

[0262] [Table 5-2]

[0263] [Table 5-3]

[0264]

[0312] Example 9: Application of identification antibodies to IHC-based evaluation of CLDN18.2 expression using tumor tissue samples of different tumor types.

[0313] In Japanese gastric cancer patients, CLDN18.2 expression was detectable in 87% of tumor samples, and moderate to strong CLDN18.2 expression was observed in 52% of tumor samples (Rohde et al, Jpn J Clin Oncol. 2019 Sep 1;49(9):870-876). Furthermore, abnormal ectopic expression of CLDN18.2 has also been reported in other cancer cells, including pancreatic, ovarian, biliary tract, and lung adenocarcinomas (see Sahin U et al. Clinical Cancer Research, 2008, 14(23): 7624-7634; Karanjawala ZE et al, Am J Surg Pathol. 2008 Feb;32(2):188-96; Micke P et al, Int J Cancer. 2014 Nov 1;135(9):2206-14; Keira Y et al, Virchows Arch. 2015 Mar;466(3):265-77).

[0265]

[0314] 14G11G2D2 was further analyzed in various related cancer tissues to confirm staining intensity, pattern, and positivity. Immunohistochemistry (IHC) was performed on 4% neutral buffered formalin-fixed, paraffin-embedded (FFPE) tumor sections of gastric cancer, pancreatic cancer, cholangiocarcinoma, and non-small cell lung cancer (NSCLC). After deparaffinization and rehydration, all sections were processed in EnVision. TM Antigen retrieval is performed by boiling at 97-99°C for 25 minutes using FLEX Target Retrieval Solution (Dako, K8002), followed by quenching, and then EnVision TM The antibodies were blocked with FLEX Peroxidase-Blocking Reagent (Dako, K8002) and incubated with appropriately diluted 14G11G2D2 antibody. Antibody binding was performed using EnVision. TM Visualize with FLEX+ and Mouse (LINKER), then use EnVision. TM FLEX / hRP and EnVision TMFLEX Substrate Working Solution (Dako, K8002) was used. Sections were counterstained with hematoxylin and mounted in permanent mount medium. All samples were analyzed as the relative percentage of positively stained tumor cells to all visible tumor cells with different intensities of membrane staining (negative (-), weak (+), medium (++), strong (++)). Only membrane staining was considered positive, and normal human stomach was used as a positive control for each stain. In gastric cancer, pancreatic cancer, cholangiocarcinoma, and NSCLC cancer tissues, 14G11G2D2 produced weak to strong membrane signals, respectively (see Figure 9), and the percentage of positive tumor cells differed individually among different tumor types (see Table 5-1 for gastric cancer, Table 5-2 for pancreatic cancer, Table 5-3 for cholangiocarcinoma, and Table 5-4 for NSCLC cancer tissue). The CLDN18.2 expression positivity and prevalence in gastric cancer, pancreatic cancer, cholangiocarcinoma, and NSCLC tissues are summarized in Table 6.

[0266]

[0315] Table 5-1 Analysis of CLDN18.2 expression in gastric cancer tissue using 14G11G2D2 mouse monoclonal antibody.

[0267] [Table 6-1]

[0268] [Table 6-2]

[0269]

[0316] Table 5-2 Analysis of CLDN18.2 expression in pancreatic cancer tissue using recombinant 14G11G2D2 mouse monoclonal antibody.

[0270] [Table 7-1]

[0271] [Table 7-2]

[0272]

[0317] Table 5-3 Analysis of CLDN18.2 expression in cholangiocarcinoma using recombinant 14G11G2D2 mouse monoclonal antibody

[0273] [Table 8-1]

[0274] [Table 8-2]

[0275]

[0318] Table 5-4 Analysis of CLDN18.2 expression in NSCLC cancer tissue using recombinant 14G11G2D2 mouse monoclonal antibody

[0276] [Table 9-1]

[0277] [Table 9-2]

[0278]

[0319] Table 6. Analysis of CLDN18.2 expression positivity and prevalence in different cancer types.

[0279] [Table 10]

[0280]

[0320] Example 10: Validation of CLDN18.2 IHC staining of stomach using biotinylated 14G11G2D2

[0321] Biotinylated 14G11G2D2 (14G11G2D2-Biotin) was prepared. Biotinamidocaproate NHS ester (Sigma, B2643-10MG) was prepared to 20 mg / mL in anhydrous DMF to serve as the stock solution. 10 μl of the stock solution was added to 1 mg of 14G11G2D2 antibody, and the mixture was gently mixed at room temperature for 1 hour before labeling. Low molecular weight reaction products were labeled using Zeba TM The substance was removed by desalting using Spin Desalting Columns (ThermoFisher, 89890) according to the manufacturer's instructions.

