HK40089087A - Anti-cldn18.2 antibodies and diagnostic uses thereof - Google Patents

Description

Anti-CLDN18.2 antibody and its diagnostic uses

Technical Field

This disclosure generally relates to a novel anti-CLDN18.2 antibody and its diagnostic applications.

Background Technology

Claudin-18 (CLDN18) molecules (Genbank accession numbers: splice variant 1 (CLDN18A1 or CLDN18.1): NP_057453, NM_016369, and splice variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, NP_001002026) are integrated transmembrane proteins with a molecular weight of approximately 27.9/27.72 kDa. CLDN18 proteins are located within tight junctions of epithelial and endothelial cells, forming a network of strands that connect intracellular granules between adjacent cells. CLDN18 and occludin are the most abundant transmembrane protein components in tight junctions. Due to their strong intercellular adhesive properties, these tight junction proteins create primary barriers to prevent and control paracellular transport of solutes and also limit the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Therefore, it plays a crucial role in the organization of the epithelial tissue structure.

While targeted therapies and immunotherapies have revolutionized systemic treatment for various cancers over the past decade, the management of advanced and/or metastatic cancers remains a significant challenge. For example, many patients with advanced gastrointestinal cancers (such as gastric, pancreatic, cholangiocarcinoma, or lung cancer) do not significantly benefit from current standard treatments. Chemotherapy remains the mainstream treatment for most patients with these advanced cancers, and its prognosis remains extremely poor. Given the highly restricted expression and frequent ectopic activation of CLDN18.2 in a wide range of tumor types, CLDN18.2 is considered a therapeutic target in the development of agents for treating solid tumors expressing CLDN18.2, including but not limited to GC/GEC, pancreatic cancer, cholangiocarcinoma, and lung cancer.

In order to screen and select patients expressing CLDN18.2, IHC assays with high specificity and affinity are required.

Summary of the Invention

In this disclosure, the articles “a/an” and “the” are used herein to refer to one or more (i.e., at least one) grammatical object of the article. For example, “a antibody” means one or more antibodies.

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.

In one aspect, this disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics:

a) It does not exhibit cross-reactivity with human CLDN18.1;

b) As measured by immunohistochemical analysis (IHC), it does not cross-react with non-cancerous cells, except for gastric epithelial cells;

c) As measured by IHC, there is no cross-reactivity with non-cancer lung tissue;

d) Capable of specifically binding to cells expressing CLDN18.2, optionally the cells expressing CLDN18.2 are pretreated to denature CLDN18.2 or otherwise prevent it from exhibiting its native conformation;

e) A fusion polypeptide containing the first extracellular loop of human CLDN18.2, as measured by surface plasmon resonance (SPR), can bind with a Kd value not exceeding 10 nM, or as measured by enzyme-linked immunosorbent assay (ELISA), can bind with an EC50 value not exceeding 20 ng/ml.

f) As measured by flow cytometry (FACS) analysis, no detectable binding was observed to human CLDN 18.2 on the cell surface;

g) As measured by ELISA, it can specifically bind to an epitope within the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19); and/or

h) The antibody does not cross-react with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples, as measured by ELISA at an antibody concentration of 1 nM or by IHC at an antibody concentration of 0.5 μg/ml.

In one aspect, this disclosure provides a separated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising:

a) Heavy chain CDR1 containing the amino acid sequence X ₁ X ₂ Y X ₃ H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX ₄ GX ₅ X ₆ X ₇ X ₈ YX ₉ EKFKG (SEQ ID NO: 12), and heavy chain CDR3 containing the amino acid sequence NYX ₁₀ STFGY (SEQ ID NO: 24); and/or

b) Light chain CDR1, which contains the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2), light chain CDR2, which contains the amino acid sequence KX ₁₁ SNRFS (SEQ ID NO: 25), and light chain CDR3, which contains the amino acid sequence FQGSHVPFT (SEQ ID NO: 6);

Where _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, _X10 is G or R and _X11 is V or I.

In some embodiments, the isolated antibody or its antigen-binding fragment comprises:

a) Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 3, and heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 5; or

b) Heavy chain CDR1, which contains SEQ ID NO: 7, heavy chain CDR2, which contains SEQ ID NO: 9, and heavy chain CDR3, which contains SEQ ID NO: 11.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises:

a) light chain CDR1, comprising the amino acid sequence of SEQ ID NO: 2; light chain CDR2, comprising the amino acid sequence of SEQ ID NO: 4; and light chain CDR3, comprising the amino acid sequence of SEQ ID NO: 6; or

b) Light chain CDR1, which contains the amino acid sequence of SEQ ID NO: 2, light chain CDR2, which contains the amino acid sequence of SEQ ID NO: 10, and light chain CDR3, which contains the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises:

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.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; or

b) The heavy chain variable region, which contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region, which contains the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibodies or antigen-binding fragments provided herein further contain one or more amino acid residue mutations, while still maintaining binding specificity for human CLDN 18.2, and optionally maintaining binding specificity for linear epitopes containing the amino acid sequence of SEQ ID NO: 19.

In some implementations, at least one of the mutations is a conservative substitution, or all of the mutations are conservative substitutions.

In some implementations, at least one of the mutations is in one or more CDR sequences in the heavy chain variable region or the light chain variable region, and/or in one or more non-CDR sequences.

In some embodiments, the antibody or its antigen-binding fragment comprises:

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% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 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) The light chain CDR3 (LCDR3) sequence, which has 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: 6, and

The antibody or its antigen-binding fragment may optionally have a binding affinity similar to or even higher than that of its parent antibody while maintaining binding specificity to CLDN18.2.

In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3, wherein HCDR1 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 1 or SEQ ID NO: 7, HCDR2 has no more than 6, 5, 4, 3, 2, or 1 amino acid mutation in SEQ ID NO: 3 or SEQ ID NO: 9, HCDR3 has no more than 6, 5, 4, 3, 2, or 1 amino acid mutation in SEQ ID NO: 5 or SEQ ID NO: 11, LCDR1 has no more than 2 or 1 amino acid mutation in SEQ ID NO: 2, LCDR2 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 4 or SEQ ID NO: 10, and LCDR3 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 6, and the antibody or its antigen-binding fragment optionally has a binding affinity similar to or even higher than that of its parent antibody while maintaining binding specificity to CLDN18.2.

In some embodiments, 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.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally comprising a heavy chain constant region and/or a light chain constant region of IgG. In some embodiments, the constant region comprises a mouse constant region, a rabbit constant region, a human constant region, or any other suitable constant region.

In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80% sequence identity with it, and/or the light chain constant region comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity with it.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, labeled antibodies, bivalent antibodies, anti-idiotype antibodies, fusion proteins, dimerizing antibodies or multimerizing antibodies, or modified antibodies (e.g., glycosylated antibodies).

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bifunctional antibodies, Fab, Fab′, F(ab′) ₂ , Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv′), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies, camelified single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein is linked to one or more portions. In some embodiments, the portions comprise radioactive isotopes, lanthanides, chemiluminescent labels, chromogenic fractions, colloidal gold particles, fluorescent labels, enzyme-substrate labels, digoxigenin labels, biotin/avidin labels, haptens, or DNA molecules or particles labeled for detection. In some embodiments, the portions comprise biotin or haptens.

In another aspect, this disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof provided herein for binding to CLDN18.2.

In another aspect, this disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein. In another aspect, this disclosure provides a vector comprising the isolated polynucleotide provided herein. In another aspect, this disclosure provides a host cell comprising the vector provided herein.

In another aspect, this disclosure provides a method for expressing the antibody or antigen-binding fragment thereof provided herein, comprising culturing the host cells provided herein under conditions of expressing the vector provided herein.

In another aspect, 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 antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to human CLDN18.2, and determining the presence or expression level of CLDN18.2 in the sample.

In another aspect, this disclosure provides a method for diagnosing CLDN18.2-related diseases or conditions (e.g., cancer) in a subject, comprising:

a) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; and

b) Determine the presence or expression level of CLDN18.2 in the sample;

When the presence of CLDN18.2 is detected or when 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 another aspect, this disclosure provides a method for determining the appropriateness of treatment with a CLDN18.2-targeting agent for a subject suffering from or at risk of developing a CLDN18.2-related disease or condition, comprising:

a) Contact the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

b) Determine the presence or expression level of CLDN18.2 in the sample;

When CLDN18.2 is detected or when its expression level reaches a threshold level, the subject is deemed suitable for treatment with a CLDN18.2-targeting agent.

When CLDN18.2 is not detected or when the expression level of CLDN18.2 is below a threshold level, the subject is deemed unsuitable for treatment with CLDN18.2-targeted agents.

In another aspect, this disclosure provides a method for predicting the therapeutic efficacy of a CLDN18.2-targeting agent in treating CLDN18.2-related diseases or symptoms in subjects, comprising:

a) Contact the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

b) Determine the presence or expression level of human CLDN18.2 in the sample;

c) Predict the therapeutic efficacy of the CLDN18.2 targeting agent.

Specifically, the presence of CLDN18.2 or the expression level of CLDN18.2 reaching a threshold level are used to predict that the CLDN18.2 targeting agent is effective in treating the subject.

When the presence of CLDN18.2 is not detected or when the expression level of CLDN18.2 is below the threshold level, it is predicted that the CLDN18.2 target is ineffective in treating the subject.

In another aspect, this disclosure provides a method for treating a subject suffering from or at risk of CLDN18.2-related disease or condition, comprising:

a) Selecting subjects suitable for the treatment, including:

i) Contact the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

ii) Determine the presence or expression level of human CLDN18.2 in the sample;

iii) When the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 in the sample reaches a threshold level, the subject is selected as a suitable candidate for treatment of the CLDN18.2-related disease or symptom;

b) Administer a therapeutically effective dose of the CLDN18.2-targeting agent to the selected subjects.

In another aspect, this disclosure provides a method for treating a subject who has cancer or is at risk of developing cancer, comprising:

a) Selecting subjects, including:

i) Contact the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

ii) Determine the presence or expression level of CLDN18.2 in the sample;

iii) When CLDN18.2 is not found or when the expression level of CLDN18.2 in the sample is below a threshold level, the subject is selected as unsuitable for treatment of the CLDN18.2 related disease or condition;

b) Administer standard treatment to the selected subjects in addition to the CLDN18.2 targeted agent.

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

In some 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 Western blotting.

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

In some embodiments, the CLDN18.2-related disease or symptom is cancer. In some embodiments, the sample comprises a tumor sample. In some embodiments, the tumor sample comprises tumor tissue or circulating tumor cells. In some embodiments, the cancer is primary cancer or metastatic cancer.

In some embodiments, the CLDN18.2-related disease or symptom is cancer. In some embodiments, the cancer is primary cancer or metastatic cancer. In some embodiments, 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.

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

In some embodiments, the CLDN18.2 target is capable of inducing cytotoxicity against cells expressing CLDN18.2. In some embodiments, the CLDN18.2 target is a therapeutic anti-CLDN18.2 antibody or a CLDN18.2 binding molecule, a cell therapy targeting CLDN18.2, a compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2.

In some embodiments, the therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule is conjugated to a cytotoxic agent. In some embodiments, the therapeutic anti-CLDN18.2 antibody is a bispecific antibody. In some embodiments, the cell therapy targeting CLDN18.2 includes chimeric antibody receptor-engineered T cells (CAR-T), genetically modified TCRT cells (TCR-T), or chimeric antibody receptor-engineered NK cells (CAR-NK) expressing a chimeric antibody receptor (CAR) that binds to CLDN18.2.

In some implementations, the subject is receiving or has received anti-cancer therapy, or has experienced a recurrence of cancer.

In another aspect, this disclosure provides a kit comprising the isolated antibody or antigen-binding fragment thereof provided herein.

In some embodiments, the kit further comprises a set of reagents for detecting complexes of the antibody or its antigen-binding fragment that binds to CLDN18.2. In some embodiments, the set of reagents comprises an anti-mouse antibody.

Attached Figure Description

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

Figure 2 shows the immunocytochemical (ICC) staining of antibodies 69H2 and 14G11 screened on cell block sections of HEK293, HEK293-CLDN18.1, and HEK293-CLDN18.2. Staining of antibody GC182 was used as a control.

Figure 3 shows the binding properties and EC50 of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182 to the hCLDN18.2 peptide (amino acid residues 28-37 of hCLDN18.2).

Figure 4 illustrates the binding characteristics and EC50 of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182 to recombinant hCLDN18.2 variant protein, as measured by ELISA, which contains the amino acid sequence of the ECL1 loop of hCLDN18.2 (SEQ ID NO: 26).

Figure 5 illustrates the binding characteristics and EC50 of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182 to recombinant hCLDN18.1 variant protein, as measured by ELISA, which contains the amino acid sequence of the ECL1 loop of hCLDN18.1 (SEQ ID NO: 27).

Figure 6 shows the binding affinity analysis of anti-CLDN18.2 antibodies 69H2F7E6, 14G11G2D2, and GC182 with the recombinant hCLDN18.2 mutant protein, as performed by ForteBio. KD, kon, and koff are shown.

Figures 7A and 7B show the immunohistochemical (IHC) analysis of selected antibodies 14G11G2D2, 69H2F7E6, and GC182 on formalin-fixed paraffin-embedded (FFPE) tissue sections of normal stomach, lung, intestine, kidney, tonsils, thyroid, breast, and skeletal muscle tissue, respectively. Arrows indicate positive staining for GC182 on normal lung tissue.

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

Figure 9 shows representative IHC images of the 14G11G2D2 antibody on various tissues of gastric cancer, pancreatic cancer, cholangiocarcinoma, and non-small cell lung cancer (NSCLC) with strong (++), medium (++), weak (+), and negative (-) staining intensities.

Figure 10 shows a comparison of IHC staining between antibody 14G11G2D2 and antibody conjugate 14G11G2D2-biotin on gastric sections.

Figure 11 shows all the sequences in this application.

Detailed Implementation

The following description of this disclosure is for illustrative purposes only, illustrating various embodiments thereof. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are also included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

definition

As used herein, unless otherwise stated herein or obviously contradicted by the context, the terms “a/an,” “the,” and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to cover both singular and plural forms.

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. A naturally occurring intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region ( _VH ) and first, second, and third constant regions ( _CH1 , _CH2 , _CH3 , respectively); mammalian light chains are classified as λ or κ, each consisting of a variable region ( _VL ) and a constant region. Antibodies are Y-shaped, with the backbone of the Y consisting of the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y contains a variable region and a first constant region of a single heavy chain, which binds to the variable and constant regions of a single light chain. 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 include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibody and antigen-binding domains disclosed in this application can be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, *Journal of Molecular Biology*, 273(4), 927(1997); Chothia, C. et al., *Journal of Molecular Biology*, Dec. 5; 186(3): 651-). 63 (1985); Chothia, C. and Lesk, AM, Journal of Molecular Biology, 196, 901 (1987); NR Whitelegg et al., Protein Engineering, v13 (12), 819-824 (2000); Chothia, C. et al., Nature, Dec 21-28; 342 (6252): 877-83 (1989); Kabat EA et 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). Three CDRs are inserted between flanking segments called framework regions (FRs), which are more conserved than CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several main antibody classes are further subdivided 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). This disclosure encompasses all antibodies and antibody derivatives as described in this application, which are covered by the term "antibody" for the purposes of this invention. The term "antibody derivative" refers to any modified form of an antibody, such as an antibody conjugate with another pharmaceutical agent, an antibody fragment, or a fusion protein containing an antibody or an antibody fragment.

As used herein, the term "antigen-binding fragment" refers to an antibody fragment comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the complete structure of a natural antibody. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab′, F(ab′) ₂ , Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv′), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibody fragments, camelified single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen that the parent antibody binds to. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular antibody.

