RU2850336C1 - Antibodies against claudin 18.2 and their diagnostic application - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention provides new antibodies against CLDN18.2.

EFFECT: detection and treatment of CLDN18.2-associated diseases, in particular cancer.

24 cl, 11 dwg, 6 tbl, 10 ex

Description

Field of technology to which the invention relates

[0001] The present invention relates to novel antibodies against CLDN18.2 and their diagnostic use.

State of the art

[0002] Claudin-18 molecule (CLDN18) (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) is an integral transmembrane protein with a molecular mass of approximately 27.9/27.72 kDa. CLDN18 proteins are localized to tight junctions of epithelia and endothelium, which form a network of intertwined filaments of intramembrane particles between adjacent cells. CLDN18 and occludin are the most prominent transmembrane protein components at tight junctions. Due to their strong intercellular adhesion properties, these tight junction proteins form a primary barrier that prevents and controls the paracellular transport of soluble substances, as well as limits the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Therefore, they are crucial in organizing the architecture of epithelial tissues.

[0003] Although targeted therapy and immunotherapy have revolutionized systemic treatment for various cancers over the past decade, treatment of advanced and/or metastatic cancers remains a major challenge. For example, many patients with common gastrointestinal cancers such as stomach cancer, pancreatic cancer, cholangiocarcinoma, and lung cancer still do not benefit significantly from current treatment standards. Chemotherapy remains the primary treatment for most patients with advanced cancer, and the prognosis for these patients remains poor. Considering the highly restricted expression level and frequent ectopic activation of CLDN18.2 in common tumor types, CLDN18.2 is considered as a therapeutic target for the development of agents for the treatment of solid tumors expressing CLDN18.2, which include, but are not limited to, gastric cancer/esophageal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, etc.

[0004] For screening and selection of patients with CLDN18.2 expression, it is desirable to perform an immunohistochemical study (IHC) with high specificity and affinity determination.

Brief summary of the invention

[0005] Throughout the present invention, the articles "a," "an," and "the" are used to denote one or more than one (i.e., at least one) grammatical object in an entry. As an example, "antibody" means one antibody or several antibodies.

[0006] The present invention relates, inter alia, to novel monoclonal antibodies against CLDN18.2 that specifically recognize CLDN18.2 without cross-reaction with CLDN18.1. The present invention also relates to nucleotide sequences encoding such antibodies against CLDN18.2 and the use of such antibodies against CLDN18.2, for example, for diagnostic purposes.

[0007] In one aspect, the present invention is an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics:

a) no cross-reactivity to human CLDN18.1;

b) absence of cross-reactivity to benign neoplasms, with the exception of gastric epithelial cells detected by immunohistochemistry (IHC);

c) absence of cross-reactivity to benign neoplasms of human lung tissue detected by IHC;

d) the ability to specifically bind to CLDN18.2-expressing cells, wherein optionally the CLDN18.2-expressing cells are pretreated such that CLDN18.2 is denatured or no longer in its native conformation;

e) the ability to bind to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 with a _Kd value of no more than 10 nM as measured by surface plasmon resonance (SPR) or an EC50 value of no more than 20 ng/ml as measured by enzyme-linked immunosorbent assay (ELISA);

f) no detection of cell surface binding of human CLDN 18.2 as measured by flow cytometry (FACS);

g) the ability to specifically bind to an epitope within the amino acid sequence DQWSTQDLYN (SEQ ID NO: 19) as determined by ELISA; and/or

h) no cross-reactivity to human CLDN18.1 in formalin-fixed paraffin-embedded (FFPE) specimen at 1 nM antibody concentration by ELISA or at 0.5 g/mL antibody concentration by IHC.

[0008] In one aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody comprises:

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

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

where X ₁ is R or T, X ₂ is N or Y, X ₃ is F or I, X ₄ is G or R, X ₅ is F or G, X ₆ is D or N, X ₇ is I or T, X ₈ is E or V, X ₉ is S or N, X ₁₀ is G or R, and X ₁₁ is V or I.

[0009] In some embodiments of the invention, the isolated antibody or antigen-binding fragment thereof comprises:

a) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO:1, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO:3, and a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO:5; or

b) a heavy chain CDR1 with the sequence presented in SEQ ID NO:7, a heavy chain CDR2 with the sequence presented in SEQ ID NO:9, and a heavy chain CDR3 with the sequence presented in SEQ ID NO:11.

[0010] In some embodiments of the invention, the antibody or antigen-binding fragment thereof provided herein further comprises:

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

b) a light chain CDR1 with the amino acid sequence presented in SEQ ID NO:2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO:10, and a light chain CDR3 with the amino acid sequence presented in SEQ ID NO:6.

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

a) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO: 1, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO: 3, a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO: 5, a light chain CDR1 with the amino acid sequence presented in SEQ ID NO: 2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO: 4, a light chain CDR3 with the amino acid sequence presented in SEQ ID NO: 6; or

b) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO:7, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO:9, a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO:11, a light chain CDR1 with the amino acid sequence presented in SEQ ID NO:2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO:10, a light chain CDR3 with the amino acid sequence presented in SEQ ID NO:6.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises:

a) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO:13 and a light chain variable region with the amino acid sequence presented in SEQ ID NO:14; or

b) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO:15 and a light chain variable region with the amino acid sequence presented in SEQ ID NO:16.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue mutations, but retains binding specificity for human CLDN 18.2, and optionally retains binding specificity for a linear epitope with the amino acid sequence presented in SEQ ID NO:19.

[0014] In some embodiments of the invention, at least one of the mutations is a conservative substitution, or all of the mutations are conservative substitutions.

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

[0016] In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises:

a) a heavy chain CDR1 (HCDR1) sequence with a degree of identity to the sequences presented in SEQ ID NO:1 or SEQ ID NO:7 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

b) a heavy chain CDR2 (HCDR2) sequence with a degree of identity to the sequences presented in SEQ ID NO:3 or SEQ ID NO:9 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

c) a heavy chain CDR3 (HCDR3) sequence with a degree of identity to the sequences presented in SEQ ID NO:5 or SEQ ID NO:11 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

d) a light chain CDR1 (LCDR1) sequence with a degree of identity to the sequence presented in SEQ ID NO:2 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

e) a light chain CDR2 (LCDR2) sequence with a degree of identity to the sequences presented in SEQ ID NO:4 or SEQ ID NO:10 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

f) a light chain CDR3 (LCDR3) sequence with a degree of identity to the sequence presented in SEQ ID NO:6 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and

while maintaining binding specificity to CLDN18.2, optionally having a binding affinity at a level similar to or even higher than that of the parent antibody.

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

[0018] In some embodiments, the variable region of the heavy chain comprises an amino acid sequence with at least 80% identity to the sequences shown in SEQ ID NO:13 or SEQ ID NO:15, and/or the variable region of the light chain comprises an amino acid sequence with at least 80% identity to the sequences shown in SEQ ID NO:14 or SEQ ID NO:16.

[0019] In some embodiments, the antibody or antigen-binding fragment provided herein further comprises an immunoglobulin constant region, optionally comprising an IgG heavy chain constant region and/or a light chain constant region. 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.

[0020] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence with at least 80% identity, and/or the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:18 or a sequence with at least 80% identity.

[0021] In some embodiments, the antibody or antigen-binding fragment provided herein 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-idiotypic antibody, a fusion protein, a dimerized or polymerized antibody, or a modified antibody (e.g., a glycosylated antibody).

[0022] In some embodiments, the antibody or antigen-binding fragment provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody (scFv), scFv dimer (bivalent diabody), multispecific antibody, camel single domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0023] In some embodiments, an antibody or antigen-binding fragment provided herein is linked to one or more molecule. In some embodiments, the molecule comprises a radioactive isotope, a lanthanide, a chemiluminescent label, a chromophore molecule, colloidal gold particles, a fluorescent label, an enzyme-substrate label, a digoxigenin label, biotin/avidin, a hapten, a DNA molecule for detecting or labeling particles. In some embodiments, the molecule comprises biotin or a hapten.

[0024] In another aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that competes for binding to CLDN18.2 with the antibody or antigen-binding fragment thereof provided herein.

[0025] In another aspect, the present invention relates to an isolated polynucleotide encoding an antibody or antigen-binding fragment as provided herein. In another aspect, the present invention relates to a vector comprising the isolated polynucleotide as provided herein. In another aspect, the present invention relates to a host cell comprising the vector as provided herein.

[0026] In another aspect, the present invention relates to a method of expressing an antibody or antigen-binding fragment as provided herein, comprising culturing a host cell as provided herein under conditions under which the vector as provided herein is expressed.

[0027] In another aspect, the present invention relates to a method for determining the presence or expression level of CLDN18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment provided herein under conditions that allow the antibody or antigen-binding fragment to specifically bind to human CLDN18.2, and determining the presence or expression level of CLDN18.2 in the sample.

[0028] In another aspect, the present invention relates to a method for diagnosing a CLDN18.2-associated disease or condition (e.g., cancer) in a subject, comprising:

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

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

c) a subject is diagnosed with a CLDN18.2-associated disease or condition (e.g., cancer) when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 reaches a threshold level.

[0029] In another aspect, the present invention relates to a method for determining the suitability of a subject having or at risk for a CLDN18.2-associated disease or condition for treatment with a CLDN18.2-targeted agent, including:

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

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

a subject is determined to be eligible for treatment with a CLDN18.2-targeted agent when the presence of CLDN18.2 is detected or when the level of CLDN18.2 expression reaches a threshold level, or

A subject is defined as ineligible for treatment with a CLDN18.2-targeted agent when the presence of CLDN18.2 is not detected or when the CLDN18.2 expression level is below a threshold level.

[0030] In another aspect, the present invention relates to a method for predicting the therapeutic efficacy of a CLDN18.2-targeted agent in the treatment of a CLDN18.2-associated disease or condition in a subject, including:

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

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

c) prediction of therapeutic efficacy of CLDN18.2-targeted agent,

A CLDN18.2-targeting agent is predicted to be effective in treating a subject when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 reaches a threshold level, or

A CLDN18.2-targeting agent is predicted to be ineffective in treating a subject when the presence of CLDN18.2 is not detected or when the level of CLDN18.2 expression is below a threshold level.

[0031] In another aspect, the present invention relates to a method of treating a subject having or at risk for a CLDN18.2-associated disease or condition, including:

a) selection of a subject suitable for treatment, including:

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

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

iii) selecting a subject as suitable for treatment of a CLDN18.2-associated disease or condition when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 in a sample reaches a threshold level;

b) administering a therapeutically effective amount of a CLDN18.2-targeting agent to the selected subject.

[0032] In another aspect, the present invention relates to a method of treating a subject having cancer or at risk of developing cancer, including:

a) selection of the subject, including:

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

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

iii) selecting a subject as ineligible for treatment of a CLDN18.2-associated disease or condition when the presence of CLDN18.2 is not detected or when the level of CLDN18.2 expression in the sample is below a threshold level;

b) administering to the selected subject a standard therapy therapeutic agent other than the CLDN18.2-targeted agent.

[0033] 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, CLDN18.2 is cell surface-associated and membrane-bound CLDN18.2.