[0281]

[0322] Immunohistochemistry (IHC) was performed on slides of 4% neutral buffered formalin-fixed, paraffin-embedded stomach samples. After deparaffinization, EnVision TM Antigen retrieval was performed by boiling in FLEX Target Retrieval Solution (Dako, K8002) at 97-99 °C for 25 minutes, followed by quenching. The antibodies were then blocked with IHC Biotin Block Kit (MaiXin, BLK-0001) as instructed, and incubated with 3 ug / mL of homemade biotinylated mouse anti-claudin 18.2 (14G11G2D2-Biotin) antibody at 37 °C for 30 minutes. Antibody binding was performed using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision. TM Visualization was performed using FLEX Substrate Working Solution (Dako, K8002). Sections were counterstained with hematoxylin and mounted using permanent mount medium.

[0282]

[0323] As shown in Figure 10, 14G11G2D2-Biotin showed the same staining pattern as 14G11G2D2 in terms of intensity and specificity. Embodiments of the Invention [Aspect 1] An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody or antigen-binding fragment exhibits one or more of the following characteristics: a) No cross-reactivity with human CLDN 18.1; b) No cross-reactivity with non-cancerous cells other than gastric epithelial cells, as measured by immunohistochemical assay (IHC); c) No cross-reactivity with non-cancerous human lung tissue as measured by IHC; d) The drug must be able to specifically bind to cells expressing CLDN18.2, and optionally, the cells expressing CLDN18.2 must be pretreated so that CLDN18.2 is denatured or no longer present in its native three-dimensional structure; e) A fusion polypeptide containing the first extracellular loop of human CLDN18.2 was found to have a K10 nM rating by surface plasmon resonance (SPR). d The substance must be able to bind at an EC50 value of 20 ng / ml or less, either by numerical value or by enzyme-linked immunosorbent assay (ELISA); f) No detectable binding to human CLDN 18.2 on the cell surface as measured by flow cytometry (FACS); g) It can specifically bind to an epitope in the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19) by ELISA measurement; and / or h) No cross-reactivity with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples at an antibody concentration of 1 nM by ELISA or 0.5 ug / ml by IHC. [Aspect 2] An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising the following: a) Heavy chain CDR1 containing the amino acid sequence X1X2YX3H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX4GX5X6X7X8YX9EKFKG (SEQ ID NO: 12), and NYX 10Heavy chain CDR3 containing the amino acid sequence of STFGY (SEQ ID NO: 24); and / or b) Light chain CDR1, KX containing the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2) 11 Light chain CDR2 containing the amino acid sequence of SNRFS (SEQ ID NO: 25), and light chain CDR3 containing the amino acid sequence of FQGSHVPFT (SEQ ID NO: 6); Here, X1 is R or T, X2 is N or Y, X3 is F or I, X4 is G or R, X5 is F or G, X6 is D or N, X7 is I or T, X8 is E or V, X9 is S or N, X 10 is G or R, and X 11 It is either V or I. [Aspect 3] An isolated antibody or its antigen-binding fragment according to Aspect 2, comprising the following: a) A light chain CDR1 containing an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 7, and / or b) A light chain CDR2 containing an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 9, and / or c) A heavy chain CDR3 containing an amino acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 11, and / or d) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, and / or e) A light chain CDR2 containing an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 10, and / or f) A light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6. [Aspect 4] An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising the following: g) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5; or h) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11. [Aspect 5] An antibody or antigen-binding fragment thereof as described in Aspect 4, comprising the following: a) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6. [Aspect 6] An antibody or antigen-binding fragment according to any one of aspects 1 to 5, comprising the following: a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6. [Aspect 7] An antibody or antigen-binding fragment according to any one of aspects 1 to 6, comprising the following: a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or b) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. [Aspect 8] An antibody or antigen-binding fragment thereof according to any one of aspects 4 to 7, further comprising one or more amino acid residue mutations while maintaining binding specificity to human CLDN 18.2. [Aspect 9] The antibody or antigen-binding fragment according to aspect 8, wherein at least one mutation is a conservative substitution, or all mutations are conservative substitutions. [Aspect 10] The antibody or antigen-binding fragment according to aspect 8 or 9, wherein at least one mutation is present in one or more CDR sequences and / or one or more non-CDR sequences in the heavy chain variable region or light chain variable region. [Aspect 11] An antibody or antigen-binding fragment thereof according to any one of aspects 8 to 10, the following: a) A heavy chain CDR1 (HCDR1) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 7, and / or b) A heavy chain CDR2 (HCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 3 or SEQ ID NO: 9, and / or c) A heavy chain CDR3 (HCDR3) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 5 or SEQ ID NO: 11, and / or d) A light chain CDR1 (LCDR1) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 2, and / or e) A light chain CDR2 (LCDR2) sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 4 or SEQ ID NO: 10, and / or f) A light chain CDR3 (LCDR3) sequence