The "Fab" in antibody refers to the monovalent antigen-binding fragment of the antibody, which consists of a single light chain (variable region and constant region) linked by disulfide bonds to a single heavy chain with a variable region and a first constant region. Fab can be obtained by digesting residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region of the antibody using papain.

“Fab′” refers to a Fab fragment containing a portion of the hinge region, which can be obtained by digesting residues near the C-terminus of the disulfide bonds between the heavy chains of the antibody hinge region with pepsin, and therefore a small number of residues (containing one or more cysteine residues) in the hinge region are different from Fab.

“F(ab′) ₂ ” refers to the dimer of Fab′, which contains two light chains and a portion of two heavy chains.

The "Fc" in antibody refers to the antibody moiety, which consists of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region of IgG and IgM contains three heavy chain constant regions (the second, third, and fourth constant regions of each chain). It can be obtained by digesting the antibody with papain. The Fc moiety of an antibody is responsible for various effector functions, such as ADCC, ADCP, and CDC, but does not play a role in antigen binding.

The term "Fv" in antibody refers to the smallest antibody fragment with a complete antigen-binding site. An Fv fragment consists of a variable region of a single light chain that binds to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment, where the bond between the variable regions of the single light chain and the single heavy chain is a disulfide bond.

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of light chain variable regions and heavy chain variable regions linked together directly or via peptide linker sequences (Huston JS et al., Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA), 85: 5879 (1988)). "scFv dimer" refers to a single-chain antibody containing two heavy chain variable regions and two light chain variable regions with linkers. In some embodiments, "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) comprising a _VH - _VL (linked by peptide linkers) dimerized with another _VH - _VL portion, such that one _VH portion coordinates with the other _VL portion, forming two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the “scFv dimer” is a bispecific bifunctional antibody comprising _VH1 - _VL2 (linked by peptide linkers) that bind to _VL1 - _VH2 (also linked by peptide linkers), such that _VH1 coordinates to _VL1 and _VH2 coordinates to _VL2 and each coordination pair has a different antigen specificity.

"Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody that consists of scFv linked to the Fc region of the antibody.

"Camelized single-domain antibody,""heavy chain antibody,""nanobody," or "HCAb" refers to an antibody containing two V and _H domains and lacking 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; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from the Camelidae family (camel, dromedary, and llama). Although they do not contain light chains, camel-like antibodies have a reliable antigen-binding repertoire (Hamers-Casterman C. et al., Nature, June 3; 363(6428): 446-8(1993); Nguyen VK. et al., Heavy-chain antibodies in Camelidae: a case of evolutionary innovation, Immunogenetics, April; 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 generated by an acquired immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub. June 15, 2007 (2007)).

A “bifunctional antibody” comprises a small antibody fragment having two antigen-binding sites, wherein the fragment contains a _VH domain ( _VH - _VL or _VL - _VH ) linked to a _VL domain in a single polypeptide chain (see, for example, Holliger P. et al., Proceedings of the National Academy of Sciences, July 15; 90(14): 6444-8(1993); EP404097; WO93/11161). Because the linker is too short, the two domains on the same chain cannot pair, thus forcing the domain to pair with a complementary domain of another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In some embodiments, two or more _VH domains are covalently linked by peptide linkers to form a bivalent or multivalent domain antibody. The two _VH domains of a bivalent domain antibody can target the same or different antigens.

"(dsFv) ₂ " refers to an Fv fragment stabilized by disulfide bonds containing three peptide chains: the two _VH moieties are linked by peptide linkers and are bound to the two _VL moieties by disulfide bridges.

"Bispecific ds bifunctional antibody" refers to a bifunctional antibody that targets two different antigens (or epitopes). It may contain _VH1 - _VL2 (also linked by a peptide linker) that is bound to _VL1 - _VH2 (linked by a peptide linker) through a disulfide bridge between _VH1 and _VL1 .

"Bispecific dsFv" or "dsFv-dsFv" refers to a disulfide-stabilized Fv fragment that targets two different antigens (or epitopes). It may consist of three peptide chains: _VH1 - _VH2 moieties, where the heavy chain is linked via a peptide linker (e.g., a long flexible linker) and paired with the _VL1 and _VL2 moieties via disulfide bridges, respectively. Each disulfide-paired heavy and light chain exhibits different antigen specificities.

As used in this application, the term "humanized" means that the antibody or antigen-binding fragment contains a CDR derived from a non-human animal, an FR region derived from a human, and, where applicable, a constant region derived from a human.

As used herein, the term "chimerism" means that a portion of the heavy chain and/or light chain is derived from one species, while the remainder of the heavy chain and/or light chain is derived from antibody or antigen-binding fragments from different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human species (such as a mouse).

As used in this application, "anti-CLDN18.2 antibody" or "antibody against CLDN18.2" means an antibody that can specifically bind to CLDN18.2 (e.g., human CLDN18.2 or non-human CLDN18.2) with sufficient affinity and can be used for, for example, diagnostic and/or therapeutic purposes.

As used in this application, 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.

As used in this application, the terms "specific binding" or "binding specificity" refer to a non-random binding reaction between two molecules, such as an antibody and an antigen.

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 amino acid (or nucleic acid) residues in a reference sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve maximum correspondence. Alignments for determining the percentage of amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., *Journal of Molecular Biology*, 215: 403-410 (1990); Stephen F. et al., *Nucleic Acids Res.*, 25: 3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D.G. et al., *Methods in Enzymology*, 266: 383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tools, or can customize the parameters suitable for alignment, for example, by selecting a suitable algorithm.

As used herein, the term "amino acid" refers to an organic compound containing amine ( _-NH₂ ) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. The names of amino acids are also represented in this disclosure using standard single-letter or three-letter codes, as summarized below.

"Conservative substitution" related to amino acid sequence refers to the replacement of amino acid residues with different amino acid residues having side chains with similar physicochemical properties. For example, conservative substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and 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 generally do not cause significant changes in protein conformation and structure, and therefore preserve the protein's biological activity.

"Separated" substances have been artificially altered from their natural state. If a "separated" composition or substance exists in nature, then the composition or substance has been altered from its original environment or removed from its original environment, or both. For example, polynucleotides or polypeptides naturally present in living organisms are not "separated," but if the same polynucleotide or polypeptide is sufficiently separated from its natural coexisting material, thus existing in a substantially pure state, then the polynucleotide or polypeptide is "separated." The terms "separated nucleic acid" and "polynucleotide" are used interchangeably and refer to the sequence of the separated nucleic acid molecule. In some embodiments, "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment with a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, or 99%, as determined by electrophoresis (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (such as ion exchange chromatography or reversed-phase HPLC).

The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, rodents (e.g., mice, rats, and guinea pigs), cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human. Unless otherwise indicated, the terms "patient," "subject," and "individual" are used interchangeably in this application.

As used herein, "effective function" refers to the biological activity resulting from the binding of the antibody's Fc region to its effectors, such as the C1 complex and Fc receptors. Exemplary effective functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody's Fc region to Fc receptors on effector cells; and antibody-dependent cell-mediated phagocytosis (ADCP), wherein nonspecific cytotoxic cells expressing FcγR recognize antibodies bound to target cells and subsequently induce phagocytosis of said target cells. Effective functions include functions that act after antigen binding and functions that act independently of antigen binding.

As used in this application, “treating” or “therapeutic treatment” for a symptom includes preventing or alleviating the symptom, slowing the onset or progression of the symptom, reducing the risk of developing the symptom, preventing or delaying the development of symptoms associated with the symptom, reducing or ending symptoms associated with the symptom, producing complete or partial remission of the symptom, curing the symptom, or a combination thereof.

"At risk" refers to subjects identified as having a higher probability of developing a disease, particularly cancer, compared to the general population; i.e., patients. Furthermore, subjects who have had or currently have a disease, particularly cancer, are subjects with an increased risk of developing the disease, and thus, such subjects may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of cancer metastasis. In the context of this invention, terms such as "protection" and "prevent/prophylactic" refer to preventing or treating, or preventing and treating the occurrence and/or spread of a disease in a subject, and in particular, to minimizing the subject's probability of developing the disease or delaying the development of the disease. For example, individuals at risk of developing cancer (as described above) would be candidates for cancer prevention therapies. Immunotherapy can be performed using any of a variety of techniques, wherein agents are used to remove antigen-expressing cells from the patient.

As described in this application, "standard treatment" includes administering standard treatments to patients. As used herein, "standard treatment" refers to a treatment method that includes medications or combinations of medications, radiation therapy (RT), surgery, or other medical interventions that are deemed appropriate, acceptable, and/or widely used by a practicing physician for a particular type of patient, disease, or clinical condition. Standard treatments used for different types of cancer are well-known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a consortium of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN), which provides detailed and up-to-date information on standard treatments for various cancers (see NCCN 2013).

The terms "effectiveness" and "efficacy" in treatment encompass both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to a drug's ability to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) produced at the cellular, organ, and/or organismal level by the administered drug.

A "therapeutic effective amount" or "therapeutic dose" of a drug or therapeutic agent (such as the antibody of this disclosure) is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or symptom, or promotes the resolution of a disease/symptom, demonstrated by a reduction in the severity of disease/symptom symptoms, an increase in the frequency and duration of disease/symptom-free phases, or prevention of impairment or disability caused by the disease/symptom. The ability of a therapeutic agent to promote disease resolution can be evaluated using a variety of methods known to a skilled practitioner, such as in human subjects during a clinical trial, in animal model systems where efficacy in humans can be predicted, or by analyzing the activity of the agent in an in vitro analysis. A therapeutically effective amount of a drug includes a "preventatively effective amount," which is any amount of drug that, when administered alone or in combination with an anti-proliferative agent to a subject at risk of developing a CLDN18.2-related disease or symptom (such as cancer) (e.g., a subject with precancerous lesions) or at risk of disease/symptom recurrence, inhibits the development or recurrence of a disease/symptom. In a preferred embodiment, the preventive effective amount completely prevents the development or recurrence of CLDN18.2-related diseases or conditions (e.g., cancer). "Inhibit" the development or recurrence of a disease/condition (e.g., cancer) means reducing the likelihood of the disease/condition developing or recurring, or completely preventing the development or recurrence of the disease/condition.

As used herein, the term "vector" refers to a carrier into which a genetic element can be operatively inserted to achieve expression of the genetic element, thereby producing a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element.

As used in this application, "host cell" means a cell in which exogenous polynucleotides and/or vectors have been introduced.

The term "CLDN18" refers to claudin 18 and any splicing variants containing CLDN18, such as CLDN18.1 and CLDN18.2. CLDN18.1 and CLDN18.2 differ in the N-terminal portion containing the first transmembrane (TM) region and loop 1, while the major protein sequence at the C-terminus is the same.

The term "CLDN18.2" refers to a Claudin-18 splice variant 2 derived from mammals (such as primates, e.g., humans, monkeys) and rodents (e.g., mice). In some embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include the human CLDN18.2 protein (NCBI reference sequence number NP_001002026.1 or SEQ ID NO: 20). Exemplary sequences of CLDN18.2 include the mouse (mice) CLDN18.2 protein (NCBI reference sequence number NP_001181852.1) and the cynomolgus macaque (Macaca fascicularis/crab-eatingmacaque) CLDN18.2 protein (NCBI reference sequence number XP_015300615.1).

The term "CLDN18.1" refers to a Claudin-18 splice variant 1 derived from mammals (such as primates (e.g., humans, monkeys) and rodents (e.g., mice)). In some embodiments, CLDN18.1 is human CLDN18.1. Exemplary sequences of human CLDN18.1 include the human CLDN18.1 protein (NCBI reference sequence number NP_057453.1 or SEQ ID NO: 21), the mouse (mice) CLDN18.2 protein (NCBI reference sequence number NP_001181851.1), and the Macaca fascicularis/crab-eating macaque CLDN18.2 protein (NCBI reference sequence number XP_005545920.1).

The terms “CLDN18.2” and “CLDN18.2” also cover variants of the exemplary sequence, such as mutants, conformational variants, isomers, allelic variants, species variants, and species homologs, especially those that are naturally occurring.

As used herein, “CLDN18.2-related disease or symptom” means any disease or symptom caused, aggravated, or otherwise associated with increased or decreased expression or activity of CLDN18.2. In some embodiments, a CLDN18.2-related symptom is, for example, cancer.

As used in this application, “cancer” means any medical condition characterized by malignant cell growth or tumors, abnormal proliferation, invasion or metastasis, and includes solid tumors and non-solid cancers (e.g., hematologic malignancies such as leukemia).

As used in this application, "solid tumor" refers to a solid mass of neoplasms and/or malignant cells.

The term "pharmaceutically acceptable" indicates that the carrier, mediator, diluent, excipient, and/or salt are generally chemically and/or physically compatible with the other components constituting the formulation and physiologically compatible with its recipients.

As used herein, the term "metastasis" or "metastatic cancer" refers to the spread of cancer cells from their original site to another part of the body. Metastasis formation is a highly complex process that depends on malignant cells detaching from the primary tumor, invading the extracellular matrix, penetrating the endothelial basement membrane into body cavities and blood vessels, and subsequently infiltrating the target organ after being transported by the bloodstream. Finally, the growth of the new tumor at the target site (i.e., secondary or metastatic tumor) depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components can remain and generate metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis" associated with metastasis distant from the primary tumor and the local lymph node system. The cells of a secondary or metastatic tumor are identical to those in the original tumor. This means, for example, if gastric cancer metastasizes to the liver, the secondary tumor is composed of abnormal gastric cells, not abnormal hepatocytes. Therefore, a tumor in the liver is referred to as metastatic gastric cancer, not liver cancer.

As mentioned in this application, the term "about" includes (and describes) the implementation of that value or parameter itself. For example, a description referring to "about X" includes a description of "X". A numerical range includes the numerical value that defines the range. Generally, the term "about" refers to the indicated value of a variable and all values within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value (whichever is greater). When the term "about" is used in the context of a time period (year, month, week, day, etc.), the term "about" means the time period plus or minus a quantity of the next next lower time period (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days, etc.), or within 10% of the indicated value, whichever is greater.

Anti-CLDN18.2 antibody

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

CLDN18.2 is a splice variant of sealing protein-18 (CLDN18). CLDN18 is a member of the family of four transmembrane proteins and has four hydrophobic regions. CLDN18 exists in several different conformations, which can be selectively targeted by antibodies (see Sahin U et al., Clinical Cancer Research, 2008, 14(23): 7624-7634). As described for most members of the CLDN family, CLDN18-conformation-1 has all four hydrophobic regions that act as transmembrane domains (TM) and has two extracellular loops (loop 1 is surrounded by hydrophobic regions 1 and 2; loop 2 is surrounded by hydrophobic regions 3 and 4). The second conformation (CLDN18-conformation-2) shows that, as described for PMP22, the second and third hydrophobic regions do not completely penetrate the plasma membrane, making the portion between the first and fourth transmembrane domains (loop D3) extracellular. The third conformation (CLDN18 conformation-3) shows a large extracellular domain with two internal hydrophobic regions surrounded by a first and a fourth hydrophobic region. Due to the classical N-glycosylation site in ring D3, the CLDN-18 topological variants CLDN18 topology-2 and CLDN18 topology-3 have additional extracellular N-glycosylation sites.

CLDN18 has two distinct splicing variants, present in both mice and humans. The splicing variants CLDN18.1 and CLDN18.2 differ in the first 21 amino acids at the N-terminus containing the first TM and loop 1, while their C-terminal protein sequences are identical. Despite a 92% amino acid sequence identity between the two isomers, their expression patterns do not overlap; CLDN18.1 is primarily expressed in normal lung tissue, while CLDN18.2 is primarily expressed in normal stomach 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.