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

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

[0036] In some embodiments, the CLDN18.2-associated disease or condition is cancer. In some embodiments, the sample is a tumor sample. In some embodiments, the tumor sample is tumor tissue or a circulating tumor cell. In some embodiments, the cancer is a primary cancer or a metastatic cancer.

[0037] In some embodiments, the CLDN18.2-associated disease or condition is cancer. In some embodiments, the cancer is primary cancer or metastatic cancer. In some embodiments, the cancer is stomach 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, spine 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, cholangiocarcinoma and/or adenocarcinoma.

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

[0039] In some embodiments, a CLDN18.2-targeted agent is capable of causing cytotoxicity in cells expressing CLDN18.2. In some embodiments, the CLDN18.2-targeted agent is a therapeutic CLDN18.2 antibody or CLDN18.2-binding molecule, a CLDN18.2-targeted cell therapy, a chemical compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2.

[0040] In some embodiments, a therapeutic CLDN18.2 antibody or CLDN18.2-binding molecule is conjugated to a cytotoxic agent. In some embodiments, a therapeutic CLDN18.2 antibody is a bispecific antibody. In some embodiments, a cellular therapy directed against CLDN18.2 comprises a CAR-T (chimeric antibody receptor T cell), a TCR-T (genetically modified TCR T cell), or a CAR-NK (chimeric antibody receptor cell) expressing a CLDN18.2-binding chimeric antibody receptor (CAR).

[0041] In some embodiments of the invention, the subject is receiving or has received anti-cancer therapy or is suffering from a recurrence of cancer.

[0042] In another aspect, the present invention relates to a kit comprising an isolated antibody or antigen-binding fragment thereof as provided herein.

[0043] In some embodiments of the invention, the kit further comprises a reagent kit for detecting a complex of an antibody or an antigen-binding fragment thereof bound to CLDN18.2. In some embodiments of the invention, the reagent kit comprises an anti-mouse antibody.

BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 shows FACS analysis of antibodies against CLDN18.2, 69H2F7E6, 14G11G2D2 binding to a HEK293-hCLDN18.2 cell and a HEK293-hCLDN18.1 cell. Antibodies 18B10D3G9F3 and EPR19202 are used as positive controls.

[0045] Figure 2 shows immunocytochemical (ICC) staining of 69H2 and 14G11 antibodies on HEK293, HEK293-CLDN18.1, and HEK293-CLDN18.2 cell blocks. GC182 antibody staining was used as a control.

[0046] Figure 3 shows the binding profiles and EC50 of anti-CLDN18.2 antibody, 69H2F7E6, 14G11G2D2, and GC182 binding to the hCLDN18.2 peptide (amino acid residues 28-37 of hCLDN18.2).

[0047] Figure 4 shows the binding profiles and EC50 of anti-CLDN18.2 antibodies, 69H2F7E6, 14G11G2D2, and GC182, binding to a recombinant hCLDN18.2 variant protein comprising the amino acid sequence of the hCLDN18.2 ECL1 loop (SEQ ID NO:26), measured by ELISA.

[0048] Figure 5 shows the binding profiles and EC50 of an antibody against CLDN18.2, 69H2F7E6, 14G11G2D2, and GC182 binding to a recombinant protein of the hCLDN18.1 variant comprising the amino acid sequence of the ECL1 loop of hCLDN18.1 (SEQ ID NO:27), measured by ELISA.

[0049] Figure 6 shows the binding affinity analysis of the anti-CLDN18.2 antibody, 69H2F7E6, 14G11G2D2, GC182, to the ForteBio recombinant hCLDN18.2 variant protein. The kDa, association rate (kon), and dissociation rate (koff) are shown.

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

[0051] Figure 8 shows IHC images of antibodies against CLDN18.2, 14G11G2D2, and EPR19202 on stomach and skeletal muscle. EPR19202 stains positively on both stomach tissue and skeletal muscle, while 14G11G2D2 stains positively only on stomach tissue.

[0052] Figure 9 shows a representative IHC image of 14G11G2D2 antibodies on various tissues of gastric cancer, pancreatic cancer, cholangiocarcinoma, and non-small cell lung cancer (NSCLC) with strong (+++), moderate (++), weak (+), and negative (-) staining.

[0053] Figure 10 shows a comparison of IHC staining between the 14G11G2D2- antibody and the 14G11G2D2-6hothh antibody conjugate on gastric sections.

[0054] Figure 11 shows all sequences in this application.

Detailed description of the invention

[0055] The following description of the disclosure is intended only to demonstrate various embodiments thereof. As such, the specific changes discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and such equivalent embodiments are intended to be included in the present description. All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

Definitions

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

[0057] The term "antibody" as used herein means any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A native, intact antibody consists of two heavy (H) and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consisting of a variable region (V _H ) and the first, second, and third constant regions ( _CH1 , _CH2 , _CH3, respectively); mammalian light chains are classified as λ or κ, each light chain consisting of a variable region (V _L ) and a constant region. An antibody is shaped like a "Y," with the Y arm consisting of the second and third constant regions of two heavy chains linked by a disulfide bond. Each arm of the Y includes the variable region and first constant region of one heavy chain, linked to the variable and constant regions of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (the light chain CDR includes LCDR1, LCDR2, and LCDR3; the heavy chain CDR includes HCDR1, HCDR2, and HCDR3). The CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified according to the numbering system of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C, Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); N. R. 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)). The three CDRs are located between flanking regions known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of antibodies include IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains. Some of the major classes of antibodies are further subdivided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgA1 (alpha1 heavy chain), or IgA2 (alpha2 heavy chain). The present invention includes all antibodies and antibody derivatives described herein, which are encompassed by the term "antibody" for the purposes of the invention. The term "antibody derivative" means any modified form of an antibody, such as a conjugate of an antibody and another agent, an antibody fragment, or a fusion protein comprising an antibody or an antibody fragment.

[0058] As used herein, the term "antigen-binding fragment" means an antibody fragment formed from an antibody fragment comprising one or more CDRs, or any other portion of the antibody that binds to an antigen, but does not include the intact native structure of the antibody. Examples of an antigen-binding fragment include, but are not limited to, a diabody, Fab, Fab', F(ab') ₂ , Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody (scFv), an scFv dimer (bivalent diabody), a multispecific antibody fragment, a camel single-domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody. The antigen-binding fragment is capable of binding to the same antigen as the parent antibody. In some embodiments, the antigen-binding fragment may include one or more CDRs of a particular antibody.

[0059] "Fab" in relation to an antibody means a monovalent antigen-binding fragment of an antibody consisting of one light chain (both variable and constant regions) linked to the variable region and the first constant region of one heavy chain by a disulfide bond. Fab can be produced by digestion of the antibody with papain at residues located proximal to the N-terminus of the disulfide bond between the heavy chains of the loop region.

[0060] "Fab" means a Fab fragment including a portion of the loop region that can be obtained by digesting an antibody with pepsin at residues located near the C-terminus of the interheavy chain disulfide bond of the loop region and thus differs from Fab by a small number of residues (including one or more cysteines) in the loop region.

[0061] "F(ab') ₂ " means a dimer of Fab', which includes two light chains and part of two heavy chains.

[0062] "Fc" in relation to an antibody means that portion of the antibody that consists of the second and third constant regions of the first heavy chain linked to the second and third constant regions of the second heavy chain by a disulfide bond. The Fc regions of IgG and IgM contain three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). It can be obtained by digestion of the antibody with papain. The Fc portion of the antibody is responsible for various effector functions, such as ADCC, ADCP, and CDC, but is not involved in antigen binding.

[0063] "Fv" in relation to an antibody means the smallest fragment of an antibody that contains a complete antigen-binding site. An Fv fragment consists of the variable region of one light chain linked to the variable region of one heavy chain. "DsFv" means a disulfide-stabilized Fv fragment in which the variable region of one light chain and the variable region of one heavy chain are linked by a disulfide bond.

[0064] "Single chain Fv antibody" or "scFv" means an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other directly or through a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)). "ScFv dimer" means a single chain consisting of two heavy chain variable regions and two light chain variable regions with a linker. In some embodiments, an "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) comprising V _H -V _L (linked by a peptide linker) dimerized with another V _H -V _L molecule such that the V _H of one molecule coordinate with the V _L of the other molecule and form two binding sites that may target the same antigens (or epitopes) or different antigens (or epitopes). In other embodiments, an "scFv dimer" is a bispecific diabody comprising V _H1 -V _L2 (linked by a peptide linker) associated with V _L1 -V _H2 (also linked by a peptide linker) such that V _H1 and V _L1 coordinate, and V _H2 and V _L2 coordinate, wherein each coordinated pair has a different antigen specificity.

[0065] "Single-knot Fv-Fc antibody" or "scFv-Fc" means an engineered antibody consisting of an scFv fused to the Fc region of an antibody.

[0066] "Camel single domain antibody,""heavy chain antibody,""nanobody," or "HCAb" means an antibody that contains two V _H domains and no light chains (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); WO 94/04678; WO 94/25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the camelid family (camels, dromedaries, and llamas). Despite the absence of light chains, camel antibodies possess a genuine repertoire of antigen-binding properties (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr; 54(1):39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of the antibody heavy chain (VHH domain) is the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0067] "Diabodies" include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V _H domain linked to a V _L domain in a single polypeptide chain (V _H -V _L or V _L -V _H ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14):6444-8 (1993); EP 404097; WO 93/11161). The two domains on one chain cannot be linked because the linker is too short, so the domains are forced to link to complementary domains on another chain, thus creating two antigen-binding sites. The antigen-binding sites may be directed to the same or different antigens (or epitopes).

[0068] "Domain antibody" means an antibody fragment comprising only the variable region of a heavy chain or the variable region of a light chain. In some embodiments, two or more V _H domains are covalently linked to a peptide linker to form a bivalent or multivalent domain antibody. The two V _H domains of a bivalent domain antibody may target the same or different antigens.

[0069] "(dsFv) ₂ " means disulfide-stabilized Fv fragments consisting of three peptide chains: two V _H molecules connected by a peptide linker and linked by disulfide bridges to two V _L molecules.

[0070] "Bispecific diabody" means a diabody that targets two different antigens (or epitopes). It may comprise V _H1 -V _L2 (linked by a peptide linker) linked to V _L1 -V _H2 (also linked by a peptide linker) via a disulfide bridge between V _H1 and V _L1 .

[0071] "Bispecific dsFv" or "dsFv-dsFv" refers to disulfide-stabilized Fv fragments that target two different antigens (or epitopes). They may comprise three peptide chains: V _H1 -V _H2 molecules, where the heavy chains are linked by a peptide linker (e.g., a long flexible linker) and connected via disulfide bridges to V _L1 and V _L2 molecules, respectively. Each disulfide-linked heavy and light chain has different antigen specificities.

[0072] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from humans, and, where applicable, constant regions derived from humans.

[0073] The term "chimeric" as used herein refers to an antibody or antigen-binding fragment that has a portion of the heavy and/or light chain derived from one species and the remainder of the heavy and/or light chain derived from another species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0074] "Anti-CLDN18.2 antibody" or "anti-CLDN18.2 antibody" as used herein means an antibody that is capable of specifically binding to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity, such as to provide diagnostic and/or therapeutic use.