having at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to sequence number 6. including, and, On the other hand, an antibody or its antigen-binding fragment that retains binding specificity to CLDN18.2 and optionally has a binding affinity at the same level as or higher than that of its parent antibody. [Aspect 12] An antibody or antigen-binding fragment thereof according to any one of aspects 8 to 11, comprising HCDR1 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 1 or SEQ ID NO: 7, HCDR2 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 3 or SEQ ID NO: 9, HCDR3 having 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 5 or SEQ ID NO: 11, LCDR1 having 2 or 1 or fewer amino acid mutations in SEQ ID NO: 2, LCDR2 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 4 or SEQ ID NO: 10, and / or LCDR3 having 3, 2, or 1 or fewer amino acid mutations in SEQ ID NO: 6, while also maintaining binding specificity to CLDN 18.2 and optionally having a binding affinity at the same level as or higher than that of the parent antibody. [Aspect 13] An antibody or antigen-binding fragment thereof according to any one of aspects 8 to 12, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and / or the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 16. [Aspect 14] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 13, further comprising an immunoglobulin constant region, optionally an IgG heavy chain constant region, and / or a light chain constant region. [Aspect 15] The antibody or antigen-binding fragment thereof according to aspect 14, wherein the constant region includes a mouse constant region, a rabbit constant region, or a human constant region. [Aspect 16] The antibody or antigen-binding fragment according to aspect 15, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17 or a sequence having at least 80% sequence identity thereto, and / or the light chain constant region comprises the amino acid sequence of SEQ ID NO: 18 or a sequence having at least 80% sequence identity thereto. [Aspect 17] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 16, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a labeled antibody, a bivalent antibody, an anti-idiotype antibody, a fusion protein, a dimerized or polymerized antibody, or a modified antibody (e.g., a glycosylated antibody). [Aspect 18] An antibody or antigen-binding fragment thereof, which is any one of aspects 1 to 17, and is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody. [Aspect 19] An antibody or antigen-binding fragment thereof, which is bound to one or more portions of aspects 1 to 18. [Aspect 20] The antibody or antigen-binding fragment thereof according to aspect 19, wherein the portion comprises a radioactive isotope, a lanthanide, a chemiluminescent label, a chromogenic portion, a gold colloid particle, a fluorescent label, an enzyme substrate label, a digoxigenin label, biotin / avidin, a hapten, or a DNA molecule for detection or particle labeling. [Aspect 21] The antibody or antigen-binding fragment thereof according to aspect 20, wherein the portion comprises biotin or a hapten. [Aspect 22] A monoclonal antibody or its antigen-binding fragment that competes with any of the antibodies or their antigen-binding fragments of aspects 1 to 21 for binding to CLDN 18.2. [Aspect 23] An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any of aspects 1 to 22. [Aspect 24] A vector comprising the isolated polynucleotide described in Aspect 23. [Aspect 25] A host cell containing the vector described in Aspect 24. [Aspect 26] A method for expressing an antibody or its antigen-binding fragment according to any one of aspects 1 to 22, comprising culturing host cells according to aspect 25 under conditions in which the vector according to aspect 24 is expressed. [Aspect 27] A method for detecting the presence or expression level of CLDN 18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment of any of aspects 1 to 22 under conditions that enable specific binding of an antibody or antigen-binding fragment thereof to human CLDN 18.2, and determining the presence or expression level of CLDN 18.2 in the sample. [Aspect 28] A method for diagnosing a CLDN18.2-related disease or condition (e.g., cancer), comprising: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment described in any of embodiments 1 to 22 under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN 18.2: and b) Determine the presence or expression level of CLDN18.2 in the sample. If CLDN18.2 is found to be present, or if the expression level of CLDN18.2 reaches a threshold level, the subject is diagnosed with a CLDN18.2-related disease or condition (e.g., cancer). [Aspect 29] A method for determining the eligibility of a subject having or at risk of having a CLDN18.2-related disease or condition for treatment with a CLDN 18.2-targeting agent, comprising: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment described in any of embodiments 1 to 22 under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN18.2; and b) Determine the presence or expression level of CLDN18.2 in the sample. If the presence of CLDN18.2 is detected, or if the expression level of CLDN18.2 reaches a threshold, the subject is determined to be eligible for treatment with a CLDN18.2-targeting agent, or A method for determining whether a subject is ineligible for treatment with a CLDN18.2-targeting agent if the presence of CLDN18.2 is not detected, or if the expression level of CLDN18.2 is below a threshold. [Aspect 30] A method for predicting the therapeutic effect of a CLDN18.2-targeted agent in the treatment of a target CLDN18.2-related disease or condition, comprising: a) Contact the sample obtained from the subject with the antibody or antigen-binding fragment described in any of embodiments 1 to 