The amino acid sequence of human sealing protein 18.2 (accession number: NP_001002026.1) (SEQ ID NO: 20):

The amino acid sequence of human sealing protein 18.1 (accession number: NP_057453.1) (SEQ ID NO: 21):

In normal tissues, CLDN18.2 expression is restricted to the basement membrane of mucosal cells and is unavailable to therapeutic antibodies. In pathological conditions (such as tumor cells), the polarity of gastric mucosal cells is disrupted and CLDN18.2 is exposed on the cell surface. CLDN18.2 protein is a pan-cancer target expressed in primary lesions and metastases of several types of human cancer, including tumors of the stomach, esophagus, pancreas, and lung, as well as human cancer cell lines (see Sahin Ugur et al., Clinical Cancer Research, 2008; 14(23); Matsuda Y et al., Cancer Science, 2007, 98(7): 1014-1019). Multiple studies have reported aberrant ectopic expression of CLDN18.2 in pancreatic cancer, ovarian cancer, cholangiocarcinoma, and lung adenocarcinoma (see, for example, Karanjawala ZE et al., *American Journal of Surgical Pathology*, February 2008; 32(2): 188-96; Micke P et al., *International Journal of Cancer*, November 1, 2014; 135(9): 2206-14; Keira Y et al., *Virchows Arch.*, March 2015; 466(3): 265-77; Coati et al., *British Journal of Cancer*). Cancer.) July 2019; 121(3): 257-263; Dottermusch et al., European Journal of Pathology November 2019; 475(5): 563-571; Rohde et al., Japanese Journal of Clinical Oncology (Jpn J Clin Oncol.) September 1, 2019; 49(9): 870-876; Woll et al., International Journal of Cancer February 1, 2014; 134(3): 731-9; Espinoza et al., Histopathology March 2019; 74(4): 597-607; Shinozaki et al., European Journal of Pathology July 2011; 459(1): 73-80).

In one aspect, this disclosure provides a monoclonal anti-CLDN18.2 antibody and its antigen-binding fragment. The monoclonal anti-CLDN18.2 antibody and antigen-binding fragment provided in this application are capable of specifically binding to CLDN18.2 (e.g., human CLDN18.2), fragments of CLDN18.2, or fusion peptides containing fragments of CLDN18.2. In some embodiments, the CLDN18.2 fragment contains a first extracellular loop of human CLDN18.2, or a sequence within the first extracellular loop of human CLDN18.2. In some embodiments, the human CLDN18.2 fragment contains the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 19. In some embodiments, the fusion peptide contains additional amino acid residues linked to the N-terminus and/or C-terminus of the CLDN18.2 fragment. In some embodiments, the CLDN18.2 fragment contained in the fusion peptide forms a loop.

The anti-CLDN18.2 antibody and antigen-binding fragment provided in this application are capable of specifically binding to cells expressing CLDN18.2. In some embodiments, the cells expressing CLDN18.2 are pretreated cells. As used with respect to cells in this application, the term "pretreated" means that the cells have been treated to denature their surface proteins (such as CLDN18.2) or otherwise prevent them from exhibiting their native conformation. For example, cells expressing CLDN18.2 may be pretreated with one or more chemicals (e.g., formalin, paraffin, or acetone) or by physical intervention (e.g., freezing or heating). In some embodiments, the cells expressing CLDN18.2 are formalin-fixed paraffin-embedded (FFPE) cells.

The binding of the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application to the antigen can be expressed as a "half-maximal effect concentration" ( _EC50 ) value, which refers to the concentration of the antibody at which 50% of its maximum effect (e.g., binding) is observed. The _EC50 value can be measured by methods known in the art, such as sandwich analyses like ELISA, Western blotting, and other binding assays. _EC50 can be measured in appropriate binding assays, wherein the binding of serially diluted antibody to the antigen is detected, and the concentration at which 50% maximum binding is determined. In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application specifically binds to human CLDN18.2, fragments of human CLDN18.2, fusion peptides containing fragments of human CLDN18.2, cells expressing human CLDN18.2, or pretreated cells expressing human CLDN18.2. In some embodiments, as measured by ELISA, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application specifically binds to the fusion polypeptide containing the first extracellular loop of human CLDN18.2 at an EC50 of no more than 50 ng/ml (e.g., no more than 40 ng/ml, no more than 35 ng/ml, no more than 30 ng/ml, no more than 20 ng/ml, no more than 18 ng/ml, no more than 16 ng/ml, no more than 15 ng/ml, no more than 14 ng/ml, no more than 13 ng/ml, no more than 12 ng/ml).

The binding affinity of the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application to human CLDN18.2 can also be characterized, for example, by _{KD ,} which is the ratio of dissociation rate to association rate ( _koff / _kon ). _KD can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance (SPR), microthermophoresis, and HPLC-MS. In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application binds to the fusion polypeptide containing the first extracellular ring of human CLDN18.2 with a _KD not exceeding ^10⁻⁶ M (e.g., not exceeding ^10⁻⁷ M, ^10⁻⁷.5 M, ^10⁻⁸ M, or ^10⁻⁸.5 M) as measured by SPR. The lower the _KD value, the higher the affinity.

In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment do not cross-react with human CLDN18.1. An anti-CLDN18.2 antibody that is "not cross-reactive with human CLDN18.1" or "does not have cross-reactivity with human CLDN18.1" is an antibody that does not produce undesirable results, such as false positives, in assays targeting human CLDN18.2 (e.g., IHC assays). In some embodiments, binding to human CLDN18.1 is undetectable in assays (e.g., IHC assays or flow cytometry). The binding level can be determined as the amount of antigen-antibody complex formed at a given concentration of antibody and a given concentration of antigen. In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application bind to human CLDN18.1 at a level or affinity lower than (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) that antibody GC182 binds to human CLDN18.1.

In some embodiments, no more than 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.3%, or 0.1% of cells expressing human CLDN18.1 were detected as positive for the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application in IHC analysis. In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application did not show detectable binding to human CLDN18.1 in IHC analysis. In some embodiments, in non-cancer human lung tissue samples, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application did not show detectable binding to human CLDN18.1 in IHC analysis.

In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application do not exhibit cross-reactivity with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples, as measured by ELISA at an antibody concentration of 1 nM or by IHC at an antibody concentration of 0.5 μg/ml. IHC can be performed according to the procedures and conditions outlined in Examples 8 or 9.

In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application do not exhibit cross-reactivity with non-cancerous tissues, except for gastric epithelial cells. In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application do not exhibit cross-reactivity with non-cancerous tissues (such as lung tissue, intestinal tissue, kidney tissue, tonsil tissue, thyroid tissue, skeletal muscle tissue, or breast tissue). This distinguishes the antibody provided in this application from existing monoclonal anti-CLDN18.2 antibodies. For example, antibody 43-14A shows binding to human CLDN18.1 and therefore cross-reactivity with non-cancerous lung tissue, see the IHC results in the Ventana specification (see Ventana CLDN18 (43-14A) analysis, reference 790-7027, 08504148001). As another example, antibody GC182 has also been found to cross-react with CLDN18.1 and therefore stain non-cancerous lung tissue (see Example 4 and Figure 2 of this disclosure). For another example, the anti-CLDN18.2 antibody EPR19202 (available from Abcam, product name ab222512) shows nonspecific binding to skeletal muscle tissue and therefore exhibits cross-reactivity with non-cancerous skeletal muscle tissue.

Exemplary anti-CLDN18.2 antibody

In some embodiments, this disclosure provides isolated antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 (e.g., human CLDN18.2), comprising:

a) Heavy chain CDR1 containing the amino acid sequence X ₁ X ₂ Y X ₃ H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX ₄ GX ₅ X ₆ X ₇ X ₈ YX ₉ EKFKG (SEQ ID NO: 12), and/or heavy chain CDR3 containing the amino acid sequence NYX ₁₀ STFGY (SEQ ID NO: 24); and/or

b) Light chain CDR1 containing the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2), light chain CDR2 containing the amino acid sequence KX ₁₁ SNRFS (SEQ ID NO: 25), and/or light chain CDR3 containing the amino acid sequence FQGSHVPFT (SEQ ID NO: 6);

Where _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, _X10 is G or R and _X11 is V or I.

In some embodiments, this disclosure provides isolated antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 (e.g., human CLDN18.2), comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs selected from the sequence set consisting of SEQ ID NO: 1-6 or the sequence set consisting of SEQ ID NO: 2, 6, 7, and 9-11.

In some embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application comprises the heavy chain CDR3 sequence of SEQ ID NO: 5 or 11. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore considered to be in the most contact with the antigen and to provide the most free energy for the antibody-antigen affinity. In addition, based on a variety of diverse mechanisms, the heavy chain CDR3 is considered to be the most diverse CDR for antigen-binding sites to date in terms of length, amino acid composition and conformation (Tonegawa S. Nature 302: 575-81 (1983)). The diversity of the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity 13: 37-45 (2000)) and the desired antigen-binding affinity (Schier R et al. Journal of Molecular Biology 263: 551-67 (1996)).

In some embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application comprises: heavy chain CDR1, which comprises an amino acid sequence selected from the following: SEQ ID NO: 1 and SEQ ID NO: 7, and/or heavy chain CDR2, which comprises an amino acid sequence selected from the following: SEQ ID NO: 3 and SEQ ID NO: 9, and/or heavy chain CDR3, which comprises an amino acid sequence selected from the following: SEQ ID NO: 5 and SEQ ID NO: 11; and/or light chain CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 2, and/or light chain CDR2, which comprises an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 10, and/or light chain CDR3, which comprises an amino acid sequence selected from SEQ ID NO: 6.

On the other hand, this disclosure provides a separated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (e.g., human CLDN18.2), comprising:

a) Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 3, and heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 5; or

b) Heavy chain CDR1, which contains SEQ ID NO: 7, heavy chain CDR2, which contains SEQ ID NO: 9, and heavy chain CDR3, which contains SEQ ID NO: 11.

In some embodiments, the antibody or its antigen-binding fragment provided in this application comprises:

a) light chain CDR1, comprising the amino acid sequence of SEQ ID NO: 2; light chain CDR2, comprising the amino acid sequence of SEQ ID NO: 4; and light chain CDR3, comprising the amino acid sequence of SEQ ID NO: 6; or

b) Light chain CDR1, which contains the amino acid sequence of SEQ ID NO: 2, light chain CDR2, which contains the amino acid sequence of SEQ ID NO: 10, and light chain CDR3, which contains the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the antibody or its antigen-binding fragment provided in this application comprises:

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.

It is known that CDRs are responsible for antigen binding; however, it has been found that not all six CDRs are necessarily essential or immutable. In other words, it is possible to substitute, alter, or modify one, two, or three of the CDRs provided above (e.g., corresponding to any one of SEQ ID NO: 1-6 or SEQ ID NO: 2, 6, 7, and 9-11) while substantially maintaining specific binding affinity to CLDN18.2. Antibodies having the modified or variant CDRs described above are also covered in this disclosure.

In some embodiments, the antibody or its antigen-binding fragment provided in this application comprises:

a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; or

b) The heavy chain variable region, which contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region, which contains the amino acid sequence of SEQ ID NO: 16.

As used in this application, 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.

As used in this application, antibody "69H2" 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.

Table 1 shows the CDR sequences of anti-CLDN18.2 antibodies 14G11 and 69H2. Heavy and light chain variable region sequences are also provided in Table 2 below.

Table 1. Sequence of the CDR region of the CLDN18.2 antibody

Table 2. Sequence of VH/VL regions of mouse/recombinant antibody

In some embodiments, the antibodies and antigen-binding fragments provided in this application contain suitable frame region (FR) sequences, provided that 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 they can be grafted with any suitable FR sequence from any suitable species (especially mice, humans, rats, and rabbits) using suitable methods known in the art (such as recombinant techniques). FR sequences can be readily identified by those skilled in the art based on the CDR sequences in Table 1 above and the variable region sequences in Table 2 above, as it is well known in the art that the CDR region in the variable region is flanked by two FR regions.

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application 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 some embodiments, the heavy chain constant region comprises an Fc region. In some embodiments, the light chain constant region comprises Cκ or Cλ.

In some embodiments, the anti-CLDN18.2 antibody and its fragment provided in this application further comprise constant regions of mouse IgG1, IgG2, IgG3, or IgG4. In some embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application comprise constant regions of mouse IgG1 isotypes. In some embodiments, the heavy chain constant region of mouse IgG1 comprises SEQ ID NO: 17 or a homologous sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and/or the light chain constant region of mouse IgG1 comprises the amino acid sequence of SEQ ID NO: 18 or a homologous sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

The anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application may be a monoclonal antibody, bispecific antibody, multispecific antibody, recombinant antibody, chimeric antibody, humanized antibody, labeled antibody, bivalent antibody, anti-idiotype antibody, fusion protein, dimerizing antibody, or multimerizing antibody, or a modified antibody (e.g., glycosylated antibody). Recombinant antibodies are antibodies prepared in vitro rather than in animals using recombinant methods.

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application is bivalent, tetravalent, hexavalent, or multivalent. Any molecule with a valence greater than bivalent is considered multivalent, encompassing, for example, trivalent, tetravalent, hexavalent, etc.

In some embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application comprises all or a portion of the heavy chain variable domains and/or all or a portion of the light chain variable domains. In one embodiment, the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application is a single-domain antibody composed of all or a portion of the heavy chain variable domains provided in this application. More information about such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

Antibody variants

The anti-CLDN18.2 antibody and its antigen-binding fragment provided in this application also cover various types of antibody 14G11 and 69H2 variants.

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application contains one or more mutations in one or more CDR sequences provided in Table 1 above, one or more mutations in one or more non-CDR sequences in the heavy chain variable region or light chain variable region provided in Table 2 above, and/or one or more mutations in the constant region (e.g., Fc region) sequence as shown in SEQ ID NO: 17 or 18, but still maintains binding specificity for CLDN18.2, specifically, binding specificity for human CLDN18.2, or more precisely, binding specificity for epitopes within the amino acid sequence of SEQ ID NO: 19. These are also referred to as variants of antibody 14G11 and 69H2 or variants of antigen-binding fragments. In some embodiments, the variants maintain binding affinity at levels similar to or even higher than their parent antibodies (e.g., antibody 14G11 or 69H2). As used in this application, “mutation” includes substitution, insertion, and/or deletion in an amino acid sequence or polynucleotide sequence. In some embodiments, at least one (or all) mutations contain conserved substitutions.

In some embodiments, the antibody variant contains a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR sequence, FR sequence, or variable region sequence of antibodies 14G11 and 69H2. In some embodiments, the antibody variant contains 1, 2, or 3 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 the sequences listed in Table 1, and the antibody or its antigen-binding fragment optionally has a binding affinity similar to or even higher than that of its parent antibody (e.g., antibody 14G11 or 69H2) while maintaining binding specificity with CLDN18.2.

In some embodiments, the antibody variant comprises 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 the sequence of SEQ ID NO: 13 or 15, and/or a light 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 the sequence of SEQ ID NO: 14 or 16, and the antibody or its antigen-binding fragment optionally has a binding affinity similar to or even higher than that of its parent antibody (e.g., antibody 14G11 or 69H2) while maintaining binding specificity with CLDN18.2. In some embodiments, a total of 1-10 amino acid residues have been mutated in the sequence selected from SEQ ID NO: 13-16. In some implementations, the mutation occurs in a region outside the CDR (i.e., in the FR). In some implementations, one or more mutations are conserved substitutions. In some implementations, all mutations are conserved substitutions.

In some embodiments, this disclosure provides variants of antibody 14G11 or 69H2, wherein the variants comprise:

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% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 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) The light chain CDR3 (LCDR3) sequence, which has 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: 6, and

The variant, while maintaining binding specificity to CLDN18.2, optionally has binding affinity similar to or even higher than that of its parent antibody (e.g., antibody 14G11 or 69H2).