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

[0076] The term "specific binding" or "specifically binds" or "binding specificity" as used herein means a non-random binding reaction between two molecules, such as between an antibody and an antigen.

[0077] "Degree (%) of sequence identity" with respect to 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 aligning the sequences and, if necessary, introducing gaps to achieve maximum correspondence. Alignment for the purpose of determining the degree of amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul S.F. et al, J. Mol. Biol., 215:403–410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, 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 software or Megalign (DNASTAR). Skilled technologists can use the default parameters provided by the tool or customize the parameters to suit alignment needs, such as by selecting a suitable algorithm.

[0078] The term "amino acid" as used herein means an organic compound containing an amine ( _-NH2 ) and a carboxyl (-COOH) functional group together with a side chain specific to each amino acid. Amino acid names are also represented in the present invention by standard one-letter or three-letter designations, which are indicated below.

[0079] A "conservative substitution" with respect to an amino acid sequence means the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and He), among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gin), among residues with acidic side chains (e.g., Asp, G1u), among amino acids with basic side chains (e.g., His, Lys, and Arg), or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, a conservative substitution generally does not cause significant changes in the conformational structure of the protein and therefore may preserve the biological activity of the protein.

[0080] An "isolated" substance has been manually altered by man from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it has been sufficiently separated from coexisting materials of its natural state so as to exist in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In some embodiments, "isolated antibody or antigen-binding fragment thereof" means an antibody or antigen-binding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% according to electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion-exchange chromatography or reversed-phase HPLC).

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

[0082] "Effector functions" as used herein refers to biological activities mediated by the binding of the Fc region of an antibody to its effectors, such as the C1 complex and the Fc receptor. Exemplary effector functions include: complement-dependent cytotoxicity (CDC), induced by the interaction of antibodies and Clq in the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC), induced by the binding of the Fc region of an antibody to the Fc receptor on an effector cell; and antibody-dependent cell-mediated phagocytosis (ADCP), where non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. Effector functions include both those that act after antigen binding and those that act independently of antigen binding.

[0083] "Treatment" or "therapy" of a condition, as used herein, includes preventing or alleviating the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or stopping symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0084] By "at risk" is meant a subject, i.e., a patient, who has a higher than normal probability of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had or currently has a disease, in particular cancer, is a subject who has an increased risk of developing the disease, since such a subject may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of developing cancer metastasis. In the context of the present invention, terms such as "protection," "prevention," "prophylaxis" mean preventing or treating, or both, ways of preventing the onset and/or spread of a disease in a subject, and, in particular, minimizing the likelihood of developing a disease in a subject or delaying the development of a disease. For example, a person at risk of developing a tumor, as described above, may be a candidate for tumor-preventive therapy. Immunotherapy can be carried out using any of a variety of techniques in which agents function to remove antigen-expressing cells from the patient's body.

[0085] "Standard of care" as described herein includes the administration of a therapeutic agent to a patient in accordance with standard clinical practice. "Standard of care" as used herein is a treatment process, including a drug or combination of drugs, radiation therapy (RT), surgery, or other medical intervention, that is recognized by practicing physicians as appropriate, accepted, and/or widely used for a particular patient type, disease, or clinical circumstance. Standard of care treatments for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), an alliance of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES ^® ), which contain detailed, up-to-date information on standard of care treatments for common types of cancer (see NCCN GUIDELINES ^® , 2013).

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

[0087] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent, such as an antibody of the present invention, is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or condition, or promotes regression of the disease/condition, as evidenced by a reduction in the severity of symptoms of the disease/condition, an increase in the frequency and duration of periods without symptoms of the disease/condition, or the prevention of worsening of the condition or disability due to the disease/condition. The ability of a therapeutic agent to promote regression of a disease can be assessed using a variety of methods known to those skilled in the art, such as in humans during clinical trials, in animal model systems predictive of efficacy in humans, or by assessing the activity of the agent in in vitro assays. A therapeutically effective amount of a drug includes a "prophylactically effective amount," meaning any amount of a drug that, when administered alone or in combination with an antineoplastic agent to a subject at risk of developing a CLDN18.2-associated disease or condition, such as cancer (e.g., a subject with a premalignant condition), or recurrence of the disease/condition, inhibits the development or recurrence of the disease/condition. In preferred embodiments, the prophylactically effective amount completely prevents the development or recurrence of a CLDN18.2-associated disease or condition (e.g., cancer). "Inhibiting" the development or recurrence of a disease/condition (e.g., cancer) means either reducing the likelihood of developing or recurring the disease/condition, or completely preventing the development or recurrence of the disease/condition.

[0088] The term "vector" as used herein means a carrier into which a genetic element can be operably inserted to provide expression of the genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element.

[0089] "Host cell" as used herein means a cell into which an exogenous polynucleotide and/or vector has been introduced.

[0090] The term "CLDN18" means claudin 18 and includes any splice variants of claudin 18, such as CLDN18.1 and CLDN18.2. CLDN18.1 and CLDN18.2 differ in the N-terminal portion, which includes the first transmembrane (TM) region and loop 1, while the main protein sequence at the C-terminus is identical.

[0091] The term "CLDN18.2" refers to splice variant 2 of claudin-18 derived from mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In some embodiments, CLDN18.2 is human CLDN18.2. An exemplary sequence of human CLDN18.2 includes the human CLDN18.2 protein (NCBI Sequence Number NP_001002026.1, or SEQ ID NO:20). An exemplary sequence of CLDN18.2 includes the Mus musculus (house mouse) CLDN18.2 protein (NCBI Sequence Number NP_001181852.1), the Macaca fascicularis (cynomolgus macaque) CLDN18.2 protein (NCBI Sequence Number XP_015300615.1).

[0092] The term "CLDN18.1" means splice variant 1 of claudin-18 derived from mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In some embodiments, CLDN18.1 is human CLDN18.1. An exemplary sequence of human CLDN18.1 includes human CLDN18.1 protein (NCBI Sequence Number NP_057453.1, or SEQ ID NO:21), Mus musculus (house mouse) CLDN18.2 protein (NCBI Sequence Number NP_001181851.1), and Macaca fascicularis (cynomolgus macaque) CLDN18.2 protein (NCBI Sequence Number XP_005545920.1).

[0093] The terms "CLDN18.2" and "CLDN18.2" also encompass variants of the exemplary sequences, such as mutants, conformational variants, isoforms, allelic variants, species variants, and species homologues, in particular those found in nature.

[0094] "CLDN18.2-associated disease or condition" as used herein means any disease or condition caused by, aggravated by, or otherwise associated with increased or decreased expression or activity of CLDN18.2. In some embodiments, the CLDN18.2-associated condition is, for example, cancer.

[0095] "Cancer" as used herein means any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration, or metastasis, and includes both solid tumors and non-solid tumors (e.g., hematological malignancies) such as leukemia.

[0096] "Solid tumor" as used herein means a solid mass of neoplastic and/or malignant cells.

[0097] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient, and/or salt as a whole are chemically and/or physically compatible with the other ingredients in the formulation, and physiologically compatible with the recipient.

[0098] The term "metastasis" or "metastatic cancer" as used herein refers to the spread of cancer cells from a primary site to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of endothelial membranes to enter body cavities and vessels, and then, after transport by the blood, penetration into target organs. Finally, the growth of a new tumor, i.e., a secondary or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, since tumor cells or components may remain and develop metastatic potential. In one embodiment of the invention, the term "metastasis" means "distant metastasis," which means a metastasis distant from the primary tumor and the regional lymph node system. The cells of a secondary or metastatic tumor are similar to the cells of the primary tumor. This means that, for example, if stomach cancer metastasizes to the liver, the secondary tumor will consist of atypical stomach cells, not atypical liver cells. In this case, the liver tumor is called metastatic stomach cancer, not liver cancer.

[0099] Reference herein to an "approximate" value or parameter includes (and describes) embodiments of the invention that are directed to that value or parameter per se. For example, a description referring to a value "about X" includes a description of "X." Numerical ranges include the numbers defining the range. The term "about" means the stated value of the variable and all values of the variable that are within the experimental error of the stated value (e.g., within a 95% confidence interval for the mean) or within 10% of the stated value, whichever is greater. When the term "about" is used in the context of a period of time (years, months, weeks, days, etc.), the term "about" means that period of time plus or minus one unit of the next lower period of time (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 stated value, whichever is greater.

[00100] Antibodies against CLDN18.2

[00101] The present invention relates to antibodies against CLDN18.2 and antigen-binding fragments thereof.

[00102] CLDN18.2 is a splice variant of claudin-18 (CLDN18). CLDN18 belongs to the tetraspanin family and has four hydrophobic regions. CLDN18 has several different conformations that can be selectively targeted by antibodies (see Sahin U et al. Clinical Cancer Research, 2008, 14(23): 7624-7634). CLDN18-Conformation-1 has all four hydrophobic regions serving as transmembrane domains (TMs) and two extracellular loops (loop 1, encompassed by hydrophobic region 1 and hydrophobic region 2; loop 2, encompassed by hydrophobic regions 3 and 4), as described for the vast majority of claudin family members. The second conformation (CLDN18-Conformation-2) implies that, as described for PMP22, the second and third hydrophobic domains do not completely cross the plasma membrane, so that the portion (loop D3) between the first and fourth transmembrane domains is extracellular. The third conformation (CLDN18-Conformation-3) has a large extracellular domain with two internal hydrophobic regions encompassed by the first and fourth hydrophobic regions. Due to the classical N-glycosylation site in the D3 loop, the topological variants CLDN18, claudin 18 topology-2 and claudin 18 topology-3, have an additional extracellular N-glycosylation site.

[00103] CLDN18 has two distinct splice variants that are present in both mice and humans. Splice variants CLDN18.1 and CLDN18.2 differ in the first 21 amino acids at the N-terminus, which includes the first TM and loop 1, while the protein sequences at the C-terminus are identical. Although the two isoforms share 92% amino acid sequence identity, their expression is mutually exclusive: CLDN18.1 is predominantly expressed in normal lung tissue, and CLDN18.2 is expressed in normal gastric tissue (see Niimi T, et al. Molecular and cellular biology, 2001, 21(21): 7380-7390.). The amino acid sequences of human CLDN18.1 and CLDN18.2 are shown below.

[00104] Human claudin 18.2 (Code: NP_001002026.1) amino acid sequence (SEQ ID NO:20):

[00105] Human claudin 18.1 (Code: NP_057453.1) amino acid sequence (SEQ ID NO:21):

[00106] In normal tissue, CLDN18.2 expression is restricted to the basement membrane of mucosal cells and is inaccessible to therapeutic antibodies. Under pathological conditions (e.g., tumor cells), gastric mucosal cell polarity 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 human cancers, including gastric, esophageal, pancreatic, and lung tumors, as well as human cancer cell lines (see Sahin Ugur et al, Clin Cancer Res 2008; 14(23); Matsuda Y et al. Cancer science, 2007, 98(7): 1014-1019.). Aberrant ectopic expression of CLDN18.2 has also been reported in pancreatic, ovarian, biliary, and lung adenocarcinomas in multiple studies (see, e.g., Karanjawala ZE et al, Am J Surg Pathol. 2008 Feb; 32(2): 188–96; Micke P et al, Int J Cancer. 2014 Nov 1; 135(9):2206–14; Keira Y et al, Virchows Arch. 2015 Mar; 466(3):265–77; Coati et al, Br J Cancer. 2019 Jul; 121(3):257–263; Dottermusch et al, Virchows Arch. 2019 Nov; 475(5):563–571; Rohde et al, Jpn J Clin Oncol. 2019 Sep l;49(9):870-876; Woll et al, Int J Cancer. 2014 Feb l;134(3):731-9; Espinoza et al, Histopathology. 2019 Mar;74(4):597-607; Shinozaki et al, Virchows Arch. 2011 Jul;459(l):73-80).