22, under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN18.2; b) Determine the presence or expression level of human CLDN18.2 in the sample; c) Predicting the therapeutic effect of CLDN18.2 targeted agents, Here, CLDN18.2-targeting agents are predicted to be effective in the treatment of the target if the presence of CLDN18.2 is detected, or if the expression level of CLDN18.2 reaches a threshold level. Here, CLDN18.2-targeting agents are predicted to be ineffective in the treatment of the target group if CLDN18.2 is not detected or if the expression level of CLDN18.2 is below the threshold. [Aspect 31] A method for treating a subject who has or is at risk of having a CLDN18.2-related disease or condition, the method comprising: a) Selecting a suitable patient for treatment, which includes: i) Contact the sample obtained from the subject with the antibody or antigen-binding fragment described in any of embodiments 1 to 22, under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN18.2; ii) To determine the presence or expression level of human CLDN18.2 in the sample; iii) If the presence of CLDN 18.2 is detected, or if the expression level of CLDN 18.2 in the sample reaches a threshold, subjects should be selected as suitable for treatment of CLDN 18.2-related diseases or conditions; b) Administer a therapeutically effective dose of CLDN18.2-targeting agent to the selected subjects. [Aspect 32] A method for treating a subject who has cancer or is at risk of cancer, comprising the following: a) Select the target, including the following: i) Contact the sample obtained from the subject with the antibody or antigen-binding fragment described in any of embodiments 1 to 22, under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN18.2; ii) Determine the presence or expression level of CLDN18.2 in the sample; iii) If CLDN18.2 is not detected, or if the expression level of CLDN18.2 in the sample is below the threshold, the subject should be selected as unsuitable for treatment with a CLDN18.2-targeting agent; b) Administer standard therapeutic agents other than CLDN18.2 targeting agents to the selected subjects. [Aspect 33] The method according to any one of aspects 27 to 32, wherein the sample is a cell sample or a tissue sample. [Aspect 34] The method according to aspect 33, wherein the sample is a fixed tissue sample, or optionally a formalin-fixed paraffin-embedded (FFPE) tissue sample. [Aspect 35] The method according to any one of aspects 27 to 34, wherein CLDN18.2 is cell surface or membrane-bound CLDN18.2. [Aspect 36] The method according to any one of aspects 27 to 35, wherein the presence or expression level of CLDN18.2 is determined by immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or immunoblotting. [Aspect 37] The method according to any one of aspects 27 to 36, wherein the expression level is quantified based on the percentage of positively stained cells in the sample. [Aspect 38] The method according to any one of aspects 27 to 36, wherein the expression level is quantified based on the staining intensity for CLDN18.2 in the sample. [Aspect 39] The method according to any one of aspects 28 to 38, wherein the disease or condition associated with CLDN18.2 is cancer. [Aspect 40] The method according to aspect 39, wherein the sample includes a tumor sample. [Aspect 41] The method according to aspect 40, wherein the tumor sample comprises tumor tissue or circulating tumor cells. [Aspect 42] The method according to aspect 41, wherein the cancer is primary cancer or metastatic cancer. [Aspect 43] The method according to any one of aspects 39 to 42, wherein the cancer is gastric cancer, lung cancer (non-small cell lung cancer or small cell lung cancer), bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, bile duct cancer and / or adenocarcinoma. [Aspect 44] The method according to aspect 43, wherein the cancer is gastric cancer, pancreatic cancer, bile duct cancer, or lung cancer (e.g., non-small cell lung cancer or small cell lung cancer). [Aspect 45] The method according to any one of aspects 29 to 44, wherein the CLDN18.2 targeting agent is capable of inducing cytotoxicity against CLDN18.2-expressing cells. [Aspect 46] The method according to any one of aspects 29 to 45, wherein the CLDN18.2 targeting agent is a therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule (e.g., an anti-CLDN18.2 antibody bound to a cytotoxic agent, or a bispecific antibody), CLDN18.2 targeted cell therapy (e.g., CAR-T, TCR-T, or CAR-NK cells expressing CLDN18.2-binding CAR), a CLDN18.2 targeted compound, or a CLDN18.2 targeted therapeutic nucleic acid. [Aspect 47] The method according to any one of aspects 27 to 46, wherein the subject is currently receiving or has previously received anticancer drug treatment, or is suffering from a recurrence of cancer. [Aspect 48] A kit comprising an isolated antibody or its antigen-binding fragment as described in any of aspects 1 to 22. [Aspect 49] The kit according to aspect 48, further comprising a reagent set for detecting a complex of an antibody or its antigen-binding fragment bound to CLDN18.2. [Aspect 50] The kit according to aspect 49, wherein the reagent set includes a secondary antibody that binds to an antibody or its antigen-binding fragment as described in any of aspects 1 to 22, and optionally the secondary antibody is detectably labeled. [Aspect 51] The kit according to aspect 48, wherein the antibody or its antigen-binding fragment is detectably labeled. [Aspect 52] The kit according to aspect 48, wherein an antibody or its antigen-binding fragment is conjugated with a portion that can be indirectly detected. [Aspect 53] The kit according to aspect 52, wherein the indirectly detectable portion contains biotin. [Aspect 54] The kit according to aspect 53, wherein the reagent set comprises detectably labeled avidin or streptavidin.