In some embodiments, the antibody variants provided in this application comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3, wherein HCDR1 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 1 or SEQ ID NO: 7; HCDR2 has no more than 6, 5, 4, 3, 2, or 1 amino acid mutation in SEQ ID NO: 3 or SEQ ID NO: 9; HCDR3 has no more than 6, 5, 4, 3, 2, or 1 amino acid mutation in SEQ ID NO: 5 or SEQ ID NO: 11; LCDR1 has no more than 2 or 1 amino acid mutation in SEQ ID NO: 2; LCDR2 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 4 or SEQ ID NO: 10; and LCDR3 has no more than 3, 2, or 1 amino acid mutation in SEQ ID NO: 7. NO: 6 contains no more than 3, 2, or 1 amino acid mutations, and the antibody variant optionally has a binding affinity similar to or even higher than that of its parent antibody (e.g., antibody 14G11 or 69H2) while maintaining binding specificity to CLDN18.2. In some embodiments, one or more mutations are conserved substitutions. In some embodiments, all mutations are conserved substitutions.

In some embodiments, the antibody variants provided in this application retain the CLDN18.2 specific binding specificity of their parent antibody, but have one or more desired properties conferred by one or more mutations. For example, the antibody variants may have increased antigen-binding affinity, improved glycosylation patterns, reduced glycosylation risk, reduced deamination, reduced or depleted one or more effector functions, pH-dependent improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or conjugation compatibility (e.g., one or more introduced cysteine residues). These variants are also referred to as affinity variants, glycosylation variants, cysteine variants, Fc variants, etc., and are described in more detail below.

a) Affinity variant

Affinity variants may contain modifications or substitutions in one or more CDR sequences as provided in Table 1 above, one or more frame (FR) sequences provided in this application, or heavy or light chain variable region sequences provided in Table 2 above.

Affinity variants maintain the CLDN18.2-specific binding affinity of the parent antibody, or even possess an enhanced CLDN18.2-specific binding affinity superior to that of the parent antibody. Various methods known in the art can be used to achieve this. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology, followed by screening for binding affinity against human CLDN18.2. As another example, computer software can be used to virtually simulate the binding of antibodies to human CLDN18.2 and identify amino acid residues on the antibody that form the binding interface. These residues can be avoided in mutations to prevent decreased binding affinity, or targeted mutations can be employed to provide stronger binding.

b) Glycosylation variants

The anti-CLDN18.2 antibody and antigen-binding fragment provided in this application also cover glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or its antigen-binding fragment.

Antibodies or their antigen-binding fragments may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue whose side chain can be linked to a carbohydrate moiety (e.g., an oligosaccharide structure). Antibody glycosylation is typically N-linked or O-linked. N-linked glycosylation refers to the linking of the carbohydrate moiety to an asparagine residue (e.g., in tripeptide sequences such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline) as a side chain. O-linked glycosylation refers to the linking of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence such that one of the tripeptide sequences described above (for N-linked glycosylation sites) or a serine or threonine residue present in the sequence (for O-linked glycosylation sites) is substituted. New glycosylation sites can be generated by introducing the tripeptide sequence or serine or threonine residues in a similar manner.

In some embodiments, the anti-CLDN18.2 antibody and antigen-binding fragment provided in this application contain a mutation in N297 (e.g., N297A, N297Q, or N297G) to remove the glycosylation site.

c) Cysteine-engineered variants

The anti-CLDN18.2 antibody and antigen-binding fragment provided in this application also cover cysteine-engineered variants containing one or more introduced free cysteine amino acid residues.

Free cysteine residues are cysteine residues that are not part of a disulfide bridge. Cysteine-engineered variants are suitable for conjugating, for example, a cytotoxic and/or imaging compound, label, or radioisotope at the engineered cysteine site via a maleic hydride or a halogenated acetyl group. Methods for engineering antibodies or their antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.

d) Fc variant

The anti-CLDN18.2 antibody and antigen-binding fragments provided in this application also cover Fc variants which contain one or more amino acid residue modifications or substitutions at their Fc region and/or hinge region, for example to provide altered effector functions such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). 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., Experimental & Molecular Medicine (Exp&Mol., Med.) (2019) 51: 138, the full text of which is incorporated herein by reference.

antigen-binding fragments

This application also provides an anti-CLDN18.2 antigen-binding fragment. In some embodiments, the antibody and antigen-binding fragment provided in this application contain 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 generated based on the anti-CLDN18.2 antibody provided in this application, which includes, for example, exemplary antibodies with CDR sequences as shown in Table 1 and their various variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.).

In some embodiments, the anti-CLDN18.2 antigen-binding fragment provided in this application is a bifunctional antibody, Fab, Fab′, F(ab′) ₂ , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv′), disulfide-stabilized bifunctional antibody (ds bifunctional antibody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bispecific antibody, multispecific antibody, camelified single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

Various techniques can be used to generate the said antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)), recombinant expression from host cells (such as E. coli) (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as described above (e.g., for ScFv), and chemically coupling two Fab′-SH fragments to form the F(ab′) ₂ fragment (Carter et al., Bio/Technology 10: 163-167 (1992)). Other techniques for generating antibody fragments will be readily apparent to those skilled in the art.

Epitope

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application binds to an epitope within the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19).

As used herein, the term "epitope" refers to a specific set of atoms or amino acids on an antigen to which an antibody binds. An epitope can be a conformational epitope or a linear epitope. In some embodiments of this disclosure, the epitope bound by the anti-CLDN18.2 antibody provided herein is linear. Those skilled in the art will recognize that it is possible, without excessive experimentation, to determine whether an antibody binds to the same, overlapping, or adjacent epitopes as the disclosed antibody (e.g., hybridoma/mouse antibodies 14G11 and 69H2).

This disclosure provides a monoclonal antibody targeting an epitope located in the N-terminal portion of CLDN18.2, suitable for detecting and identifying cells expressing CLDN18.2 without cross-reactivity with CLDN18.1. There are eight distinct amino acids between amino acids 28-70 (i.e., the N-terminal portion containing the first transmembrane (TM) region and loop 1) in the sequences of human CLDN18.1 and CLDN18.2, while the C-terminal sequences of human CLDN18.1 and CLDN18.2 are identical. The linear epitope located in the N-terminus of human CLDN18.2 (amino acids 28-37 in 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) Dec 1, 2008). However, to date, no monoclonal antibodies targeting the peptide fragment have been reported, and to the best of the inventors’ knowledge, this disclosure is the first to provide a monoclonal antibody that binds to the peptide fragment of SEQ ID NO: 19.

On the other hand, this disclosure provides monoclonal antibodies or antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof provided in this application (such as 14G11 and 69H2) for binding to CLDN18.2.

As used herein, the term "competitive binding" refers to two antigen-binding proteins (e.g., antibodies), meaning that one antigen-binding protein blocks or reduces the binding of another protein to an antigen (e.g., human CLDN18.2) to any detectable extent, as determined by a competitive binding assay. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIA), direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, the assay involves using purified antigen or cells carrying antigen bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of labeling that binds to the solid surface or cells in the presence of the test antibody. Typically, an excess of the test antibody is present. If two antibodies compete to bind to CLDN18.2, then both antibodies bind to the same or overlapping epitopes, or to adjacent epitopes that are close enough to the epitope to which the other antibody binds to to cause steric hindrance. Typically, when an excess of the competitive antibody is present, it will 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%, 85%-90%, or more.

Polynucleotide and Recombinant Methods

This disclosure provides isolated polynucleotides encoding anti-CLDN18.2 antibodies and their antigen-binding fragments. As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless otherwise stated, a particular polynucleotide sequence also implicitly encompasses its conserved modification variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or deoxyinosine residue (see Batzer et al., Nucleic Acid Research 19:5081 (1991); Ohtsuka et al., Journal of Biochemistry 260:2605-2608 (1985); and Rossolini et al., Molecular Cell Probes 8:91-98 (1994)).

DNA encoding monoclonal antibodies can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody. The encoding DNA can also be obtained through synthetic methods.

This disclosure provides vectors (e.g., expression vectors) containing the isolated polynucleotides provided in this application. The vectors can be used to transform, transduce, or transfect host cells to express the genetic elements they carry within the host cells. Examples of vectors include plasmids, phagemids, granules, and artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), bacteriophages (such as λ phage or M13 phage), and animal viruses. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, optional elements, and reporter genes. Additionally, the vector may contain an origin of replication. The vector may also contain materials that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coatings. The vector can be an expression vector or a cloning vector.

In some embodiments, the vector provided in this application is an expression vector. In some embodiments, the expression vector provided in this application comprises a polynucleotide encoding the antibody or its antigen-binding fragment provided in this application, at least one promoter operatively linked to the polynucleotide sequence (e.g., SV40, CMV, EF-1α), and at least one select marker.

Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40), λ phages and M13 phages, and plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, and pFU. SE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2 , pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

Vectors containing polynucleotide sequences encoding antibodies or their antigen-binding fragments can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors of this application are prokaryotic, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli); Enterobacter; Erwinia; Klebsiella; Proteus; and Salmonella (e.g., Salmonella typhimurium). typhimurium); Serratia, such as Serratia marcescans; and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeasts) are suitable cloning or expression hosts for anti-CLDN18.2 antibody encoding vectors. Saccharomyces cerevisiae or common baking yeasts are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and applicable to this application, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermogenlerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP 402,226); and *Pichia pastoris* (EP 402,226). 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as hosts of Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

Suitable host cells for expressing the glycosylated antibodies or antigen fragments provided in this application are derived from multicellular organisms, such as invertebrate cells, including plant and insect cells. Various baculovirus strains and variants, and corresponding permissible insect host cells from the following hosts have been identified: fall armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Several publicly available viral strains for transfection are available, such as the L-1 variant of the alfalfa silver-striped armyworm (Autographa californica) NPV and the Bm-5 virus strain of the silkworm NPV, and these viruses can be used in accordance with the present invention, particularly for transfecting fall armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, the greatest interest lies in vertebrate cells, and the culture and propagation of vertebrate cells (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines include: monkey kidney CV1 line (COS-7, ATCCCRL 1651) transformed with SV40; human embryonic kidney line (subcloned to grow 293 or 293 cells in suspension culture; Graham et al., *Journal of General Virology* 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., *Proceedings of the National Academy of Sciences* 77:4216 (1980)); mouse supporting cells (TM4, Mather, *Biol. Reprod.* 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC CCL 10). 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals of the New York Academy of Sciences 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NSO, 293, and their derivatives.

The host cells are transformed with the expression or cloning vector described above for generating anti-CLDN18.2 antibodies and cultured in a modified conventional nutrient medium to induce promoters, select transformants, or amplify genes encoding desired sequences. In another embodiment, the antibody can be generated via homologous recombination known in the art.

Host cells used to produce the antibodies or antigen-binding fragments provided in this application can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma), are suitable for culturing host cells. In addition, any of the culture media described in Ham et al., Enzymatic Methods 58:44 (1979); Barnes et al., Analytical Biochemistry 102:255 (1980); U.S. Patent Nos. 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 Re. 30,985 can be used as a culture medium for host cells. Any of these culture media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN ^™ drug), trace elements (defined as inorganic compounds typically present in micromolar concentrations), and glucose or an equivalent energy source. Any other necessary supplements may also be included in appropriate concentrations known to a person skilled in the art. Culture conditions, such as temperature and pH, are those already used with the host cells selected for expression and will be obvious to a person skilled in the art.

When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If antibodies are produced intracellularly, then as a first step, microparticle debris (host cell fragments or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Biotechnology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of *E. coli*. In simple terms, the cell paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and benzyl sulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. In the case of antibody secretion into the culture medium, the supernatant from the expression system is generally first concentrated using a commercially available protein concentrator filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in either of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

Anti-CLDN18.2 antibodies and their antigen-binding fragments prepared from cells can be purified using, for example, hydroxyapatite 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.

In some implementations, 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., *Journal of Immunological Methods* 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., *European Journal of Molecular Biology (EMBO J.)* 5: 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices can also be used. Mechanically stable matrices, such as controlled-pore glass or poly(divinyl styrene)benzene, can achieve faster flow rates and shorter processing times than agarose. When the antibody contains a CH3 domain, Bakerbond ABX ^™ resin (JTBaker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification, such as ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE ^™ chromatography, anion or cation exchange resin (such as polyaspartic acid column) chromatography, chromatographic polymerization, SDS-PAGE, and ammonium sulfate precipitation, may also be available depending on the antibody to be recovered.

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

This disclosure provides a method for expressing the anti-CLDN18.2 antibody or its antigen-binding fragment provided in this application, which includes culturing the host cells provided in this application under conditions expressing the vector provided in this application.

Conjugate

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment is linked to or conjugated to one or more moieties. Examples of said moieties include, but are not limited to, therapeutic agents (e.g., DNA alkylating agents, topoisomerase inhibitors, tubulin binders, or other anticancer drugs), detectable markers, pharmacokinetic modified moieties (e.g., polymers with extended half-life, such as PEG), or purified moieties (e.g., magnetic beads or nanoparticles).

The linking of a portion to an antibody or its antigen-binding fragment can be direct, or via a linker, or via another portion, such as through covalent binding, affinity binding, insertion, coordination binding, complexation, association, blending, or addition, and other methods. In some embodiments, the antibody or its antigen-binding fragment is linked to one or more portions via a linker. In some embodiments, the linker is an hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

In some embodiments, the antibody or antigen-binding fragment provided in this application may be engineered to contain specific sites, in addition to the epitope-binding portion, that can be used for linkage with one or more portions. For example, the site may contain one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent bonding with the portion.

In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment is linked to one or more portions, said portions being used to (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, such as fluorescence resonance energy transfer (FRET); (iii) influence mobility by charge, hydrophobicity, shape or other physical parameters, such as electrophoretic mobility; or (iv) provide a capture portion, such as affinity, antibody/antigen or ion complex.

The term "parts" includes, but is not limited to, labels or portions that can be directly detected by imaging, enzyme reactions, etc. (such as radioactive isotopes, lanthanides, chemiluminescent labels, chromogenic fractions, enzyme labels, colloidal gold particles, and fluorescent labels), as well as portions that can be indirectly detected, for example, through molecular interactions. Examples of indirectly detectable labels include biotin/avidin, biotin/streptococcal, digoxigenin labels, haptens, DNA molecules and particle labels for detection (paramagnetic particles, metal particle labels, magnetic particle labels, and polymeric particle labels, etc.).

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

Fluorescent or luminescent labels include, but are not limited to, rare earth chelating agents (europium chelating agents), fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, fluorescein, dansyl, umbelliferone, fluorescein, luminol labeling, isoluminol labeling, aromatic acridine ester labeling, imidazole labeling, acridine onion salt labeling, oxalate labeling, jellyfish luminescent protein labeling, 2,3-dihydrophthalazinedione, Texas Red, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores (such as SPECTRUM and SPECTRUM) and/or derivatives of any one or more of the above. For example, fluorescent labels can be conjugated to antibodies using techniques disclosed, for example, in the aforementioned "Recent Immunology Protocols". Fluorescence can be quantified using a fluorometer.

Another type of useful label is the enzyme-substrate label. A variety of enzyme-substrate labels are available (see U.S. Patent No. 4,275,149). Enzymes generally catalyze chemical changes in chromogenic substrates that can be measured using various techniques. For example, an enzyme can catalyze a color change in a substrate that can be measured spectrophotometrically. Alternatively, an enzyme can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying fluorescence changes have been described above. Chemiluminescent substrates become electronically excited through a chemical reaction, subsequently emitting light that can be measured (e.g., using a chemiluminometer) or that supplies energy to a fluorescent acceptor.