[00107] In one aspect, the present invention provides anti-CLDN18.2 monoclonal antibodies and antigen-binding fragments thereof. The anti-CLDN18.2 monoclonal antibodies and antigen-binding fragments provided herein are capable of specifically binding to CLDN18.2 (e.g., human CLDN18.2), a fragment of CLDN18.2, or a fusion polypeptide comprising a fragment of CLDN18.2. In some embodiments, the fragment of CLDN18.2 comprises the first extracellular loop of human CLDN18.2 or a sequence within the first extracellular loop of human CLDN18.2. In some embodiments, the fragment of human CLDN18.2 comprises the amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:19. In some embodiments, the fusion polypeptide comprises additional amino acid residues attached to the N-terminus and/or C-terminus of the CLDN18.2 fragment. In some embodiments, the CLDN18.2 fragment contained in the fusion polypeptide forms a loop.

[00108] The CLDN18.2 antibodies and antigen-binding fragments provided herein are capable of specifically binding to CLDN18.2-expressing cells. In some embodiments, the CLDN18.2-expressing cells are pretreated cells. The term "pretreated" as used herein with respect to cells means that the cells have been treated such that their surface proteins, such as CLDN18.2, are denatured or are no longer in their native conformation. For example, CLDN18.2-expressing cells can be pretreated with one or more chemicals, such as formalin, paraffin, or acetone, or a physical treatment, such as freezing or heating. In some embodiments, the CLDN18.2-expressing cells are formalin-fixed paraffin-embedded (FFPE) cells.

[00109] The binding of the CLDN18.2 antibodies and antigen-binding fragments provided herein to an antigen can be represented by an "half-maximal effective concentration" ( _EC50 ) value, which refers to the concentration of the antibody at which 50% of its maximal effect (e.g., binding) is observed. The _EC50 value can be measured by methods known in the art, for example, by sandwich assays such as ELISA, Western blot, and other quantitative measurements of the extent of binding. The EC50 can be measured in a corresponding quantitative measurement of the extent of binding, where serial dilutions of antibodies are tested for binding to the antigen, and the concentration at 50% of the maximal binding is determined. In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein specifically binds to human CLDN18.2, a fragment of human CLDN18.2, a fusion polypeptide comprising a fragment of human CLDN18.2, human CLDN18.2-expressing cells, or pre-treated human CLDN18.2-expressing cells. In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein specifically binds to a fusion polypeptide comprising 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) as measured by ELISA.

[00110] The binding affinity to human CLDN18.2 of the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein can also be characterized, for example, by _KD , which is the ratio of the rate of dissociation to the rate of association ( _koff / _kon ). _KD can be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance (SPR), microscale thermophoresis, and HPLC-MS. In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein binds to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 with a _KD of no more than ^10-6 M (e.g., no more than ^{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.

[00111] In some embodiments, anti-CLDN18.2 antibodies and antigen-binding fragments thereof do not cross-react with human CLDN18.1. An anti-CLDN18.2 antibody that "does not cross-react with" or "does not cross-react with" human CLDN18.1 is an antibody that does not produce undesirable results, such as false positive results, in detection assays (e.g., IHC) for human CLDN18.2. In some embodiments, binding to human CLDN18.1 is not detected in a detection assay (e.g., IHC or flow cytometry). The level of binding can be determined as the level of antigen-antibody complex that is formed at a given concentration of antibody and a given concentration of antigen. In some embodiments, the CLDN18.2 antibodies and antigen-binding fragments thereof provided herein bind to human CLDN18.1 at a level or affinity lower (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% lower) than the binding of the GC182 antibody to human CLDN18.1.

[00112] In some embodiments, no more than 5%, 4%, 3%, 2%, 1%, 0.8%), 0.5%, 0.3%, or 0.1% of human CLDN18.1-expressing cells are detected positive in an IHC assay using the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein. In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein do not show detectable binding to human CLDN18.1 in an IHC assay. In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein do not show detectable binding to human CLDN18.1 in a human lung tissue sample with a benign tumor in an IHC assay.

[00113] In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein do not have cross-reactivity to human CLDN18.1 in a formalin-fixed paraffin-embedded (FFPE) sample at an antibody concentration of 1 nM as measured by ELISA or at an antibody concentration of 0.5 μg/mL as measured by IHC assay. The IHC assay can be performed according to the procedures and conditions described in Example 8 or 9.

[00114] In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein do not have cross-reactivity to benign tumor cells, except for gastric epithelial cells. In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein do not have cross-reactivity to human benign tumor tissues, such as lung tissue, intestinal tissue, kidney tissue, lymphoid tissue, thyroid tissue, skeletal muscle tissue, or breast tissue. This distinguishes the antibodies provided herein from existing anti-CLDN18.2 monoclonal antibodies. In an example, the 43-14A antibody was shown to bind to human CLDN18.1 and therefore exhibit cross-reactivity with human benign lung tissue; the IHC result is presented in the Ventana package insert (see Ventana CLDN18(43-14A) Assay, Ref. 790-7027, 08504148001). In another example, the GC182 antibody was also found to cross-react with CLDN18.1 and therefore stain human benign lung tissue (see Example 4 and Figure 2 of the present invention). In another example, the anti-CLDN18.2 antibody EPR19202 (available from Abcam as ab222512) was found to bind non-specifically to skeletal muscle tissue and therefore exhibit cross-reactivity with benign skeletal muscle tissue.

[00115] Illustrative Anti-CLDN18.2 Antibody

[00116] In some embodiments, the present invention provides isolated antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 (e.g., human CLDN18.2), including

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

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

where X ₁ is R or T, X ₂ is N or Y, X ₃ is F or I, X ₄ is G or R, X ₅ is F or G, X ₆ is D or N, X ₇ is I or T, X ₈ is E or V, X ₉ is S or N, X ₁₀ is G or R, and X ₁₁ is V or I.

[00117] In some embodiments, the present invention 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 set of sequences presented in SEQ ID NOs: 1-6, or selected from the set of sequences presented in SEQ ID NOs: 2, 6, 7, and 9-11.

[00118] In some embodiments, the CLDN18.2 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence shown in SEQ ID NO:5 or 11. The heavy chain CDR3 regions are located in the center of the antigen-binding site and are therefore believed to have the greatest contact with the antigen and provide the greatest free energy for the affinity of the antibody for the antigen. The heavy chain CDR3 is also believed to be the most diverse CDR of the antigen-binding site to date in length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81 (1983)). Heavy chain CDR3 diversity is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13:37–45 (2000)), as well as the desired antigen-binding affinity (Schier R, etc. J Mol Biol. 263:551–67 (1996)).

[00119] In some embodiments, the CLDN18.2 antibodies and antigen-binding fragments provided herein comprise: a heavy chain CDR1 with an amino acid sequence selected from: SEQ ID NO:1 and SEQ ID NO:7, and/or a heavy chain CDR2 with an amino acid sequence selected from: SEQ ID NO:3 and SEQ ID NO:9, and/or a heavy chain CDR3 with an amino acid sequence selected from: SEQ ID NO:5 and SEQ ID NO:11; and/or a light chain CDR1 with an amino acid sequence selected from: SEQ ID NO:2, and/or a light chain CDR2 with an amino acid sequence selected from: SEQ ID NO:4 and SEQ ID NO:10, and/or a light chain CDR3 with an amino acid sequence selected from: SEQ ID NO:6.

[00120] In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (e.g., human CLDN18.2), wherein the antibody comprises:

a) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO:1, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO:3, and a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO:5; or

b) a heavy chain CDR1 with the sequence presented in SEQ ID NO:7, a heavy chain CDR2 with the sequence presented in SEQ ID NO:9, and a heavy chain CDR3 with the sequence presented in SEQ ID NO:11.

[00121] In some embodiments, an antibody or antigen-binding fragment thereof provided herein comprises:

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

b) a light chain CDR1 with the amino acid sequence presented in SEQ ID NO:2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO:10, and a light chain CDR3 with the amino acid sequence presented in SEQ ID NO:6.

[00122] In some embodiments, an antibody or antigen-binding fragment thereof provided herein comprises:

a) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO: 1, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO: 3, a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO: 5, a light chain CDR1 with the amino acid sequence presented in SEQ ID NO: 2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO: 4, a light chain CDR3 with the amino acid sequence presented in SEQ ID NO: 6; or

b) a heavy chain CDR1 with the amino acid sequence presented in SEQ ID NO: 7, a heavy chain CDR2 with the amino acid sequence presented in SEQ ID NO: 9, a heavy chain CDR3 with the amino acid sequence presented in SEQ ID NO: 11, a light chain CDR1 with the amino acid sequence presented in SEQ ID NO: 2, a light chain CDR2 with the amino acid sequence presented in SEQ ID NO: 10, a light chain CDR3 with the amino acid sequence presented in SEQ ID NO: 6.

[00123] While CDRs are known to be responsible for antigen binding, it has been found that not all 6 CDRs are necessarily essential or immutable. In other words, 1, 2, or 3 of the CDRs presented above (e.g., corresponding to any of SEQ ID NOs: 1-6, or SEQ ID NOs: 2, 6, 7, and 9-11) can be replaced, changed, or modified while still substantially maintaining the specific binding affinity for CLDN18.2. Antibodies having such modified or variant CDRs are also included in the present invention.

[00124] In some embodiments, an antibody or antigen-binding fragment thereof provided herein comprises:

a) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO:13 and a light chain variable region with the amino acid sequence presented in SEQ ID NO:14; or

b) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO:15 and a light chain variable region with the amino acid sequence presented in SEQ ID NO:16.

[00125] The antibody "14G11" as used herein means a murine antibody having a heavy chain variable region of SEQ ID NO:13 and a light chain variable region of SEQ ID NO:14.

[00126] The antibody "69H2" as used herein means a murine antibody having a heavy chain variable region of SEQ ID NO:15 and a light chain variable region of SEQ ID NO:16.

[00127] Table 1 shows the CDR sequences of the CLDN18.2 antibodies 14G11 and 69H2. The heavy chain and light chain variable region sequences are also shown below in Table 2.

[00130] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences if the antibodies and antigen-binding fragments thereof 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 to any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant methods. The FR sequences can be readily identified by one of skill in the art based on the CDR sequences from Table 1 above and the variable region sequences from Table 2 above, since it is well known in the art that the CDR region is flanked by two FR regions in the variable region.

[00131] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein further comprises an immunoglobulin constant region. The constant region optionally comprises an IgG heavy chain constant region and/or a light chain constant region. The heavy chain constant region comprises the CH1, loop, 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 a Cκ or Cλ.