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to CLDN18.2, the following: a) X 1 X 2 YX 3 heavy chain CDR1 comprising the amino acid sequence of H (SEQ ID NO: 8), WIYPX 4 GX 5 X 6 X 7 X 8 YX 9 heavy chain CDR2 comprising the amino acid sequence of EKFKG (SEQ ID NO: 12), and NYX 10 heavy chain CDR3 comprising the amino acid sequence of STFGY (SEQ ID NO: 24); and b) Light chain CDR1, KX containing the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2) 11 Light chain CDR2 containing the amino acid sequence of SNRFS (SEQ ID NO: 25), and light chain CDR3 containing the amino acid sequence of FQGSHVPFT (SEQ ID NO: 6); This includes, where X 1 is R or T, X 2 is N or Y, X 3 is F or I, X 4 is G or R, X 5 is F or G, X 6 is D or N, X 7 is I or T, X 8 is E or V, X 9 is S or N, X 10 is G or R, and X 11 is V or I, and The antibody or its antigen-binding fragment is a) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 5, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6; or b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 9, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, An antibody or its antigen-binding fragment, including the above.

2. The antibody or antigen-binding fragment according to claim 1, comprising the following: a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or b) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence of SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising an immunoglobulin constant region.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a heavy chain constant region and / or a light chain constant region of IgG.

5. The antibody or antigen-binding fragment according to claim 3, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17 or a sequence having at least 80% sequence identity thereto, and / or the light chain constant region comprises the amino acid sequence of SEQ ID NO: 18 or a sequence having at least 80% sequence identity thereto.

6. Monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, labeled antibodies, bivalent antibodies, anti-idiotype antibodies, fusion proteins, dimerized or polymerized antibodies, or modified antibodies, or diabolic, Fab, Fab', F(ab') 2 Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 An antibody or antigen-binding fragment according to any one of claims 1 to 5, which is a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), or a multispecific antibody.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the modified antibody is a glycosylated antibody.

8. An antibody or antigen-binding fragment thereof from any one of claims 1 to 7, wherein one or more portions are bound to a radioactive isotope, lantanide, chemiluminescent label, chromogenic portion, gold colloid particle, fluorescent label, enzyme substrate label, digoxigenin label, biotin / avidin, hapten, or DNA molecule for detection or particle labeling.

9. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising an isolated polynucleotide as described in claim 9.

11. A host cell comprising the vector according to claim 10.

12. A method for expressing the antibody or antigen-binding fragment according to claim 1, comprising culturing a host cell containing the vector under conditions in which the vector containing the isolated polynucleotide encoding the antibody or antigen-binding fragment according to claim 1 is expressed.

13. A method for detecting the presence or expression level of CLDN 18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment according to any of claims 1 to 8 under conditions that enable specific binding of an antibody or antigen-binding fragment thereof to human CLDN 18.2, and determining the presence or expression level of CLDN 18.2 in the sample.

14. A method for assisting in the diagnosis of a CLDN18.2-related disease or condition, comprising the following: a) Contacting a sample obtained from the subject with the antibody or antigen-binding fragment described in any one of claims 1 to 8 under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN 18.2: and b) Determine the presence or expression level of CLDN18.2 in the sample.

15. A pharmaceutical composition for use in a method of treating a subject having or at risk of having a CLDN18.2-related disease or condition, comprising a CLDN18.2 targeting agent, wherein the method comprises: a) Selecting a suitable patient for treatment, which includes: i) Contacting a sample obtained from a subject with the antibody or antigen-binding fragment described in any one of claims 1 to 8, under conditions that enable specific binding of the antibody or antigen-binding fragment to CLDN18.2; ii) Determining the presence or expression level of human CLDN18.2 in the sample; and iii) If the presence of CLDN 18.2 is detected, or if the expression level of CLDN 18.2 in the sample reaches a threshold, subjects should be selected as suitable for treatment of CLDN 18.2-related diseases or conditions; and b) Administer a therapeutically effective dose of CLDN18.2-targeting agent to the selected subjects.

16. The method according to claim 13 or 14, wherein the sample is a cell sample or a tissue sample.

17. The method according to claim 16, wherein the sample comprises tumor tissue or circulating tumor cells.

18. The method according to any one of claims 13, 14, 16, and 17, wherein CLDN18.2 is cell surface or membrane-bound CLDN18.2, and the presence or expression level of CLDN18.2 is determined by immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or immunoblotting, and the expression level is quantified based on the percentage of positively stained cells in the sample or the staining intensity for CLDN18.2 in the sample.

19. The method according to any one of claims 14 and 16-18, wherein the disease or condition associated with CLDN18.2 is cancer.

20. The method according to claim 19, wherein the cancer is primary cancer or metastatic cancer.

21. The method according to claim 19 or 20, wherein the cancer is stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, bile duct cancer and / or adenocarcinoma.

22. The method according to claim 21, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

23. The method according to claim 21, wherein the esophageal cancer is esophageal adenocarcinoma or gastric / gastroesophageal junction (G / GEJ) cancer.

24. The pharmaceutical composition according to claim 15, wherein the CLDN18.2 targeting agent is capable of inducing cytotoxicity against CLDN18.2-expressing cells, and the CLDN18.2 targeting agent is a therapeutic anti-CLDN18.2 antibody or CLDN18.2-binding molecule, CLDN18.2-targeted cell therapy, CLDN18.2-targeted compound, or CLDN18.2-targeted therapeutic nucleic acid.

25. The pharmaceutical composition according to claim 24, wherein the therapeutic anti-CLDN18.2 antibody or CLDN18.2-binding molecule is an anti-CLDN18.2 antibody conjugated to a cytotoxic agent, or a bispecific antibody.

26. The pharmaceutical composition according to claim 24, wherein the CLDN18.2-targeted cell therapy is CAR-T, TCR-T, or CAR-NK cells expressing CLDN18.2-binding CAR.

27. The method according to any one of claims 14 and 16 to 23, wherein the subject is currently receiving or has previously received anticancer drug treatment, or is suffering from a recurrence of cancer.

28. A kit comprising an isolated antibody or its antigen-binding fragment according to any one of claims 1 to 8.

29. The kit according to claim 28, further comprising a reagent set for detecting a complex of the antibody or its antigen-binding fragment bound to CLDN18.2, wherein the reagent set comprises a secondary antibody bound to the antibody or its antigen-binding fragment.

30. The kit according to claim 29, wherein the secondary antibody is detectably labeled.

31. The kit according to claim 28, wherein the antibody or its antigen-binding fragment is detectably labeled, or the antibody or its antigen-binding fragment is conjugated with an indirectly detectable portion.

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