Those skilled in the art can use many enzyme-substrate combinations (see U.S. Patents 4,275,149 and 4,318,980), such as: (i) horseradish peroxidase (HRP) having catalase as a substrate, wherein the catalase oxidizes dye precursors, such as 3,3′-diaminobenzidine (DAB), which produces a brown final product; 3-amino-9-ethylcarbazole (AEC), which forms a rose-red final product upon oxidation; 4-chloro-1-naphthol (CN), which precipitates as a blue final product; and p-phenylenediamine dihydrochloride/catechol, which produces a blue-black product; o-phenylenediamine (OPD) and 3,3′,5,5-diaminobenzidine (DDP). (ii) alkaline phosphatase (AP) and p-nitrophenyl phosphate, naphthol AS-MX phosphate, Solid Red TR and Solid Blue BB, naphthol AS-BI phosphate, naphthol AS-TR phosphate, 5-bromo-4-chloro-3-indophenol phosphate (BCIP), Solid Red LB, Solid Garnet Red GBC, Nitroblue Tetrazol (NBT) and Iodonitrotetrazol Violet (INT); and (iii) β-D-galactosidase (β-D-Gal) having a chromogenic substrate (e.g., p-nitrophenyl-P-D-galactosidase) or a fluorescent substrate (e.g., 4-methylumbellatus-P-D-galactosidase).

Other applicable enzyme markers include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase (HRPO)), glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for enzyme-antibody conjugation are described in O'Sullivan et al., "Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay," Enzymological Methods (J. Langbne and H. Van Vunakis, eds.), Academic Press, New York, 73: 147-166 (1981).

Testing and Usage Methods for CLDN18.2

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 antigen-binding fragment thereof provided in this application under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2 (e.g., human CLDN18.2), and determining the presence or expression level of CLDN18.2 in the sample.

On the other hand, methods for diagnosing CLDN18.2-related diseases or conditions (e.g., cancer) in subjects are provided, including:

a) Contacting a sample obtained from the subject with the antibody or its antigen-binding fragment provided in this application under conditions that allow the antibody or its antigen-binding fragment to specifically bind to CLDN18.2; and

b) Determine the presence or expression level of CLDN18.2 in the sample;

When the presence of CLDN18.2 is detected, or when 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 some embodiments, the sample is not gastric epithelial tissue.

On the other hand, a method is provided for determining the appropriateness of treatment with a CLDN18.2-targeting agent for a subject who has a CLDN18.2-related disease or symptom, or who is at risk of having said disease or symptom, comprising:

a) Contacting a sample obtained from the subject with the antibody or its antigen-binding fragment provided in this application under conditions that allow the antibody or its antigen-binding fragment to specifically bind to CLDN18.2; and

b) Determine the presence or expression level of CLDN18.2 in the sample;

When CLDN18.2 is detected or when its expression level reaches a threshold level, the subject is deemed suitable for treatment with a CLDN18.2-targeting agent.

When CLDN18.2 is not detected or when the expression level of CLDN18.2 is below a threshold level, the subject is deemed unsuitable for treatment with CLDN18.2-targeted agents.

On the other hand, methods for predicting the therapeutic efficacy of CLDN18.2-targeting agents in treating CLDN18.2-related diseases or symptoms in subjects include:

a) Contact the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

b) Determine the presence or expression level of human CLDN18.2 in the sample; and

c) Predict the therapeutic efficacy of the CLDN18.2 targeting agent.

Specifically, the presence of CLDN18.2 or the expression level of CLDN18.2 reaching a threshold level are used to predict that the CLDN18.2 targeting agent is effective in treating the subject.

When the presence of CLDN18.2 is not detected or when the expression level of CLDN18.2 is below the threshold level, it is predicted that the CLDN18.2 target is ineffective in treating the subject.

In another aspect, a method of treating a subject suffering from or at risk of CLDN18.2-related disease or condition includes:

a) Selecting subjects suitable for the treatment, including:

i) Contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided in this application under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

ii) Determine the presence or expression level of human CLDN18.2 in the sample; and

iii) When the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 in the sample reaches a threshold level, the subject is selected as suitable for treatment with the CLDN18.2 target agent; and

b) Administer a therapeutically effective dose of the CLDN18.2-targeted agent to the selected subjects.

In another aspect, methods for treating subjects who have cancer or are at risk of developing cancer include:

a) Selecting subjects, including:

i) Contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided in this application under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2;

ii) Determine the presence or expression level of CLDN18.2 in the sample; and

iii) When CLDN18.2 is not detected or when the expression level of CLDN18.2 in the sample is below a threshold level, the subject is selected as unsuitable for treatment with CLDN18.2-targeted agents; and

b) Administer standard treatment to the selected subjects in addition to the CLDN18.2 targeted agent.

According to the present invention, "sample" can be any sample applicable under this disclosure, particularly biological samples such as tissue samples, comprising body fluids and/or cell samples, and which can be obtained by conventional means, such as by tissue biopsy, including drill biopsy, and by obtaining blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the present invention, the term "sample" also includes processed samples, such as eluted portions or isolates of biological samples, such as nucleic acid and peptide/protein isolates.

Any biological sample suspected of containing CLDN18.2 can be detected using the methods provided in this application. In some embodiments, suitable samples may be cell or tissue samples obtained from the subject to be tested. For example, the sample may comprise normal and cancerous tissues of the stomach, lungs, breast, colon, kidney, bone, brain, muscle, pancreas, bladder, ovary, uterus, and heart, embryonic, or placental tissues. Preferably, the sample contains cells or tissues of the organ to be examined, for example, for the diagnosis of cancer. For example, if the cancer to be diagnosed is gastric cancer, then the sample may contain cells or tissues obtained from the stomach. In some embodiments, the sample is a tumor sample. In some embodiments, the tissue sample is a sample with CLDN18.2-related diseases or symptoms.

Suitable samples can be bodily samples derived from patients who have or are expected to have tumors or cancer cells. Bodily samples can be any tissue sample (such as blood), tissue samples obtained from primary tumors or metastases, or any other sample containing tumors or cancer cells. The term "primary tumor" refers to a tumor that grows at the site of origin of the cancer. The term "metastasis" refers to a secondary tumor that grows at a site different from the site of origin of the cancer.

The source of tissue or cell samples can 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, samples are obtained from in vitro tissue or cell cultures.

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

In some embodiments, cell samples are generated from cell blocks. A "cell block" is a method for preparing cytological material so that it can be processed, sectioned, stained, and considered as a histological segment. It can provide diagnostic information in addition to that obtained from cytological slides. In some embodiments, cell blocks may be prepared from residual fluid, sputum, urinary sediment, gastrointestinal fluid, cell scrapings, or fine-needle aspirate. Cells are concentrated or piled up by centrifugation or membrane filtration.

Many methods have been developed for preparing cell blocks. Representative procedures include fixation deposition, bacterial agar, or membrane filtration. In the fixation deposition method, cell deposits are mixed with a fixative (such as Bouin's solution, picric acid, or buffered formalin), and the mixture is then centrifuged to aggregate the fixed cells. The aggregates are collected and placed in a tissue cassette, which is then placed in a container with an additional fixative and processed into a tissue sample. The agar method is very similar, but the aggregates are cut in half and the cut side is placed in a drop of molten agar on a glass slide. The agar is allowed to harden, and any excess agar is then trimmed off. Alternatively, the aggregates can be suspended directly in 2% liquid agar at 65°C and the sample centrifuged. The agar-coated cell aggregates are then solidified at 4°C for one hour. The solid agar can be removed from the centrifuge tube and cut in half. It is then placed in a tissue cassette and the tissue is processed. In any of these procedures, centrifugation can be replaced with membrane filtration. Any of these processes can be used to produce a “cell block sample.”

In some embodiments, cell blocks can be prepared using specialized resins comprising Lowicryl resin, LR White, LR Gold, Unicryl, and MonoStep. These resins have low viscosity and can be polymerized at low temperatures using ultraviolet (UV) light. The embedding process relies on gradually cooling the sample during dehydration, transferring the sample to the resin, and polymerizing the blocks at a final low temperature using an appropriate UV wavelength.

Cell block sections can be stained with hematoxylin-eosin, Hoechst stain, or DAPI for cell morphology examination, while additional sections are used to examine CLDN18.2 by exposing the sections to anti-CLDN18.2 antibody (i.e., primary antibody) under suitable conditions for a sufficient period of time to allow the antibody to bind to the CLDN18.2 protein in the cell block sections. Unbound and excess primary antibody can be washed away.

In some embodiments, the sample may contain, for example, preservatives, anticoagulants, buffers, nutrients, antibiotics, etc. In some embodiments, the sample has been exposed to and/or contains one or more fixatives. Exemplary fixatives suitable for the methods provided in this application include formalin, glutaraldehyde, osmium tetroxide, acetic acid, ethanol, acetone, picric acid, chloroform, potassium dichromate, and mercuric chloride, and/or stabilized by microwave heating or freeze-drying.

In some embodiments, the sample comprises 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 mm, preferably 4-40 μm, which are mounted and dried on a microscope slide. Examples of paraffin include, but are not limited to, pararaplast, brolooid, and tissuemay. For 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 in this application.

In some implementations, the deparaffin-free sample may be further processed to allow antigen retrieval. Antigen retrieval refers to any technique in which epitope masking is reversed and epitope-antibody binding is restored. While fixation is essential for the preservation of tissue morphology, this process can also negatively impact antibody binding and detection. Fixation can alter the biochemistry of proteins, causing the epitope of interest to be masked and no longer bind to antibodies. Epitope masking can be caused by cross-linking of amino acids within the epitope, cross-linking of unrelated peptides at or near the epitope, alteration of the epitope conformation, or alteration of the electrostatic charge of the antigen. The need for antigen retrieval depends on a variety of variables, including but not limited to the target antigen, the antibody used, the tissue type, and the method and duration of fixation. Antigen retrieval techniques generally include protease-induced epitope retrieval (PIER, using enzymes such as proteinase K, trypsin, and/or pepsin) and heat-induced epitope retrieval (HIER, using a microwave oven, pressure cooker, vegetable steamer, autoclave, or water bath).

In some embodiments, the presence or expression level of cell surface or membrane-bound CLDN18.2 is detected or determined in the methods provided in this application. The phrase "cell surface or membrane-bound CLDN18.2" means that CLDN18.2 is bound to and located at the cell's plasma membrane, wherein at least a portion of CLDN18.2 is exposed to the extracellular space of the cell and is available from outside the cell (e.g., by an extracellular antibody). In some embodiments, the extracellularly exposed portion of CLDN18.2 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 located within the tight junctions of epithelium and endothelium and is considered to be non-obtainable from outside the cell by antibodies, and therefore not considered cell surface or membrane-bound CLDN18.2.

The presence or expression level of CLDN18.2 protein in a sample can be determined based on the presence or amount of a complex of the antibody or its antigen-binding fragment disclosed in this application bound to the CLDN18.2 antigen. Any suitable method can be used to detect antibody-antigen complexes, for example by immunoassays such as immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), Western blotting (e.g., Western proteoblotting), flow cytometry (e.g., FACS ^™ ), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA). For a review of immunology and immunoassay procedures, see *Basic and Clinical Immunology* (eds. Stites and Terr, 7th ed., 1991). Furthermore, immunoassays can be performed in any of several configurations, which are fully reviewed in *Enzyme Immunoassay* (ed. Maggio, 1980); and ibid., Harlow and Lane. For a review of general immunoassays, see Methods in Cell Biology: Antibodies in Cell Biology, Vol. 37 (edited by Asai, 1993); Basic Clinical Immunology (edited by Stites and Terr, 7th edition, 1991).

In some embodiments, the antibody or antigen-binding fragment disclosed in this application is detectably labeled (e.g., a primary antibody) or unlabeled but can react with a detectably labeled second molecule (e.g., a detectably labeled secondary antibody).

In some embodiments, the presence or expression level of CLDN18.2 protein in a sample is determined by IHC or ICC. IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section (e.g., cells of the tissue mentioned in this application). Immunohistochemical staining is widely used to diagnose abnormal cells, such as those seen in cancerous tumors. In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment disclosed in this application can be used as a primary antibody in IHC or ICC. Generally, IHC or ICC can be performed to directly or indirectly detect 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.

To allow direct detection of antigens (e.g., CLDN18.2), the anti-CLDN18.2 antibody disclosed in this application or its antigen-binding fragment linked to a detectable marker can be used, which allows direct observation without further antibody interaction.

For indirect detection of antigens (e.g., CLDN18.2), the anti-CLDN18.2 antibody disclosed herein, or its antigen-binding fragment thereof, can be used as an unlabeled antibody that reacts with a second molecule of a detectable label (e.g., a detectable-labeled secondary antibody). For example, the anti-CLDN18.2 antibody or its antigen-binding fragment disclosed herein may be unlabeled and further contacted with a secondary antibody conjugated to a detectable label (e.g., an anti-allotype antibody) to allow indirect detection of the antigen. As another example, the anti-CLDN18.2 antibody provided herein may be conjugated to biotin, which may react with a detectable-labeled avidin, or vice versa. Biotin selectively binds to avidin and thus allows indirect detection of the antibody-antigen complex. In yet another example, the anti-CLDN18.2 antibody provided herein may be conjugated to a hapten, which may react with an anti-hapten antibody linked to a detectable label as described herein. Exemplary types of labels have been described above in this application, and any suitable detectable label may be used.

ICC or IHC analysis can be performed using automated pathology systems that may include automated staining (routine staining agents, histochemical techniques, immunostaining agents); automated in situ hybridization systems; automated slide preparation (coverslips, slide drying); and integrated slide and cassette labeling, as described by Roia et al., “Review of imaging solutions for integrated, quantitative immunohistochemistry in the Pathology daily practice,” *Folia Histochemica et Cytobiologica*, Vol. 47, No. 3, pp. 349-354, 2009.

Exemplary IHC analyses can be performed using the commercially available Dako EnVision ^™ FLEX detection system, intended for use with Dako automated staining instruments (Dako, Glostrup, Denmark, an Agilent Technologies company). These reagents can be used directly with other automated staining instruments or for manual staining without the use of an automated staining instrument.

After the staining process is completed, the CLDN18.2 staining of the sample (e.g., slide) is analyzed by a person (e.g., a pathologist) or by a computer programmed to distinguish between specific and non-specific staining results. The sample can be viewed directly under low, medium (10-20×), and high (40-60×) microscopes, or analyzed by observing high-resolution images of slides acquired at low, medium, and high magnification.

The presence of CLDN18.2 expression in a sample can be confirmed by the presence of positively stained cells, such as cells with at least partial membrane staining of any intensity of anti-CLDN18.2 antibody staining. In some embodiments, a normal sample can be used as a control sample, and the presence of CLDN18.2 expression in the 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 and control samples are comparable in sample type, for example, both are fixed tissue samples. If the test sample shows an increase in staining intensity or an increase in the number of positively stained cells compared to the control sample, then the test sample can be determined to be positive for CLDN18.2 expression.

In some implementations, 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.

In some implementations, 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 cells with at least partial membrane staining of any intensity of 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 regions under a microscope and calculate or estimate the percentage of CLDN18.2-positive cells. If the sample is highly heterogeneous, the sample can be partitioned, each region scored separately, and then combined into a single set of percentage values.