[00132] In some embodiments, the anti-CLDN18.2 antibodies and fragments thereof provided herein further comprise a constant region of a mouse IgG1, IgG2, IgG3, or IgG4. In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of a mouse IgG1 isotype. In some embodiments, the murine IgG1 heavy chain constant region 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, and/or the murine IgG1 light chain constant region comprises the amino acid sequence of SEQ ID NO: 18 or a homologous sequence thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[00133] The CLDN18.2 antibodies or antigen-binding fragments provided herein are 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-idiotypic antibody, a fusion protein, a dimerized or polymerized antibody, or a modified antibody (e.g., a glycosylated antibody). A recombinant antibody is an antibody produced in vitro using recombinant techniques rather than in animals.

[00134] In some embodiments, the CLDN18.2 antibodies or antigen-binding fragments thereof provided herein are divalent, tetravalent, hexavalent, or multivalent. Any molecule that is more than divalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[00135] In some embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein comprise all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. In one embodiment, the anti-CLDN18.2 antibodies and antigen-binding fragments provided herein are a single-domain antibody that consists of all or a portion of a heavy chain variable domain as provided herein. More detailed information regarding such a single-domain antibody is available in the art (see, for example, U.S. Patent No. 6,248,516).

[00136] Antibody variants

[00137] The CLDN18.2 antibodies and antigen-binding fragments thereof provided herein also include various variants of the 14G11 and 69H2 antibodies.

[00138] In some embodiments, the CLDN18.2 antibody or antigen-binding fragments thereof provided herein comprise one or more mutations in one or more CDR sequences provided in Table 1 above, one or more non-CDR sequences of the heavy chain variable region or the light chain variable region provided in Table 2 above, and/or constant region sequences (e.g., an Fc region) provided in SEQ ID NO: 17 or 18, while maintaining binding specificity for CLDN18.2, in particular for human CLDN18.2, or more particularly for an epitope within the amino acid sequence of SEQ ID NO: 19. These are also referred to as variants of the 14G11 and 69H2 antibodies, or variant antigen-binding fragments. In some embodiments, variants retain binding affinity similar to or even higher than that of the parent antibody (e.g., antibodies 14G11 or 69H2). "Mutations" or "mutated" as used herein refer to substitutions, insertions, and/or deletions in an amino acid sequence or polynucleotide sequence. In some embodiments, at least one (or all) mutation(s) comprise a conservative substitution.

[00139] In some embodiments, antibody variants include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in total in the CDR sequences, in the FR sequences, or in the variable region sequences of antibodies 14G11 and 69H2. In some embodiments, antibody variants comprise 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 to the one or more listed in Table 1, and yet retain binding specificity for CLDN18.2, optionally having a binding affinity that is similar to or even higher than that of the parent antibody (e.g., antibody 14G11 or 69H2).

[00140] In some embodiments, antibody variants comprise 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 to the sequence(s) 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 to that(s) of SEQ ID NO: 14 or 16, and still retains binding specificity with CLDN18.2, optionally having a binding affinity at a level similar to or even higher than that of the parent antibody (e.g., antibody 14G11 or 69H2). In some embodiments, a total of 1 to 10 amino acid residues in a sequence selected from SEQ ID NOs: 13-16 are mutated. In some embodiments, the mutations occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, one or more mutations are conservative substitutions. In some embodiments, all mutations are conservative substitutions.

[00141] In some embodiments, the present invention relates to a variant of the 14G11 or 69H2 antibody, wherein the variant comprises:

a) a heavy chain CDR1 (HCDR1) sequence with a degree of identity to the sequences presented in SEQ ID NO:1 or SEQ ID NO:7 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

b) a heavy chain CDR2 (HCDR2) sequence with a degree of identity to the sequences presented in SEQ ID NO:3 or SEQ ID NO:9 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

c) a heavy chain CDR3 (HCDR3) sequence with a degree of identity to the sequences presented in SEQ ID NO:5 or SEQ ID NO:11 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

d) a light chain CDR1 (LCDR1) sequence with a degree of identity to the sequence presented in SEQ ID NO:2 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

e) a light chain CDR2 (LCDR2) sequence with a degree of identity to the sequences presented in SEQ ID NO:4 or SEQ ID NO:10 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or

f) a light chain CDR3 (LCDR3) sequence with a degree of identity to the sequence presented in SEQ ID NO:6 of at least about 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and

while maintaining binding specificity to CLDN18.2, optionally having a binding affinity at a level similar to or even higher than that of the parent antibody (e.g., antibody 14G11 or 69H2).

[00142] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 having no more than 3, 2, or 1 amino acid mutations in SEQ ID NO: 1 or SEQ ID NO: 7, HCDR2 having no more than 6, 5, 4, 3, 2, or 1 amino acid mutations in SEQ ID NO: 3 or SEQ ID NO: 9, HCDR3 having no more than 6, 5, 4, 3, 2, or 1 amino acid mutations in SEQ ID NO: 5 or SEQ ID NO: 11, LCDR1 having no more than 2 or 1 amino acid mutations in SEQ ID NO: 2, LCDR2 having no more than 3, 2, or 1 amino acid mutations in SEQ ID NO: 4 or SEQ ID NO: 10, and/or LCDR3 having no more than 3, 2, or 1 amino acid mutations in SEQ ID NO: 6, and while maintaining binding specificity to CLDN18.2, and, optionally, having a binding affinity at a level similar to or even higher than that of the parent antibody (e.g., antibody 14G11 or 69H2). In some embodiments, one or more mutations are conservative substitutions. In some embodiments, all mutations are conservative substitutions.

[00143] In some embodiments, antibody variants provided herein retain the CLDN18.2 binding specificity of their parent antibodies, but have one or more desirable properties conferred by mutation. For example, antibody variants may have improved antigen binding affinity, an improved glycosylation pattern, a reduced glycosylation risk, reduced deamination, reduced or attenuated effector function, improved pH-dependent binding to the FcRn receptor, an increased pharmacokinetic half-life, pH sensitivity, and/or conjugation compatibility (e.g., one or more engineered cysteine residues), and more. Such variants are also known as affinity variants, glycosylation variants, cysteine variants, Fc variants, and so on, which are described in more detail below.

[00144] a) Affine variant

[00145] An affinity variant may comprise modifications or substitutions in one or more of the CDR sequences presented in Table 1 above, one or more of the framework (FR) sequences presented herein, or the heavy or light chain variable region sequences presented in Table 2 above.

[00146] An affinity variant retains the specific binding affinity to CLDN18.2 of the parent antibody or even has an improved specific binding affinity to CLDN18.2 compared to the parent antibody. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology and then tested for binding affinity to human CLDN18.2. In another example, computer software can be used to virtually simulate the binding of antibodies to human CLDN18.2 and determine the amino acid residues of the antibodies that form the binding interface. Such residues can either be deleted by mutation to prevent a decrease in binding affinity or targeted for mutation to ensure stronger binding.

[00147] b) Glycosylation option

[00148] The CLDN18.2 antibodies and antigen-binding fragments provided herein also include a glycosylation variant that can be produced to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[00149] An antibody or antigen-binding fragment thereof may include one or more modifications that introduce or remove a glycosylation site. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies typically occurs at either an N-linkage or an O-linkage. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is replaced. A new glycosylation site can be created similarly by introducing such a tripeptide sequence or serine or threonine residue.

[00150] In some embodiments, the CLDN18.2 antibodies and antigen-binding fragments provided herein comprise a mutation at N297 (e.g., N297A, N297Q, or N297G) to remove the glycosylation site.

[00151] c) Cysteine-modified variant

[00152] The CLDN18.2 antibodies and antigen-binding fragments provided herein also include a cysteine variant that includes one or more introduced free cysteine amino acid residues.

[00153] A free cysteine residue is a residue that is not part of a disulfide bridge. A cysteine-modified variant is useful for conjugation, for example, with a cytotoxic and/or imaging compound, label, or radioisotype, including at the site of the introduced cysteine, for example, via maleimide or haloacetyl. Methods for modifying antibodies or antigen-binding fragments thereof to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.

[00154] d) Option Fc

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

[00155] Antigen-binding fragments

[00156] Also provided herein are antigen-binding fragments against CLDN18.2. In some embodiments, the antibodies and antigen-binding fragments provided herein comprise all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CLDN18.2 antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences are shown in Table 1 and various variants thereof (such as affinity variants, glycosylation variants, Fc variants, cysteine-modified variants, etc.).

[00157] In some embodiments, the anti-CLDN18.2 antigen-binding fragment provided herein is a diabody, Fab, Fab', F(ab') ₂ , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single chain antibody (scFv), scFv dimer (bivalent diabody), bispecific antibody, multispecific antibody, camelid single domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[00158] Various methods can be used to produce such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from a phage display library as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Other methods for producing antibody fragments will be apparent to one of skill in the art.

[00159] Epitope

[00160] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein binds to an epitope within the amino acid sequence DQWSTQDLYN (SEQ ID NO:19).

[00161] The term "epitope" as used herein refers to a specific group of atoms or amino acids of an antigen to which an antibody binds. An epitope may be a conformational epitope or a linear epitope. In some embodiments of the present disclosure, the epitopes bound by the CLDN18.2 antibodies provided herein are linear. Those skilled in the art will appreciate that it can be determined without undue experimentation whether an antibody binds to the same, an overlapping, or an adjacent epitope as an antibody of the present invention (e.g., the hybridoma/mouse antibodies 14G11 and 69H2) by determining whether they compete for binding to the CLDN18.2 antigenic polypeptide.

[00162] The present invention relates to monoclonal antibodies directed against a specific epitope located in the N-terminal portion of CLDN18.2, which are useful for the detection and identification of cells expressing CLDN18.2 without cross-reaction with CLDN18.1. According to the sequences of human CLDN18.1 and CLDN18.2, there are 8 different amino acids located between amino acids 28-70 (i.e., the N-terminal portion including the first transmembrane (TM) region and loop 1), while the C-terminal sequences of human CLDN18.1 and CLDN18.2 are identical. A linear epitope located at the N-terminus of human CLDN18.2 (amino acids 28-37 in the first extracellular domain, i.e., SEQ ID NO:19) was presented by Sahin U et al. (See Sahin Ugur et al, Clin Cancer Res 2008; 14(23) December 1, 2008). However, to date, no monoclonal antibodies directed to such a peptide fragment have been reported, and, to the best of the inventors' knowledge, the present disclosure provides for the first time monoclonal antibodies that bind to the peptide fragment of SEQ ID NO:19.

[00163] In another aspect, the present disclosure provides monoclonal antibodies or antigen-binding fragments thereof that compete for binding to CLDN18.2 with an antibody or antigen-binding fragment thereof provided herein, such as 14G11 and 69H2.

[00164] The term "compete for binding" as used herein with respect to two antigen-binding proteins (e.g., antibodies) means that one antigen-binding protein blocks or reduces the binding of the other to the 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 radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (ELISA), and sandwich assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, such an assay utilizes purified antigen bound to a solid surface or cells bearing the antigen, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of a test antibody. Typically, the test antibody is present in excess. When two antibodies compete for binding to CLDN18.2, the two antibodies bind to the same or an overlapping epitope, or to an adjacent epitope close enough to the epitope bound by the other antibody to create steric hindrance. Typically, when a competing antibody is present in excess, it inhibits (e.g., reduces) 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.