In some implementations, expression levels are quantified based on the staining intensity of CLDN18.2 (e.g., membrane-bound CLDN18.2) in the sample. For example, an intensity score can be calculated based on the staining intensity within the range of 0 (no staining), 1+ (weak staining), 2+ (obvious staining), 3+ (strong staining), and 4+ (very strong/saturated signal), multiplied by the percentage of cells stained at each intensity (0 to 100%), such as a 4-point HSCORE (see McCarty, K.S. Jr. et al., Cancer Research 46(Supplement 8): 4244s-4248s (1986) for details). As another example, an Alfred score can be calculated based on a total score (TS, range 0-8) by adding the proportion score (PS) to the intensity score (IS). PS represents the proportion of positive tumor cells in the range of 0 to 5 (0 = no positive cells, 1 = 1/100 cells are positive, 2 = 1/10 cells are positive, 3 = 1/3 cells are positive, 4 = 2/3 cells are positive, 5 = all tumor cells are positive). IS refers to the average staining intensity of positive tumor cells in the range of 0-3 (0 = negative, 1 = weak, 2 = moderate staining, 3 = strong staining) (see Alfred DC et al., Modern Pathology, 11: 155-168 (1998) for details).

In some implementations, the sample is evaluated by two independent observers and then the percentages or scores are integrated. In other implementations, appropriate software is used to identify or score positive and negative cells.

In some implementations, the content of CLDN18.2 can be determined, for example, by normalization against a control value or a standard curve. The control value can be predetermined from a negative control sample or a blank control sample, or determined simultaneously.

In some implementations, for diagnostic or clinical applications, the expression level of CLDN18.2 in the test sample (e.g., exposed on the cell surface) is compared to a threshold level.

The term "threshold level" or "threshold" for CLDN18.2 expression refers to an expression level that allows differentiation between positive and negative CLDN18.2 expression on cell surfaces, or an expression level that allows the determination or exclusion of CLDN18.2-related conditions (e.g., cancer) or the onset or risk of cancer in a subject, or a threshold level that allows monitoring of treatment response in subjects receiving cancer treatment. In some implementations, the threshold is determined relative to a control expression level.

For example, the threshold could be a level greater than that that would score a sample as having positive expression of CLDN18.2 and therefore be suitable for treatment with a CLDN18.2-targeting agent. If the CLDN18.2 level in a sample reaches or exceeds the threshold, it can indicate the presence of CLDN18.2-related disease or symptom and/or the likelihood of a response to a CLDN18.2-targeting agent.

The threshold level can be determined by a person skilled in the art by considering a variety of factors, including, for example, sample type, detection method, disease or symptom to be diagnosed and/or CLDN18.2 target agent to be used.

In some implementations, the presence of CLDN18.2 or cells expressing CLDN18.2 and/or an increase in the amount of CLDN18.2 or cells expressing CLDN18.2, compared to a threshold level, such as compared to subjects without CLDN18.2-related conditions (e.g., non-cancer subjects), indicates the presence or risk (i.e., disease potential) of CLDN18.2-related diseases or conditions (e.g., cancer) in the patient.

In some embodiments, the threshold level is the percentage (%) of CLDN18.2 positive cells with at least partial membrane staining of any intensity. In one embodiment, the threshold level for the percentage (%) of CLDN18.2 positive cells is 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

In some embodiments, the sample is obtained from a subject suffering from or at risk of developing 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 cells of a diseased tissue or organ compared to a state in healthy or non-cancerous tissue or organ other than the stomach. The increase may, for example, be at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, or even more. In some embodiments, CLDN18.2 expression in cells of a diseased tissue or organ is above the detection limit and/or sufficiently high to allow the added CLDN18.2-specific antibody to bind to the cells. In some embodiments, the increased expression of CLDN18.2 is found only in diseased tissue, while CLDN18.2 expression in normal tissue is undetectable.

In some implementations, CLDN18.2-related diseases or symptoms are characterized by increased expression of CLDN18.2 on the cell surface or bound to the membrane.

In some embodiments, the CLDN18.2-related disease or symptom is cancer. In one embodiment, cancer cells express or aberrantly express CLDN18.2, while the corresponding normal cells do not express CLDN18.2 or express it in low amounts. CLDN18.2, as a tight junction protein, is believed to be a good therapeutic target for CLDN18.2-related diseases (such as tumors) and can therefore be used to select patients suitable for treatment with CLDN18.2-targeting agents. Unlike normal epithelial tissue (except gastric epithelial cells) where CLDN binds to form classical tight junctions, CLDN expressed in tumor cells typically does not form said classical tight junctions, and therefore, tumor cells may have exposed, and extracellular antibody-bound, and immunotherapeutic free CLDN.

CLDN18.2 is a valuable target for the prevention and/or treatment of primary tumors such as 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, ductal carcinoma, and lobular carcinoma), liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, hepatic cancer, and head-neck cancer. Cancer, gallbladder 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, cholangiocarcinoma and/or adenocarcinoma, and/or their metastases, especially gastric cancer metastases such as Krukenberg tumor, peritoneal metastases, and lymph node metastases.

The samples are preferably cancer cells or tissues, and especially selected from the group consisting of tumorigenetic gastric, esophageal, pancreatic, lung, ovarian, colon, liver, head and neck, and gallbladder cancer cells or tissues.

In some implementations, cancers include, but are not limited to, renal cell carcinoma, gastric cancer, mesothelioma, melanoma, cervical cancer, thymic carcinoma, myeloma, mycoses fungoids, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoma, basal cell carcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, and classical Hodgkin's lymphoma. Lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell-rich/histiocytic B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumor, EBV-positive and negative PTLD and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, HHV8-related primary lymphoma Exudative lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplastic syndrome, primary CNS lymphoma, spinal cord tumors, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

In some implementations, the cancer is cancer that expresses CLDN18.2. The presence or expression level of CLDN18.2 in the 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.

As used in this application, "CLDN18.2 target agent" refers to one or more agents that target the CLDN18.2 protein or nucleic acid (DNA or mRNA) in cells, tissues, or living organisms. CLDN18.2 target agents can reduce/eliminate the expression of the CLDN18.2 gene product, inhibit/disrupt CLDN18.2 signaling, or induce cytotoxicity against cells that abnormally express CLDN18.2.

In some embodiments, the CLDN18.2 target includes a therapeutic anti-CLDN18.2 antibody, a CLDN18.2 binding molecule, a cell therapy targeting CLDN18.2, a compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2. In some embodiments, the CLDN18.2 target is capable of inducing cytotoxicity against cells expressing CLDN18.2.

In some embodiments, the CLDN18.2 target comprises a therapeutic anti-CLDN18.2 antibody or a CLDN18.2 binding molecule. In some embodiments, the therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule can induce ADCC, CDC, or ADCP in cells expressing CLDN18.2. Alternatively, the therapeutic anti-CLDN18.2 antibody or CLDN18.2 binding molecule can be conjugated to a cytotoxic agent, for example, to form an antibody-drug conjugate (ADC). In some embodiments, the cytotoxic agent can be any agent that is harmful to cells or can damage or kill cells. In some embodiments, the cytotoxic agent is optionally a toxin, a chemotherapeutic agent (such as a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binder, a growth inhibitor, or other anticancer drug), or a radioactive isotope. 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.

In some embodiments, the CLDN18.2 targeting agent comprises a cell therapy targeting CLDN18.2. In some embodiments, the CLDN18.2-targeting cell therapy comprises chimeric antibody receptor-engineered T cells (CAR-T), genetically modified TCR T cells (TCR-T), or chimeric antibody receptor-engineered NK cells (CAR-NK) expressing a chimeric antibody receptor (CAR) that binds to CLDN18.2. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines the antigen-binding domain of an antibody with one or more signaling domains that activate T cells. Immune cells such as T cells and natural killer (NK) cells can be genetically engineered to express CARs or genetically modified TCRs (see D. Li et al., *SigTransduct Target Therapy* 4, 35 (2019); S. Kloess et al., *Transfus Med Hemother* 46: 4-13 (2019); Wang W. et al., *Cancer Letters* 472: 175-180 (2020)). T cells expressing CARs are called CAR-T cells. CARs can mediate antigen-specific cellular immune activity in T cells, enabling CAR-T cells to eliminate cells expressing target antigens (such as tumor cells). In one embodiment, the binding of the CAR-T cells provided in this application to CLDN18.2 expressed on cells (such as cancer cells) causes the proliferation and/or activation of the CAR-T cells, wherein the activated CAR-T cells can release cytotoxic factors, such as perforin, granzyme and granzyme, and induce cell lysis and/or apoptosis of cancer cells.

In some implementations, the therapeutic nucleic acid targeting CLDN18.2 may be a short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), or short hairpin RNA (shRNA) molecule capable of mediating RNA interference (RNAi) against the CLDN18.2 gene sequence or another gene sequence in cells expressing CLDN18.2.

In some embodiments, the CLDN18.2 target promotes cancer regression in the subject. In a preferred embodiment, a therapeutically effective amount of the CLDN18.2 target promotes cancer regression to the point of cancer elimination. "Promoting cancer regression" means, alone or in combination with an anti-regenerative agent, administering an effective amount of the drug that results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free phases, or prevention of impairment or disability caused by disease.

In some implementations, the subject is receiving or has received anticancer therapy, or has experienced a recurrence of cancer. Anticancer therapy includes, but is not limited to, chemotherapy agents, anticancer drugs, radiation therapy, immunotherapy, anti-angiogenic agents, targeted therapy, cell therapy, gene therapy, hormone therapy, palliative care, surgery for treating cancer (e.g., tumor resection), or one or more antiemetics or other treatments for complications caused by chemotherapy.

A relapse occurs when a subject is again affected by a symptom that previously affected them. For example, a patient who has had cancer, successfully treated it, and then develops the disease again. This newly acquired disease can be considered a relapse. However, according to this disclosure, a relapse of cancer can, but does not necessarily, occur at the site of the original cancer. Therefore, for example, if a patient has had a gastric tumor and has received successful treatment, a relapse could be the appearance of a gastric tumor or a tumor in a location different from the stomach. Tumor relapse also includes the appearance of a tumor in a location different from the original tumor and the appearance of a tumor in the location of the original tumor. Preferably, the original tumor that the patient has already treated is a primary tumor, and the tumor in a location different from the original tumor is a secondary tumor or a metastasis.

Reagent test kit

In some embodiments, this disclosure provides a kit comprising the isolated antibody or its antigen-binding fragment provided in this application. In some embodiments, the kit disclosed in this application is a diagnostic kit. The kit can be used to detect the presence or amount of CLDN18.2 in biological samples, or can be used in the diagnostic methods provided in this application.

In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that is optionally labeled and provided in this application. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an indirect detectable portion.

In some embodiments, the kit further comprises a set of reagents for detecting complexes of the antibody or its antigen-binding fragment bound to CLDN18.2, which can be used for a variety of detection assays, including, for example, immunoassays such as IHC, ICC, or ELISA (sandwich or competitive forms).

In another embodiment of the invention, reagents for performing immunohistochemistry on FFPE tumor tissue sections are provided in the kit along with instructions for performing IHC analysis.

Any of the indirect detectable markers or portions disclosed in this application may be used. In some embodiments, the indirect detectable portion comprises biotin. In said embodiments, the reagent set comprises avidin or streptavidin as a detectable marker.

In some embodiments, the detectable marker is conjugated to the antibody or its antigen-binding fragment provided in this application. In some embodiments, the kit comprises the isolated antibody or its antigen-binding fragment provided in this application and a secondary antibody conjugated to the detectable marker. In some embodiments, the secondary antibody comprises an antibody that specifically binds to a primary antibody (e.g., the antibody or its antigen-binding fragment provided in this application). In some embodiments, the secondary antibody may be an anti-mouse antibody, an anti-rabbit antibody, or an anti-human antibody.

The detectable label may need to be combined with one or more components, such as buffers, antibody-enzyme conjugates, enzyme substrates, etc., before use, and these reagents may be included in the kit. For example, when the detectable portion contains an enzyme, the kit will contain the substrate and cofactor required for the enzyme (e.g., a substrate precursor providing the detectable chromophore or fluorophore). Additionally, other reagents may be included, such as blocking reagents to reduce nonspecific binding to solid-phase surfaces, washing reagents, enzyme substrates, etc. The relative amounts of various reagents can vary widely to provide reagent concentrations in solution that substantially optimize analytical sensitivity. Specifically, reagents may be provided in dry powder (usually lyophilized) form, containing excipients that, upon dissolution, will provide a reagent solution with an appropriate concentration. Instructions in the form of inserts or labels, indicating guidance for the preparation of the detection and/or analytical system, may also be included in the kit.

The kit components can be pre-attached to a solid support or applied to the surface of the solid support during kit use. The solid surface can be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides.

The container used for the kit may typically comprise at least one vial, test tube, flask, bottle, syringe, or other suitable container in which one or more of the detection compositions may be placed, and preferably appropriately aliquoted. The kits disclosed in this application will also typically include components for tightly containing one or more vials for commercial sale, such as injection-molded or blow-molded plastic containers in which one or more desired vials are held. Where the kit contains radiolabeled, chromogenic, fluorescent, or other types of detectable labels or detection components, the labeling reagent may be provided in the same container as the detection composition itself, or alternatively, may be placed in a second, different container component in which this second composition may be placed and appropriately aliquoted. Alternatively, the detection reagent may be prepared in a single container component, and in most cases, the kit will also typically include components for tightly containing one or more vials for commercial sale and/or for convenient packaging and shipping.

Apparatus or apparatus is also provided for performing the detection or monitoring methods described in this application. The apparatus may include a chamber or tube for sample input, a fluid handling system optionally including valves or pumps to guide sample flow through the apparatus, a filter optionally for separating plasma or serum from blood, a mixing chamber for adding a capture agent or detection reagent, and a detection device optionally for detecting the amount of a detectable label bound to the capture agent immune complex. Sample flow may be passive (e.g., by capillary action, hydrostatic force, or other force that does not require further manipulation of the apparatus after sample application) or active (e.g., by applying a force generated by a mechanical pump, electroosmotic pump, centrifugal force, or increased gas pressure), or a combination of active and passive forces.

Example

While this disclosure has been specifically shown and described with reference to specific embodiments (some of which are preferred embodiments), those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as disclosed herein.

Example 1: Preparation of antigens for immunization

1. Design of antigenic peptides

To restrict the antibody epitope to CLDN18.2 rather than CLDN18.1, a unique peptide targeting CLDN18.2 (Genbank accession number: NP_001002026) was designed by MabSpace Biosciences (Suzhou) Co., Limited. The coding sequence of this peptide (SEQ ID No. 19: DQWSTQDLYN) is located at amino acid residues Asp28-Asn37 (D28 to N37) of CLDN18.2.

2. Peptide synthesis

The antigenic peptide was synthesized at SANGON BIOTECH (Shanghai) and used for animal immunization.

Example 2: Antibody Production

1. Immunization and hybridoma fusion

Using CFA as an adjuvant, 6-8 week old mice of different strains were immunized with 40 μg/well of mouse peptide (conjugated with KLH). Three days after the initial immunization, each mouse received 11 booster injections (multiple subcutaneous injections) three times a week for four weeks, followed by two booster injections at one-week intervals. To determine serum titer, 100 μl/well of serially diluted mouse serum was added to the antigen peptide-coated plate, followed by incubation at 4°C for 30 minutes. The plate was washed three times with wash buffer, then 100 μl/well of goat anti-mIgG-HRP was added, and incubation was repeated at 4°C. After washing with wash buffer, TMB was added to react with the HRP on the plate. The OD450 nm of the plate was then read using a microplate reader, and the titer was analyzed using Graphpad Prism 6 software. Mice with higher titers were selected for the following fusion steps.

2. Integration

Four days prior to fusion, each mouse was boosted with an intraperitoneal immunization of 20 μg of peptide. On the day of fusion, the spleen was aseptically removed and subsequently processed into a single-cell suspension. Red blood cells were lysed to obtain spleen cells. Live log-phase myeloma cells (SP2/0) were mixed with mouse spleen cells at a 1:1 ratio in fusion medium, followed by electrofusion for 1 minute. The cells were resuspended and cultured in 96-well plates at 37°C in a 5% _CO2 incubator. After 7 days of culture, the growth medium was replaced with fresh growth medium. Screening of the hybridoma supernatant began 2-3 days after replacing the growth medium with fresh growth medium.