[00165] Polynucleotide and recombinant methods

[00166] The present invention relates to isolated polynucleotides encoding antibodies against CLDN18.2 and antigen-binding fragments thereof. The term "nucleic acid" or "polynucleotide" as used herein means deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise specified, a particular polynucleotide sequence also includes conservatively altered variants thereof (e.g., degenerate substitutions in codons), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly specified sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J Biol Chem. 260:2605–2608 (1985); and Rossolini et al., Mol Cell. Probes 8:91–98 (1994)).

[00167] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[00168] The present invention relates to vectors (e.g., expression vectors) comprising an isolated polynucleotide as provided herein. The vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as the yeast artificial chromosome (YAC), the bacterial artificial chromosome (BAC), or the P1-derived artificial chromosome (PAC), bacteriophages such as the lambda phage or the M13 phage, and animal viruses. The vector may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The vector may also include materials that facilitate its entry into the cell, including, but not limited to, a viral particle, a liposome, or a protein coat. The vector can be an expression vector or a cloning vector.

[00169] In some embodiments, the vectors provided herein are expression vectors. In some embodiments, the expression vector provided herein comprises a polynucleotide encoding the antibodies or antigen-binding fragments thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide sequence, and at least one selection marker.

[00170] Examples of vectors include, but are not limited to, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpesvirus (e.g., a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, a papovavirus (e.g., SV40), a lambda phage, and an M13 phage, 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, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p5TV-L, pProl8, pTD, pRSlO, pLexA, pACT2.2, pCMV-SCRIPT.RTM.., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[00171] Vectors comprising a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors provided herein include prokaryotic, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, enterobacteria such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus; salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans, and Shigella, as well as bacilli such as B. subtilis and B. licheniformis; Pseudomonas, such as P.aeruginosa, and Streptomyces.

[00172] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expressing vectors encoding an anti-CLDN18.2 antibody. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic microbial host. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906), K. thermotolerans and K. marxianus; Yarrowia (EP 402.226); Pichia pastoris (EP 183.070); Candida; Trichoderma reesia (EP 244.234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A.niger.

[00173] Suitable host cells for expressing the glycosylated antibodies or antigen fragment provided herein are derived from multicellular organisms, such as invertebrate cells, for example, plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (codling moth), and Bombyx mori. Various transfection virus strains are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus of the present invention, in particular for transfecting Spodoptera frugiperda cells. Cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco plants can also be used as hosts.

[00174] However, vertebrate cells are of greatest interest, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol. 36:59 (1977)); newborn hamster kidney cells (NHK, ATCC CCL 10); Chinese hamster ovary/-DНFR cells (CHO, Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); Mouse Sertoli cells (TM4, Mather, Biol Reprod. 23: 243–251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); brown rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y.Acad Sci 383:44–68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some preferred embodiments, the host cell is a cultured mammalian cell line such as CHO, BHK, NS0, 293 and derivatives thereof.

[00175] Host cells are transformed with the above-described expression or cloning vectors to produce antibodies against CLDN18.2 and cultured in a conventional nutrient medium modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, the antibody can be produced by homologous recombination known in the art.

[00176] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth Enz. 58:44 (1979), Barnes et al., Anal Biochem. 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 No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented, if necessary, with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffering agents (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^™ ), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary additives can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, and the like are consistent with those previously used with the host cell selected for expression and will be readily apparent to one of ordinary skill in the art.

[00177] Using recombinant methods, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the first step is to remove the particles, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 min. Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatants of such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. A protease inhibitor, such as PMSF, can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of additional contaminants.

[00178] Anti-CLDN18.2 antibodies and antigen-binding fragments thereof obtained from cells can be purified, for example, by hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification method.

[00179] In some embodiments, protein A immobilized on a solid phase is used for the immunoaffinity purification of an antibody and an antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc domain immunoglobulin present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J Immunol Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices exist. Mechanically stable matrices, such as controlled-pore glass or poly(styrene divinyl)benzene, allow for higher flow rates and shorter processing times compared to agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Other protein purification methods, such as ion-exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica chromatography, SEPHAROSE™ heparin chromatography, anion- or cation-exchange resin chromatography (e.g., polyaspartic acid), chromatofocusing, protein polyacrylamide gel electrophoresis, and ammonium sulfate precipitation, are also available depending on the antibody being extracted.

[00180] Following any pre-purification step, the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH in the range of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0-0.25 M salt).

[00181] The present invention relates to methods for expressing an anti-CLDN18.2 antibody or antigen-binding fragment thereof as provided herein, comprising culturing a host cell as provided herein under conditions under which the vector as provided herein is expressed.

[00182] Conjugates

[00183] In some embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof are linked or conjugated to one or more molecules. Examples of such compounds include, but are not limited to, a therapeutic agent (e.g., a DNA alkylator, a topoisomerase inhibitor, a tubulin-binding agent, or other anti-cancer drugs), a detectable label, a pharmacokinetic modifying compound (e.g., a polymer such as PEG that increases half-life), or a purification compound (e.g., a magnetic bead or nanoparticle).

[00184] The molecule may be attached to the antibodies or antigen-binding fragments thereof directly or through a linker or through another molecule, such as by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, mixing, or addition, among other methods. In some embodiments, the antibodies or antigen-binding fragments thereof are linked to one or more molecules through a linker. In some embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[00185] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific regions outside the epitope-binding portion that can be used to bind to one or more molecules. For example, such a region may include one or more reactive amino acid residues, such as, for example, cysteine or histidine residues, to facilitate covalent binding to a substance.

[00186] In some embodiments, anti-CLDN18.2 antibodies or antigen-binding fragments thereof are linked to one or more compounds that function to (i) provide a detectable signal; (ii) interact with a second label to alter the detectable signal provided by the first or second label, such as FRET (fluorescence resonance energy transfer); (iii) influence mobility, such as electrophoretic mobility, through charge, hydrophobicity, shape, or other physical parameters; or (iv) provide molecule capture, such as antibody/antigen affinity or ion complexation.

[00187] Such molecules include, but are not limited to, labels or molecules that are directly detectable (e.g., a radioactive isotope, a lanthanide, a chemiluminescent label, a chromophore, an enzyme label, colloidal gold particles, and a fluorescent label) by imaging, an enzymatic reaction, etc., as well as molecules that are indirectly detectable, such as by molecular interaction. Examples of an indirectly detectable label include biotin/avidin, biotin/streptavidin, a digoxigenin label, a hapten, a DNA molecule for detection, and particle labels (paramagnetic particles, metal particle labels, magnetic particle labels, polymer particle labels, etc.).

[00188] Examples of radioactive isotopes include S, C, I, H, and I. An antibody can be labeled with a radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed Wiley-Interscience, New York, NY, Pubs. (1991), and the radioactivity can be measured using a scintillation counter. Other radionuclides include ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ 1, ³⁵ S, ³ H, ⁹⁹ Tc, ⁹⁰ Y, ¹¹¹ In, ¹¹² In, ³² P, ¹⁴ C, ¹⁵ 0, ¹³ N, ¹⁸ F, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ⁵¹ Cr, ⁵⁷ To, ²²⁵ Ra, ⁶⁰ Co, ⁵⁹ Fe, ⁵⁷ Se, ¹⁵² Eu, ⁶⁴ Cu, ⁶⁷ Cu, ²¹⁷ Ci, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ²³⁴ Th, ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ⁵² Tr and ⁵⁶ Fe.

[00189] Fluorescent or luminescent labels include, but are not limited to, rare earth metal chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, aromatic acridinium ester label, imidazole label, acridimium salt label, oxalate ester label, aequorin label, 2,3-dihydrophthalazinediones, Texas red, dansyl, lissamine, umbelliferone, phycocritterin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE ^® and SPECTRUM ^GREEN® and/or derivatives of any one or more of the above. Fluorescent labels can be conjugated to the antibody using methods disclosed, for example, in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorimeter.

[00190] Another type of useful label is an enzyme-substrate label. Various enzyme-substrate labels exist (see U.S. Patent No. 4,275,149). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using various methods. For example, the enzyme may catalyze a color change in the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may change the fluorescence or chemiluminescence of the substrate. Methods for quantifying the change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited as a result of the chemical reaction and can then emit light, which can be measured (e.g., using a chemiluminometer), or donate energy to a fluorescent acceptor.

[00191] Numerous enzyme and substrate combinations are available to those skilled in the art (see U.S. Patent Nos. 4,275,149 and 4,318,980), such as: (i) horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as, for example, 3,3' diaminobenzidine (DAB), which produces a brown end product; 3-amino-9-ethylcarbazole (ABS), which upon oxidation produces a pink-red end product; 4-chloro-l-napthol (CN), which precipitates as a blue end product; and p-phenylenediamine dihydrochloride/pyrocathecol, which produces a blue-black product; orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB); (ii) alkaline phosphatase (AP) and p-nitrophenyl phosphate, naphthol AS-MX phosphate, Fast Red TR and Fast Blue BB, naphthol AS-BI phosphate, naphthol AS-TR phosphate, 5-bromo-4-chloro-3-indoxyl phosphate (BCIP), Fast Red LB, Fast Garnet GBC, nitroblue tetrazolium (NBT), and iodonitrotetrazolium violet (INT); and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase).

[00192] Other useful enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Methods for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langbne & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

[00193] CLDN18.2 Detection and Methods of Use

[00194] The present invention relates to methods for determining the presence or expression level of CLDN18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment provided herein under conditions that allow the antibody or antigen-binding fragment to specifically bind to CLDN18.2 (e.g., human CLDN18.2), and determining the presence or expression level of CLDN18.2 in the sample.

[00195] In another aspect, methods are provided for diagnosing a CLDN18.2-associated disease or condition (e.g., cancer) in a subject, comprising:

(a) contacting a sample obtained from a subject with an antibody or antigen-binding fragment thereof as provided herein under conditions that allow specific binding of the antibody or antigen-binding fragment thereof to CLDN18.2; and

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

A subject is diagnosed with a CLDN18.2-associated disease or condition (e.g., cancer) when the presence of CLDN18.2 is detected or when the CLDN18.2 expression level reaches a threshold level. In some embodiments, the sample is not gastric epithelial tissue.

[00196] In another aspect, methods for determining the suitability of a subject having or at risk for a CLDN18.2-associated disease or condition are provided for treatment with a CLDN18.2-targeted agent, including:

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

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

a subject is determined to be eligible for treatment with a CLDN18.2-targeted agent when the presence of CLDN18.2 is detected or when the level of CLDN18.2 expression reaches a threshold level, or

A subject is defined as ineligible for treatment with a CLDN18.2-targeted agent when the presence of CLDN18.2 is not detected or when the CLDN18.2 expression level is below a threshold level.