Example 3: Combining screening, subcloning of positive hybridoma clones, and small-scale antibody production

1. Combination screening via ELISA analysis

Hybridoma supernatant was collected for antigen binding screening. 100 μl/well of hybridoma supernatant was added to the antigen peptide-coated plate and incubated at 4°C for 1 hour. The plate was washed three times with wash buffer, followed by the addition of 100 μl/well of goat anti-mIgG-HRP and incubation at 4°C. After washing with wash buffer, TMB was added to react with the HRP on the plate. The OD490 nm of the plate was then read using a microplate reader. Positive clones that bound to the ELISA were selected and amplified. Two days later, the binding of the selected clones' hybridoma supernatant was tested in the same manner, and subcloning of positive clones was performed.

2. Subcloning of positive hybridoma clones

Cells from positive hybridoma wells with the desired binding properties were selected and limitedly diluted in 96-well plates. The diluted cells were allowed to grow for 7 days. Once sufficient cell quality was achieved, the supernatant from each well was collected and re-screened using the same ELISA binding assay described above.

The clones exhibiting the highest cell-binding activity in each 96-well plate were amplified and then subjected to a second round of limiting dilution into 96-well plates, with 200 μl of hybridoma growth medium per well. After 7 days, the cell supernatant in the 96-well plates was analyzed using the same ELISA binding assay. Subcloning was performed more than twice until more than 90/96 wells showed a positive binding signal. Two subclones with the highest binding activity (i.e., 69H2 and 14G11) were identified from each clone, amplified, and cultured to produce purified antibodies. Isotype determination was performed using standard methods.

3. Small-scale antibody production

Hybridoma cells were inoculated and cultured for 14 days. Monoclonal antibodies (mAbs) were purified from the hybridoma cell cultures by protein A affinity chromatography (Bio-Rad).

After chromatographic purification, these mAbs were prepared by dialysis in PBS, followed by a filtration step.

Example 4: Antibody Immunocytochemical Screening on Cell Blocks

1. Preparation of cell block sections

HEK293-human CLDN18.2 cells (hereinafter referred to as HEK293-CLDN18.2) and HEK293-human CLDN18.1 cells (hereinafter referred to as HEK293-CLDN18.1) were constructed by Mabpharm (Suzhou) Co., Ltd. In short, HEK293 cells (Shanghai Institutes for Biological Sciences, catalog number GNhu43) were transfected with pcDNA3.1/hCLDN18.2 or pcDNA3.1/hCLDN18.1 plasmids, and G418 selection was used to obtain stable expression cell lines HEK293-CLDN18.2 or HEK293-CLDN18.1. Expression of CLDN18.1 in HEK293-CLDN18.1 and expression of CLDN18.2 in HEK293-CLDN18.2 were tested and confirmed using FACS with positive control antibodies. As shown in Figure 1, HEK293-CLDN18.1 cells showed a positive binding signal of antibody EPR19203 (available from Abcam, product name ab222513) to CLDN18.1, and HEK293-CLDN18.2 cells showed a positive binding signal of antibody 18B10 (see PCT application PCT/CN2019/101563) specifically binding to human CLDN18.2.

HEK293 cells and 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) for 30 min at room temperature. 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 mixture was dehydrated in a gradient of alcohols and cleared in xylene. After infiltrating paraffin at 60 °C, the agar-cell mixture was embedded in paraffin according to standard procedures, sliced into sections 3 μm thick, and immediately mounted onto positively charged glass slides.

2. Antibody immunocytochemical screening on cell block slices

To screen for CLDN18.2-specific and sensitive antibodies, immunocytochemistry (ICC) was performed on paraffin-embedded (FFPE) HEK293, HEK293-CLDN18.2, and HEK293-CLDN18.1 cell sections. After deparaffining and rehydration, all sections were boiled in EnVision ^™ FLEX target retrieval solution (Dako, K8002) at 97–99 °C for 25 min, followed by quenching, blocking with EnVision ^™ FLEX peroxidase blocking agent (Dako, K8002), and incubated with appropriately diluted antibodies for antigen retrieval. Antibody binding was observed using EnVision ^™ FLEX+ mice (LINKER), followed by EnVision ^™ FLEX/hRP and EnVision ^™ FLEX substrate working solution (Dako, K8002). Finally, the sections were counterstained with hematoxylin and mounted with a permanent mounting agent. No significant differences in staining patterns were observed, and the staining intensity varied from weak to strong among the antibodies.

As shown in Table 3 and Figure 2, clones of the 1 nM antibodies 69H2 and 14G11 stained strongly and specifically on the surface of HEK293-CLDN18.2, but were negative on HEK293 and HEK293-CLDN18.1. Further titration to 0.5 nM was performed to test sensitivity. 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.

Staining with the anti-CLDN18.2 antibody GC182 was used as a control, and it showed positive staining on both HEK293-CLDN18.2 and HEK293-CLDN18.1 cells. Antibody GC182 possesses 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 Mabsys Biopharmaceuticals according to the sequence disclosed in WO2013167259. The results in Table 3 indicate that antibody GC182 cross-reacts with CLDN18.1 and therefore is not specific for CLDN18.2.

GC182 heavy chain variable region sequence (SEQ ID NO: 22):

GC182 light chain variable region sequence (SEQ ID NO: 23):

Table 3 Antibody screening on cell block sections by immunocytochemical staining.

Example 5: Cloning and recombinant production of the selected antibody

Production of recombinant antibodies

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

The heavy and light chains of the 14G11 and 69H2 antibodies are linked to the mouse IgG1 heavy chain constant region and κ light chain constant region, respectively, as shown below:

Mouse IgG1 heavy chain constant region (SEQ ID NO: 17):

Mouse κ light chain constant region (SEQ ID NO: 18):

Expression and purification of recombinant antibodies

ExpiCHO cells were transfected using the ExpiCHO transfection kit with equal volumes of DNA from the heavy and light chain vectors. Transfected cells were cultured in shaking flasks at 125 rpm in a 37°C, 8% _CO2 incubator. Cell cultures were collected on day 10, and the collected antibodies were purified by affinity chromatography. The resulting antibodies were analyzed, and purity levels were determined using SDS-PAGE and size exclusion chromatography (TSK gel G3000SWXL, TOSOH).

Example 6: Evaluation of the combination selectivity of hCLDN18.2 and hCLDN18.1 by ELISA and FACS analysis

Specific binding to hCLDN18.2 peptide (aa28-37) was analyzed by ELISA.

1 μg/ml peptide (100 μL/well) was plated onto a high-binding transparent polystyrene 96-well plate and blocked with blocking buffer. Serially diluted antibody (150-0.0732 ng/ml) in blocking buffer was then added, and the plate was incubated at room temperature for 1.5 hours. The plate was washed 6 times with washing buffer, followed by the addition of 100 μL/well of goat pAb targeting Ms IgG (HRP), and incubated at room temperature for 1.5 hours. After washing with washing buffer, TMB was added to react with the HRP on the plate. The OD450 nm of the plate was then read using a SpectraMax i3x (Molecular Devices). 14G11G2D2 and 69H2F7E6 showed high affinity and specific binding to the peptide, while GC182 did not recognize the epitope in the linear peptide. (Figure 3)

Recombinant anti-sealing protein 18.2 antibody and recombinant hCLDN18.2 and hCLDN18.1 proteins were analyzed by ELISA. Combination Selectivity Analysis

This analysis used the human recombinant CLDN18.2 (N-6His) variant (obtained from Novoprotein, catalog number NC101) and the human recombinant CLDN18.1 (N-8His) variant (obtained from Novoprotein, catalog number CR54). The human recombinant CLDN18.2 (N-6His) variant is a fusion polypeptide containing the extracellular domain of human CLDN 18.2 (residues Ala24-Ala81, SEQ ID NO: 26), with a sequence flanking the extracellular domain that can fold or associate to allow the extracellular domain of human CLDN 18.2 to form a loop. Similarly, the human recombinant CLDN18.1 (N-8His) variant is a fusion polypeptide containing the extracellular domain of human CLDN 18.1 (residues Asp28-Leu76, SEQ ID NO: 27), with a sequence flanking the extracellular domain that can fold or associate to allow the extracellular domain of human CLDN 18.1 to form a loop.

Spread 1 μg/ml of recombinant human CLDN18.2 (N-6His) variant or recombinant human CLDN18.1 (N-8His) variant (100 μL/well) onto a high-binding transparent polystyrene 96-well plate and block with blocking buffer. Add serially diluted antibody (100-0.0244 ng/ml) to the blocking buffer and incubate at room temperature for 1.5 h. Wash the plate 6 times with washing buffer (PBS + 0.1% Tween), then add 100 μl/well of goat pAb targeting Ms IgG (HRP) and incubate at room temperature for 1.5 h. After washing with washing buffer, add TMB to react with the HRP on the plate. Subsequently, read the OD450 nm of the plate using a SpectraMax i3x (Molecular Devices). Both 14G11G2D2 and 69H2F7E6 specifically bound to recombinant human CLDN18.2 (N-6His) with high affinity, with EC50 values of 12.75 ng/ml and 13.87 ng/ml, respectively (Figure 4). Neither 14G11G2D2 nor 69H2F7E6 showed specific binding to recombinant human CLDN18.1 (N-8His) protein (Figure 5). As a control, GC182 did not show specific binding to either recombinant human CLDN18.2 (N-6His) or recombinant human CLDN18.1 (N-8His) (Figures 4 and 5). This indicates that 14G11G2D2 and 69H2F7E6 bind to epitopes within the extracellular domain of CLDN18.2, but GC182 does not recognize these epitopes.

FACS analysis on HEK293-hCLDN18.2 and HEK293-hCLDN 18.1 cell lines

HEK293-hCLDN18.2 or HEK293-hCLDN18.1 cells in logarithmic growth phase were collected, washed, and resuspended. Diluted antibody (20 μg/mL) in blocking buffer was added, and the cells were incubated at 4°C for 1 hour. Cells were then washed twice with blocking buffer, followed by the addition of secondary antibody (goat anti-mouse IgG (H+L) cross-adsorption secondary antibody, Alexa Fluor 488, ThermoFisher) in blocking buffer. Cells were incubated at 4°C for 1 hour, washed twice with blocking buffer, and resuspended in blocking buffer. Cells were then transferred to FACS tubes, and antibody binding to cells was detected using flow cytometry (BDAccuri C6). Antibodies 18B10 and EPR19203 were used as positive controls for the analysis on HEK-hCLDN18.2 and HEK-hCLDN 18.1 cells, respectively. As shown in Figure 1, antibodies 14G11G2D2 and 69H2F7E6 cannot bind to human sealing protein 18.2 or sealing protein 18.1, which are expressed on the cell surface in their native conformation.

Example 7: Binding affinity determination of recombinant anti-sealing protein 18.2 and recombinant hCLDN18.2 protein by ForteBio

100 nM of recombinant human CLDN18.2 (N-6His) (Novoprotein, NC101) protein in 1×Kinetics buffer (PBS + 0.1% BSA + 0.02% Tween 20) was loaded onto a pre-wetted Ni-NTA biosensor (PALL, 18-5101) for 200 seconds. After baseline equilibration with 1×Kinetics buffer (60 seconds), the sensor was immersed in serially diluted antibodies (50 nM, 25 nM) in 1×Kinetics buffer for 200 seconds to determine association (kon), followed by dissociation in 1×Kinetics buffer for 200 seconds (koff). The biosensor was regenerated in regeneration buffer for 5 seconds, followed by neutralization in neutralization buffer for 5 seconds, repeated 3 times. All procedures were performed at 30 °C using Octet RED 96 (PALL). Binding affinity KD was calculated and analyzed using the ratio of koff to kon (Figure 6). 69H2F7E6 displays a KD value of 1.48 nM, while 14G11G2D2 displays a KD value of 0.213 nM.

Example 8: Validation of antibody specificity using normal tissue

CLDN18.2 is a highly selective gastric lineage antigen, its expression limited to short-lived differentiated epithelial cells of the gastric mucosa, while CLDN18.1 expression is limited to the lung. Based on this, selected antibodies were analyzed in various relevant normal tissues to confirm specific binding to CLDN18.2 rather than CLDN18.1. Immunohistochemistry (IHC) was performed on paraffin-embedded (FFPE) sections of normal intestine, kidney, tonsils, thyroid, skeletal muscle, stomach, lung, and breast tissue fixed in 4% neutral buffered formalin. After deparaffining and rehydration, all sections were boiled in EnVision ^™ FLEX target retrieval solution (Dako, K8002) at 97–99 °C for 25 min, followed by quenching, blocking with EnVision ^™ FLEX peroxidase blocking agent (Dako, K8002), and incubated with appropriately diluted antibodies for antigen retrieval. Antibody binding was observed using EnVision ^™ FLEX+ mice (LINKER), followed by EnVision ^™ FLEX/hRP and EnVision ^™ FLEX substrate working solution (Dako, K8002). Finally, sections were counterstained with hematoxylin and mounted with a permanent mounting medium.

Clones 14G11G2D2 and 69H2F7E6 were selected for further specific analysis on normal FFPE tissues. Both 69H2F7E6 and 14G11G2D2 showed good staining intensity, pattern, and selectivity. Both 69H2F7E6 and 14G11G2D2 showed strong staining intensity in FFPE gastric sections, but staining in lung, intestine, kidney, skeletal muscle, tonsils, thyroid, and breast sections was negligible (see Table 4 and Figures 7A and 7B). All stained cells were epithelial cells, not other cell types, including lymphocytes, blood vessels, fibroblasts, or smooth muscle cells. In contrast, the anti-CLDN18.2 antibody GC182 showed strong and moderate staining intensity in FFPE gastric and lung sections, respectively, confirming its binding to CLDN18.2 and CLDN18.1. In comparison, 14G11G2D2 performed better than 69H2F7E6, and 14G11G2D2 was selected as the candidate for further IHC testing. The results of IHC staining analysis are shown in Table 4 and Figures 7A and 7B.

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

Table 4. Specificity analysis on normal gastric, intestinal, kidney, skeletal muscle, and lung FFPE tissues.

Example 9: Evaluation of CLDN18.2 expression based on IHC using identified antibodies in tumor tissue samples from different tumor types.

It has been reported that the expression rate of CLDN18.2 in Japanese patients with gastric cancer was detectable in 87% of tumor samples, and moderate to strong CLDN18.2 expression was observed in 52% of tumor samples (Rohde et al., Japanese Journal of Clinical Oncology, September 1, 2019; 49(9): 870-876). In addition, there are reports indicating that aberrant ectopic expression of CLDN18.2 has appeared in other cancer cells, including pancreatic cancer, ovarian cancer, bile duct cancer and lung adenocarcinoma (Sahin U et al. Clinical Cancer Research, 2008, 14(23): 7624-7634; Karanjawala ZE et al. American Journal of Surgical Pathology, February 2008; 32(2): 188-96; Micke P et al. International Journal of Cancer, November 1, 2014; 135(9): 2206-14; Keira Y et al. European Journal of Pathology, March 2015; 466(3): 265-77).