[00197] In another aspect, methods are provided for predicting the therapeutic efficacy of a CLDN18.2-targeted agent in treating a CLDN18.2-associated disease or condition in a subject, comprising:

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

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

c) predicting the therapeutic efficacy of a CLDN18.2-targeted agent, the CLDN18.2-targeted agent is predicted to be effective in treating a subject when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 reaches a threshold level, or

when the CLDN18.2-targeting agent is predicted to be ineffective in treating the subject, when the presence of CLDN18.2 is not detected, or when the expression level of CLDN18.2 is below a threshold level.

[00198] In another aspect, methods for treating a subject having or at risk of developing a CLDN18.2-associated disease or condition are provided, comprising:

a) selection of the subject suitable for treatment, including:

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

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

iii) selecting a subject as ineligible for treatment with a CLDN18.2 targeted agent when the presence of CLDN18.2 is detected or when the level of CLDN18.2 expression in the sample reaches a threshold level; and

b) administering a therapeutically effective amount of a CLDN18.2-targeting agent to the selected subject.

[00199] In another aspect, methods of treating a subject having or at risk of developing cancer are provided, comprising:

a) selection of the subject, including:

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

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

iii) selecting a subject as ineligible for treatment with a CLDN18.2 targeted agent if the presence of CLDN18.2 is not detected or the level of CLDN18.2 expression in the sample is below a threshold; and

b) administering to the selected subject a standard therapy therapeutic agent other than the CLDN18.2-targeted agent.

[00200] According to the invention, a "sample" may be any sample useful in accordance with the present invention, in particular a biological sample, such as a tissue sample, including body fluids, and/or a cellular sample, and may be obtained in a conventional manner, for example, by tissue biopsy, including punch biopsy, and by collecting blood, bronchial aspirate, sputum, urine, feces, or other body fluids. According to the invention, the term "sample" also includes processed samples, such as fractions or isolates of biological samples, for example, nucleic acid and peptide/protein isolates.

[00201] Any biological sample suspected of containing CLDN18.2 can be detected using the methods provided herein. In some embodiments, a suitable sample may be a cell sample or a tissue sample obtained from a subject to be detected. For example, a sample may include normal and cancerous tissue from the stomach, lung, breast, colon, kidney, bone, brain, muscle, pancreas, bladder, ovary, uterus, as well as cardiac, fetal, or placental tissue. Preferably, the sample comprises cells or tissue from an organ to be examined, such as to be diagnosed for cancer. For example, if the cancer to be diagnosed is gastric cancer, the sample may comprise cells or tissue obtained from the stomach. In some embodiments, the sample is a tumor sample. In some embodiments, the tissue samples are samples with a CLDN18.2-associated disease or condition.

[00202] A suitable sample may be a body sample obtained from a patient that contains or is suspected of containing tumor or cancer cells. A body sample may be any tissue sample, such as blood, a tissue sample obtained from a primary tumor or from tumor metastases, or any other sample containing tumor or cancer cells. The term "primary tumor" means a tumor growing at the site of cancer origin. The term "metastatic tumor" means a secondary tumor growing at a site other than the site of cancer origin.

[00203] The source of the tissue or cell sample may be solid tissue obtained from a fresh, frozen, and/or preserved organ or tissue sample, biopsy, or aspirate; blood or any blood components; biological fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells obtained at any time during pregnancy or development of the subject. In some embodiments, the sample is obtained from in vitro tissue or cell culture.

[00204] Examples of samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed paraffin-embedded (FFPE) tumor samples or frozen tumor samples.

[00205] In some embodiments of the invention, a cell sample is obtained from a cell block. A "cell block" is a method of preparing cytological material so that it can be processed, sectioned, stained, and viewed as a histological section. It can provide diagnostic information in addition to that obtained from a cytological preparation. In some embodiments, cell blocks can be prepared from residual effusions, sputum, urine sediment, gastrointestinal fluids, cell scrapings, or fine-needle aspirates. The cells are concentrated or packaged by centrifugation or membrane filtration.

[00206] Several methods for preparing cell blocks have been developed. Representative procedures include the fixed pellet, bacterial agar, or membrane filtration methods. In the fixed pellet method, the cell pellet is mixed with a fixative such as Bouins, picric acid, or buffered formalin, and the mixture is then centrifuged to pellet the fixed cells. The pellets are collected and placed in a tissue cassette, which is placed in a jar with additional fixative and processed as a tissue sample. The agar method is very similar, but the pellet is cut in half and the cut side is placed in a drop of molten agar on a glass slide. The agar is allowed to solidify, and then excess agar is trimmed off. Alternatively, the pellet can be directly suspended in 2% liquid agar at 65°C and the sample centrifuged. The cell pellet with agar is allowed to solidify within 1 hour at 4°C. The solid agar can be removed from the centrifuge tube and cut in half. This is placed in a tissue cassette, and the tissue processing process is complete. Centrifugation can be replaced in any of these procedures with membrane filtration. Either process can be used to obtain a "cell block sample."

[00207] In some embodiments, cell blocks can be prepared using a specialized resin, including Lowicryl, LR White, LR Gold, Unicryl, and MonoStep resins. These resins have low viscosity and can polymerize at low temperatures and under the influence of ultraviolet (UV) light. The embedding process is based on gradual cooling of the sample during dehydration, transfer of the sample to the resin, and polymerization of the block at a final low temperature at an appropriate UV wavelength.

[00208] Sections of the cell block can be stained with hematoxylin and eosin, Hoechst stain, or DAPI for cytomorphological examination, and additional sections are used for CLDN18.2 analysis by exposure to anti-CLDN18.2 antibodies (i.e., the primary antibody) for a sufficient period of time and under suitable conditions to allow the antibody to bind to the CLDN18.2 protein in the sections of the cell block. Unbound and excess primary antibody can be washed and removed.

[00209] In some embodiments, the sample may contain, for example, preservatives, anticoagulants, buffers, nutrients, antibiotics, or the like. In some embodiments, the sample has been exposed to and/or contains one or more fixatives. Exemplary fixatives suitable for the methods described herein include formalin, glutaraldehyde, osmium tetroxide, acetic acid, ethanol, acetone, picric acid, chloroform, potassium dichromate, and mercuric chloride, and/or stabilization by microwave heating or freezing.

[00210] 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 millimeters in size, and preferably 4-40 micrometers in size, and are mounted and dried on a microscope slide. Examples of paraffin include, but are not limited to, Paraplast, Broloid, and Tissuemay. For a fixed tissue sample, such as an FFPE tissue sample, the sample may be deparaffinized prior to contact with the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein.

[00211] In some embodiments, the deparaffinized sample may be further processed to extract the antigen. Antigen retrieval refers to any technique that reverses epitope masking and restores epitope binding to the antibody. While fixation is necessary to preserve tissue morphology, this process may also negatively impact antibody binding and detection. Fixation may alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the antibody. Epitope masking may be caused by cross-linking of amino acids within the epitope, cross-linking of unbound peptides within or near the epitope, altering the conformation of the epitope, or altering 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 methods typically 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).

[00212] In some embodiments, the presence or expression level of cell surface or membrane-bound CLDN18.2 is detected or determined in the methods provided herein. The phrase "cell surface or membrane-bound CLDN18.2" means that CLDN18.2 is associated with and located on the plasma membrane of a cell, wherein at least a portion of CLDN18.2 is exposed to the extracellular space of the cell and is accessible from outside the cell, such as by antibodies outside the cell. In some embodiments, the portion of CLDN18.2 that is exposed extracellularly includes at least 4, at least 8, at least 10, at least 12, or at least 20 amino acid residues. In normal tissues, with the exception of gastric epithelial cells, CLDN18.2 is located within the epithelial-endothelial tight junctions and is thought to be inaccessible to antibodies from outside the cells and is therefore considered non-cell surface or membrane-associated CLDN18.2.

[00213] The presence or level of expression of CLDN18.2 protein in a sample can be determined based on the presence or level of a complex of CLDN18.2 antigen bound by an antibody or antigen-binding fragment thereof disclosed herein. Any suitable method can be used to detect the antibody-antigen complex, such as immunoassay, such as immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS™), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). A review of immunological and immunoassay procedures is provided in Basic and Clinical Immunology (Stites & Terr eds., ^7th ed 1991). Furthermore, the immunoassay can be performed in any of several configurations, which are discussed in detail in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra. For reviews of general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., ^7th ed. 1991).

[00214] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are detectably labeled (e.g., a primary antibody) or are not labeled but can react with a second molecule that is detectably labeled (e.g., a detectably labeled secondary antibody).

[00215] 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 site, such as in the cells of the tissues mentioned herein. Immunohistochemical staining is widely used in the diagnosis of atypical cells, such as those found in cancer tumors. In some embodiments, the CLDN18.2 antibodies or antigen-binding fragments thereof provided herein can be used as a primary antibody in IHC or ICC. In general, IHC or ICC can be performed to directly detect or indirectly detect CLDN18.2 protein (e.g., human CLDN18.2 protein) in a sample, and CLDN18.2 expression can be assessed using appropriate imaging equipment.

[00216] For direct detection of an antigen (e.g., CLDN18.2), the CLDN18.2 antibodies or antigen-binding fragments thereof provided herein can be used, which are linked to a detectable label, allowing for direct visualization without the need for further antibody interaction.

[00217] For indirect detection of an antigen (e.g., CLDN18.2), CLDN18.2 antibodies or antigen-binding fragments thereof provided herein that are not labeled but can react with a second molecule that is detectably labeled (e.g., a detectively labeled secondary antibody) can be used. For example, anti-CLDN18.2 antibodies or antigen-binding fragments thereof provided herein can be unlabeled and then contacted with a secondary antibody (e.g., an anti-isotype antibody) conjugated to a detectable label to indirectly detect the antigen. For example, an anti-CLDN18.2 antibody provided herein can be conjugated to biotin, which can react with avidin labeled with a detectable substance, or vice versa. Biotin selectively binds to avidin and, therefore, allows for indirect detection of the antibody-antigen complex. In another example, the anti-CLDN18.2 antibody described herein can be conjugated to a small hapten that can react with an anti-hapten antibody linked to detectable labels, as described herein. Exemplary label types are described above, but any suitable detectable label can be used.

[00218] IHC or ICC analysis can be performed using an automated pathology system, which may include automated staining (conventional stains, histochemistry, immunostainers); automated in situ hybridization systems; automated slide preparation (coverslip, slide drying), and integrated labeling of slides and cassettes, as described in Roja et al., Review of imaging solutions for integrated, quantitative immunohistochemistry in the Pathology daily practice, Folia Histochemica et Cytobiologica, Vol. 47, No. 3, 349-354, 2009.

[00219] The example IHC assay may use the commercially available Dako En Vision™ FLEX detection system, which is designed for use with the Dako Autostainer (Dako, an Agilent Technologies Company, G1ostrup, Denmark). These reagents may be used with other autostainers or for manual staining without the use of an autostainer.

[00220] After the staining process is complete, the specimen (e.g., a glass slide) is analyzed for CLDN18.2 staining, either by a human subject, such as a pathologist, or by a computer programmed to distinguish between specific and nonspecific staining results. Analysis can be performed directly by viewing the specimen through a microscope at low, medium (10-20x), and high power (40-60x), or by viewing high-resolution images acquired at low, medium, and high power.