In addition, 14G11G2D2 was analyzed on various relevant cancer tissues to ensure staining intensity, pattern, and positivity. Immunohistochemistry (IHC) was performed on paraffin-embedded (FFPE) tumor sections of gastric cancer, pancreatic cancer, cholangiocarcinoma, and non-small cell lung cancer (NSCLC) fixed in 4% neutral buffered formalin. After deparaffining and rehydration, all sections were boiled at 97–99°C for 25 minutes in EnVision ^™ FLEX target retrieval solution (Dako, K8002), followed by quenching, blocking with EnVision ^™ FLEX peroxidase blocking agent (Dako, K8002), and incubated with appropriately diluted 14G11G2D2 antibody for antigen retrieval. Antibody binding was observed using EnVision ^™ FLEX+ mice (LINKER), followed by EnVision ^™ FLEX/hRP and EnVision ^™ FLEX substrate working solution (Dako, K8002). Finally, sections were counterstained with hematoxylin and mounted with a permanent mounting agent. All samples were analyzed by the relative proportion of positively stained tumor cells to all visible tumor cells stained with membranes of varying intensities (negative (-), weak (+), medium (++), strong (+++)). Only membrane staining was considered positive, with normal human stomach serving as a positive control for each stain. 14G11G2D2 produced weak to strong membrane signals in gastric cancer, pancreatic cancer, cholangiocarcinoma, and NSCLC cancer tissues, respectively (see Figure 9). The percentage of positive tumor cells varied among individuals and 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 tissues). The positivity and incidence of CLDN18.2 expression in gastric cancer, pancreatic cancer, cholangiocarcinoma, and NSCLC cancer tissues are summarized in Table 6.

Table 5-1 Analysis of CLDN18.2 expression in gastric cancer tissues using recombinant 14G11G2D2 mouse monoclonal antibody.

Table 5-2 Analysis of CLDN18.2 expression in pancreatic cancer tissues using recombinant 14G11G2D2 mouse monoclonal antibody.

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

Table 5-4 Analysis of CLDN18.2 expression in NSCLC cancerous tissues using recombinant 14G11G2D2 mouse monoclonal antibody

Table 6. Positive and incidence analysis of CLDN18.2 expression in different cancer types

Example 10: Verification of CLDN18.2 in the stomach using biotinylated 14G11G2D2 IHC staining.

Preparation of biotinylated 14G11G2D2 (14G11G2D2-Biotin). In short, a stock solution of 20 mg/mL biotinamidocaproate NHS ester (Sigma, B2643-10MG) was prepared in anhydrous DMF. 10 μl of the stock solution was added for every 1 mg of the 14G11G2D2 antibody to be labeled, and the mixture was gently mixed at room temperature for 1 hour. The product was then desalted on a Zeba ^™ rotary desalting column (ThermoFisher, 89890) according to the manufacturer's instructions to remove low molecular weight reaction products.

Immunohistochemistry (IHC) was performed on slides of paraffin-embedded gastric samples fixed in 4% neutral buffered formalin. After deparaffinization and rehydration, all slides underwent antigen retrieval by boiling in EnVision ^™ FLEX target retrieval solution (Dako, K8002) at 97–99 °C for 25 min, followed by quenching and blocking with an IHC biotin blocking kit (MaiXin, BLK-0001) according to the manufacturer's instructions, and incubated at 37 °C for 30 min with 3 μg/mL of self-generated biotinylated monoclonal mouse anti-blocking protein 18.2 (14G11G2D2-biotin) antibody. Antibody binding was observed using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision ^™ FLEX substrate working solution (Dako, K8002). Finally, sections were counterstained with hematoxylin and mounted with a permanent mounting medium.

As shown in Figure 10, 14G11G2D2-Biotin exhibits the same staining pattern as 14G11G2D2 in terms of intensity and specificity.

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Claims (54)

1. A separated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein said antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics: a) It does not exhibit cross-reactivity with human CLDN 18.1; b) As measured by immunohistochemical analysis (IHC), it does not cross-react with non-cancerous cells, except for gastric epithelial cells; c) As measured by IHC, there is no cross-reactivity with non-cancer lung tissue; d) Capable of specifically binding to cells expressing CLDN18.2, optionally the cells expressing CLDN18.2 being pretreated to denature CLDN18.2 or otherwise prevent it from exhibiting its native conformation; e) A fusion polypeptide containing the first extracellular loop of human CLDN18.2, as measured by surface plasmon resonance (SPR), can bind with a Kd value not exceeding 10 nM, or as measured by enzyme-linked immunosorbent assay (ELISA), can bind with an EC50 value not exceeding 20 ng/ml. f) As measured by flow cytometry (FACS) analysis, no detectable binding was observed to human CLDN 18.2 on the cell surface; g) As measured by ELISA, it can specifically bind to an epitope within the amino acid sequence of DQWSTQDLYN (SEQ ID NO: 19); and/or h) The antibody does not cross-react with human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) samples, as measured by ELISA at an antibody concentration of 1 nM or by IHC at an antibody concentration of 0.5 μg/ml. 2. An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a) Heavy chain CDR1 containing the amino acid sequence X ₁ X ₂ Y X ₃ H (SEQ ID NO: 8), heavy chain CDR2 containing the amino acid sequence WIYPX ₄ GX _s X ₆ X ₇ X ₈ Y X ₉ EKFKG (SEQ ID NO: 12), and heavy chain CDR3 containing the amino acid sequence NYX ₁₀ STFGY (SEQ ID NO: 24); and/or b) Light chain CDR1, which contains the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2), light chain CDR2, which contains the amino acid sequence KX ₁₁ SNRFS (SEQ ID NO: 25), and light chain CDR3, which contains the amino acid sequence FQGSHVPFT (SEQ ID NO: 6); Where _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, _X10 is G or R and _X11 is V or I. 3. The isolated antibody or its antigen-binding fragment according to claim 2, comprising: a) Heavy chain CDR1, comprising an amino acid sequence selected from the following: SEQ ID NO: 1 and SEQ ID NO: 7, and/or b) Heavy chain CDR2, comprising an amino acid sequence selected from the following: SEQ D NO: 3 and SEQ ID NO: 9, and/or c) Heavy chain CDR3, comprising an amino acid sequence selected from the following: SEQ ID NO: 5 and SEQ ID NO: 11; and/or d) Light chain CDR1, which contains the amino acid sequence of SEQ ID NO: 2, and/or e) Light chain CDR2, comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 10, and/or f) Light chain CDR3, which contains the amino acid sequence of SEQ ID NO: 6. 4. An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: g) Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1; heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 3; and heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 5; or h) Heavy chain CDR1, which contains the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2, which contains the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3, which contains the amino acid sequence of SEQ ID NO: 11. 5. The antibody or antigen-binding fragment thereof according to claim 4, further comprising: a) light chain CDR1, comprising the amino acid sequence of SEQ ID NO: 2; light chain CDR2, comprising the amino acid sequence of SEQ ID NO: 4; and light chain CDR3, comprising the amino acid sequence of SEQ ID NO: 6; or b) Light chain CDR1, which contains the amino acid sequence of SEQ ID NO: 2, light chain CDR2, which contains the amino acid sequence of SEQ ID NO: 10, and light chain CDR3, which contains the amino acid sequence of SEQ ID NO: 6. 6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, 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, 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. 7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; or b) The heavy chain variable region, which contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region, which contains the amino acid sequence of SEQ ID NO: 16. 8. The antibody or antigen-binding fragment thereof according to any one of claims 4-7, further comprising one or more amino acid residue mutations, but still maintaining binding specificity to human CLDN 18.2. 9. The antibody or antigen-binding fragment thereof according to claim 8, wherein at least one of the mutations is a conserved substitution, or all of the mutations are conserved substitutions. 10. The antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein at least one of the mutations is in one or more CDR sequences of the heavy chain variable region or the light chain variable region, and/or in one or more non-CDR sequences. 11. The antibody or antigen-binding fragment thereof according to any one of claims 8-10, comprising: 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% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 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) The light chain CDR3 (LCDR3) sequence, which has 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: 6, and The antibody or its antigen-binding fragment may optionally have a binding affinity similar to or even higher than that of its parent antibody while maintaining binding specificity to CLDN18.2. 12. The antibody or antigen-binding fragment thereof according to any one of claims 8-11, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3, wherein HCDR1 has no more than 3, 2 or 1 amino acid mutations in SEQ ID NO: 1 or SEQ ID NO: 7, HCDR2 has no more than 6, 5, 4, 3, 2 or 1 amino acid mutations in SEQ ID NO: 3 or SEQ ID NO: 9, HCDR3 has no more than 6, 5, 4, 3, 2 or 1 amino acid mutations in SEQ ID NO: 5 or SEQ ID NO: 11, LCDR1 has no more than 2 or 1 amino acid mutations in SEQ ID NO: 2, LCDR2 has no more than 3, 2 or 1 amino acid mutations in SEQ ID NO: 4 or SEQ ID NO: 10, and LCDR3 has no more than 3, 2 or 1 amino acid mutations in SEQ ID NO: 6, and wherein the antibody or antigen-binding fragment thereof optionally has a binding affinity similar to or even higher than that of its parent antibody while maintaining binding specificity to CLDN18.2. 13. The antibody or antigen-binding fragment thereof according to any one of claims 8-12, 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. 14. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising an immunoglobulin constant region, optionally comprising a heavy chain constant region and/or a light chain constant region of IgG. 15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the constant region comprises a mouse constant region, a rabbit constant region, or a human constant region. 16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80% sequence identity with it, and/or the light chain constant region comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80% sequence identity with it. 17. The antibody or antigen-binding fragment thereof according to any one of the preceding claims 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 dimerizing antibody or a multimerizing antibody, or a modified antibody (e.g., a glycosylated antibody). 18. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a bifunctional antibody, Fab, Fab′, F(ab′) ₂ , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv′), disulfide-stabilized bifunctional antibody (ds bifunctional antibody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelified single-domain antibody, nanobody, domain antibody or bivalent domain antibody. 19. An antibody or an antigen-binding fragment thereof according to any one of the preceding claims, wherein it is linked to one or more portions. 20. The antibody or antigen-binding fragment thereof according to claim 19, wherein the portion comprises a radioactive isotope, a lanthanide element, a chemiluminescent label, a chromogenic portion, colloidal gold particles, a fluorescent label, an enzyme-substrate label, a digoxigenin label, a biotin/avidin label, a hapten, or a DNA molecule or particle label for detection. 21. The antibody or antigen-binding fragment thereof according to claim 20, wherein the portion comprises biotin or a hapten. 22. A monoclonal antibody or an antigen-binding fragment thereof, which competes with an antibody or an antigen-binding fragment thereof according to any one of the preceding claims for binding to CLDN18.2. 23. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of the preceding claims. 24. A vector comprising the isolated polynucleotide according to claim 23. 25. A host cell comprising the vector according to claim 24. 26. A method for expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1-22, comprising culturing a host cell according to claim 25 under conditions of expressing a vector according to claim 24. 27. A method for detecting the presence or expression level of CLDN18.2 in a sample, comprising contacting the sample with an antibody or an antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or the antigen-binding fragment thereof to specifically bind to human CLDN18.2, and determining the presence or expression level of CLDN18.2 in the sample. 28. A method for diagnosing a CLDN18.2-related disease or symptom (e.g., cancer) in a subject, comprising: a) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; and b) Determine the presence or expression level of CLDN1 8.2 in the sample; When the presence of CLDN18.2 is detected or when 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). 29. A method for determining the appropriateness of treatment with a CLDN18.2-targeting agent in a subject suffering from or at risk of developing a disease or symptom related to CLDN18.2, comprising: a) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; b) Determine the presence or expression level of CLDN18.2 in the sample; When CLDN18.2 is detected or when its expression level reaches a threshold level, the subject is deemed suitable for treatment with a CLDN18.2-targeting agent. When CLDN18.2 is not detected or when the expression level of CLDN18.2 is below a threshold level, the subject is deemed unsuitable for treatment with CLDN18.2-targeted agents. 30. A method for predicting the therapeutic efficacy of a CLDN18.2-targeting agent in treating CLDN18.2-related diseases or symptoms in subjects, comprising: a) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; b) Determine the presence or expression level of human CLDN18.2 in the sample; c) Predict the therapeutic efficacy of the CLDN18.2 targeting agent. Specifically, the presence of CLDN18.2 or the expression level of CLDN18.2 reaching a threshold level are used to predict that the CLDN18.2 targeting agent is effective in treating the subject. When the presence of CLDN18.2 is not detected or when the expression level of CLDN18.2 is below the threshold level, it is predicted that the CLDN18.2 target is ineffective in treating the subject. 31. A method of treating a subject suffering from or at risk of developing a CLDN18.2-related disease or condition, comprising: a) Selecting subjects suitable for the treatment, including: i) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; ii) Determine the presence or expression level of human CLDN18.2 in the sample; iii) When the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 in the sample reaches a threshold level, the subject is selected as a suitable candidate for treatment of the CLDN18.2-related disease or symptom; b) Administer a therapeutically effective dose of the CLDN18.2-targeted agent to the selected subjects. 32. A method of treating a subject who has cancer or is at risk of developing cancer, comprising: a) Selecting subjects, including: i) Contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-22 under conditions that allow the antibody or antigen-binding fragment thereof to specifically bind to CLDN18.2; ii) Determine the presence or expression level of CLDN18.2 in the sample; iii) When CLDN18.2 is not found or when the expression level of CLDN18.2 in the sample is below a threshold level, the subject is selected as unsuitable for treatment with CLDN18.2-targeted agents; b) Administer standard treatment to the selected subjects in addition to the CLDN18.2 targeted agent. 33. The method according to any one of claims 27-32, wherein the sample is a cell sample or a tissue sample. 34. The method of claim 33, wherein the sample is a fixed tissue sample, optionally a formalin-fixed paraffin-embedded (FFPE) tissue sample. 35. The method according to any one of claims 27-34, wherein the CLDN18.2 is a cell surface or membrane-bound CLDN18.2. 36. The method according to any one of claims 27-35, 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 Western blotting. 37. The method according to any one of claims 27-36, wherein the expression level is quantified based on the percentage of positively stained cells in the sample. 38. The method according to any one of claims 27-36, wherein the expression level is quantified based on the staining intensity of CLDN18.2 in the sample. 39. The method according to any one of claims 28-38, wherein the CLDN18.2 related disease or symptom is cancer. 40. The method of claim 39, wherein the sample comprises a tumor sample. 41. The method of claim 40, wherein the tumor sample comprises tumor tissue or circulating tumor cells. 42. The method of claim 41, wherein the cancer is a primary cancer or a metastatic cancer. 43. The method according to any one of claims 39-42, 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. 44. The method of claim 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). 45. The method according to any one of claims 29-44, wherein the CLDN18.2 target agent is capable of inducing cytotoxicity against cells expressing CLDN18.2. 46. The method according to any one of claims 29-45, wherein the CLDN18.2 target is a therapeutic anti-CLDN18.2 antibody or a CLDN18.2 binding molecule (e.g., an anti-CLDN18.2 antibody conjugated with a cytotoxic agent, or a bispecific antibody), a cell therapy targeting CLDN18.2 (e.g., CAR-T, TCR-T, or CAR-NK cells expressing a CAR that binds to CLDN18.2), a compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2. 47. The method according to any one of claims 27-46, wherein the subject is receiving or has received anticancer therapy, or has experienced a recurrence of cancer. 48. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-22. 49. The kit of claim 48, further comprising a set of reagents for detecting complexes of the antibody or its antigen-binding fragment bound to CLDN18.2. 50. The kit of claim 49, wherein the reagent set comprises a secondary antibody that binds to an antibody or an antigen-binding fragment thereof according to any one of claims 1-22, optionally said secondary antibody being detectably labeled. 51. The kit of claim 48, wherein the antibody or its antigen-binding fragment is detectably labeled. 52. The kit of claim 48, wherein the antibody or its antigen-binding fragment is conjugated to an indirectly detectable portion. 53. The kit of claim 52, wherein the indirect detectable portion comprises biotin. 54. The kit of claim 53, wherein the reagent set comprises a detectable labeled avidin or streptavidin.