[00221] The presence of CLDN18.2 expression in a sample can be confirmed by the presence of a positively stained cell, such as a cell having at least partial membrane staining of any intensity for staining with an anti-CLDN18.2 antibody. In some embodiments, a normal sample can be used as a control sample, and the presence of CLDN18.2 expression in a test sample can be determined relative to the control sample. The term "normal" as used in the term "normal sample" or "normal tissue" means 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, such as both being a fixed tissue sample. If the test sample shows an increase in staining intensity or the number of positively stained cells compared to the control sample, then the test sample can be determined as positive for CLDN18.2 expression.

[00222] In some embodiments of the invention, the level of CLDN18.2 expression can be quantified using any suitable methods known in the art, such as by determining the relative proportion of positively stained cells and the intensity of staining on the cell membrane.

[00223] In some embodiments, the level of CLDN18.2 expression is determined based on the percentage of positively stained cells (i.e., CLDN18.2-positive cells) in a sample. A CLDN18.2-positive cell is a cell that has at least partial membrane staining of any intensity for staining with an anti-CLDN18.2 antibody. For example, to assess CLDN18.2 expression in a sample, an observer may examine the number of membrane-bound CLDN18.2+ cells in one or more selected fields under a microscope and calculate or estimate the percentage of cells positive for CLDN18.2. If the sample is highly heterogeneous, the sample may be divided into zones, and each zone is assessed separately and then combined into a single set of percentage values.

[00224] In some embodiments, the expression level is determined based on the intensity of CLDN18.2 staining (e.g., membrane-bound CLDN18.2) in a sample. For example, an intensity score, such as a 4-point HSCORE, can be calculated based on staining intensity ranging from 0 (no staining), 1+ (weak staining), 2+ (distinct staining), 3+ (strong staining), and 4+ (extremely strong/saturated staining) and multiplied by the percentage of cells stained at each intensity (0 to 100%) (see McCarty, K.S. Jr, et al, Cancer Res. 46(suppl 8):4244s-4248s (1986) for details). As another example, the Alfred score can be calculated from the total score (TS, range 0 to 8) by adding the proportion score (PS) and the intensity score (IS). PS represents the proportion of positive tumor cells, ranging from 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 represents the mean staining intensity of positive tumor cells, ranging from 0 to 3 (0 = negative, 1 = weak, 2 = intermediate staining, 3 = strong staining) (see details in Alfred DC et al. Mod Pathol. 11: 155–168 (1998)).

[00225] In some embodiments of the invention, samples are evaluated by two observers working independently of each other, and the percentages or scores are subsequently combined. In some other embodiments of the invention, the identification of positive and negative cells is performed or evaluated using appropriate software.

[00226] In some embodiments, the CLDN18.2 level may be determined, for example, by normalization to a control value or to a standard curve. The control value may be predetermined or determined simultaneously with a negative control sample or a blank control sample.

[00227] In some embodiments, for diagnostic or clinical use, the level of CLDN18.2 expression (e.g., on the cell surface) in a test sample is compared to a threshold level.

[00228] "Threshold level" or "threshold value" with respect to CLDN18.2 expression means an expression level that distinguishes positive from negative expression of CLDN18.2 on a cell surface, or an expression level that excludes or excludes a CLDN18.2-associated condition, such as cancer, or the onset or risk of onset of cancer in a subject, or a threshold level that monitors the response to treatment in a subject receiving cancer treatment. In some embodiments, the threshold level is determined relative to a control expression level.

[00229] For example, the threshold may be a level above which a sample is assessed as having positive CLDN18.2 expression and therefore suitable for treatment with a CLDN18.2 targeted agent. If the level of CLDN18.2 in a sample reaches or exceeds the threshold level, this may indicate the presence of a CLDN18.2-associated disease or condition, and/or the likelihood of a response to a CLDN18.2 targeted agent.

[00230] The threshold level can be determined by one of skill in the art taking into account a variety of factors, including, for example, the type of sample, the detection method, the disease or condition being diagnosed, and/or the CLDN18.2 targeting agents used.

[00231] In some embodiments, the presence of CLDN18.2 or CLDN18.2-expressing cells and/or a number of CLDN18.2 or CLDN18.2-expressing cells increased relative to a threshold level, such as compared to a subject without a CLDN18.2-associated condition (e.g., a non-cancer subject), indicates the presence of or risk (i.e., potential for developing) a CLDN18.2-associated disease or condition (e.g., cancer) in a patient.

[00232] In some embodiments, the cutoff level is the percentage of CLDN18.2 positive cells (%) that have at least partial membrane staining of any intensity. In one embodiment, the cutoff level of 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%.

[00233] In some embodiments, the sample is obtained from a subject who has or is at risk of developing a CLDN18.2-associated disease or condition. The term "CLDN18.2-associated disease or condition" as provided herein means a disease or condition characterized by increased expression of CLDN18.2 in cells of a diseased tissue or organ compared to the condition in healthy or non-cancerous tissue or organ other than the stomach. The increase can be, for example, an increase of at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, or even more. In some embodiments, the expression of CLDN18.2 in cells of a diseased tissue or organ is above the limit of detection and/or is high enough to allow binding by antibodies specific for CLDN18.2 added to the cells. In some embodiments of the invention, an increase in CLDN18.2 expression is detected only in diseased tissue, while CLDN18.2 expression is not detected in normal tissue.

[00234] In some embodiments, the CLDN18.2-associated disease or condition is characterized by increased expression of cell surface or membrane-bound CLDN18.2.

[00235] In some embodiments of the invention, the disease or condition associated with CLDN18.2 is cancer. In one embodiment, cancer cells express or aberrantly express CLDN18.2, while corresponding normal cells do not express CLDN18.2 or express CLDN18.2 at a lower level. It is believed that CLDN18.2, as a tight junction protein, may be a good therapeutic target for a CLDN18.2-associated disease, such as a tumor, and therefore can be used to select patients suitable for treatment with CLDN18.2 targeted agents. Unlike normal epithelial tissue (except gastric epithelial cells), in which claudins associate to form classical tight junctions, claudins expressed in tumor cells often do not form such classical tight junctions, and as a result, tumor cells likely have free claudins that are exposed and accessible to extracellular antibody binding and immunotherapy.

[00236] 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 carcinoma, ductal carcinoma, and lobular breast carcinoma), liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, liver cancer, head cervical cancer, gallbladder cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, cancer gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, cholangiocarcinoma and/or adenocarcinoma, and/or their metastases, in particular gastric cancer metastases such as Krukenberg tumors, peritoneal metastases and lymph node metastases.

[00237] The samples are preferably cancer cells or tissues and in particular are selected from the group consisting of tumor cells or tissues of stomach, esophagus, pancreas, lung, ovary, colon, liver, neck and head and gallbladder cancer.

[00238] In some embodiments, the cancer includes, but is not limited to, renal cell carcinoma, gastric carcinoma, mesothelioma, melanoma, cervical cancer, thymic carcinoma, myeloma, mycotic fungoids, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, basal cell carcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell tumors, EBV-positive and -negative PTLD, and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal cord tumor, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[00239] In some embodiments of the invention, the cancer is a cancer that expresses CLDN18.2. The presence or level of expression of CLDN18.2 in a sample indicates whether the cancer cells express CLDN18.2 and, therefore, whether the cancer is likely to respond to treatment with an agent targeting CLDN18.2.

[00240] "CLDN18.2 targeted agent" as used herein means one or more agents that target CLDN18.2 protein or nucleic acids (DNA or mRNA) in a cell, tissue, or living organism. The CLDN18.2 targeted agent may reduce/eliminate expression of the CLDN18.2 gene product, inhibit/disrupt CLDN18.2 signaling, or cause cytotoxicity in cells that atypically express CLDN18.2.

[00241] In some embodiments, a CLDN18.2 targeting agent comprises a therapeutic CLDN18.2 antibody, a CLDN18.2 binding molecule, a cell therapy targeting CLDN18.2, a chemical compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2. In some embodiments, a CLDN18.2 targeting agent is capable of inducing cytotoxicity in cells expressing CLDN18.2.

[00242] In some embodiments, a CLDN18.2 targeting agent comprises a therapeutic CLDN18.2 antibody or CLDN18.2 binding molecule. In some embodiments, a therapeutic CLDN18.2 antibody or CLDN18.2 binding molecule can induce ADCC, CDC, or ADCP on CLDN18.2-expressing cells. In another embodiment, a therapeutic CLDN18.2 antibody or CLDN18.2 binding molecule can be conjugated to a cytotoxic agent, such as 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 a toxin, a chemotherapeutic agent (such as DNA alkylators, a topoisomerase inhibitor, a tubulin-binding agent, a growth inhibitor, or other anticancer drugs), or a radioactive isotope. In other embodiments, the therapeutic CLDN18.2 antibody may be a bispecific antibody that binds to different antigens or different epitopes on the CLDN18.2 protein.

[00243] In some embodiments, a CLDN18.2-targeted agent comprises a CLDN18.2-targeted cell therapy. In some embodiments, the cell therapy directed at CLDN18.2 comprises CAR-T (chimeric receptor-based antibodies), TCR-T (genetically modified TCR T cells), or CAR-NK (chimeric NK cell-based antibodies) expressing a chimeric antibody receptor (CAR) that binds CLDN18.2. Chimeric antigen receptors (CARs) are modified chimeric receptors that combine the antigen-binding domain of an antibody with one or more signaling domains for T cell activation. Immune cells such as T cells and natural killer (NK) cells can be genetically modified to express CARs or genetically modified TCRs (see D. Li et al, Sig Transduct Target Ther 4, 35 (2019); S. Kloess et al, Transfus Med Hemother; 46:4–13 (2019); Wang W. et al, Cancer Letters, 472: 175–180 (2020) for details). T cells expressing CARs are called CAR-T cells. CARs can mediate antigen-specific cellular immune activity in T cells, allowing CAR-T cells to kill cells (e.g., tumor cells) expressing the target antigen. In one embodiment of the invention, binding of CAR-T cells provided herein to CLDN18.2 expressed on cells, such as cancer cells, results in proliferation and/or activation of said CAR-T cells, wherein the activated CAR-T cells can release cytotoxic factors, such as perforin, granzymes and granu lysin, and initiate cytolysis and/or apoptosis of cancer cells.

[00244] In some embodiments of the invention, a therapeutic nucleic acid targeting CLDN18.2 may be a short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), and short hairpin RNA (shRNA) capable of RNA interference (RNAi) against a CLDN18.2 gene sequence or another gene sequence in a cell expressing CLDN18.2.

[00245] In some embodiments, a CLDN18.2 targeted agent promotes cancer regression in a subject. In preferred embodiments, a therapeutically effective amount of a CLDN18.2 targeted agent promotes cancer regression up to and including eradication of the cancer. "Promoting cancer regression" means that administration of an effective amount of the agent, alone or in combination with an antineoplastic agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one symptom of the disease, an increase in the frequency and duration of symptom-free periods, or a prevention of worsening of the condition or disability due to disease.

[00246] In some embodiments, the subject is receiving or has received anti-cancer therapy or is suffering from a recurrence of cancer. Anti-cancer therapy includes, for example, a chemotherapeutic agent, an anti-cancer drug, radiation therapy, immunotherapy, an anti-angiogenesis agent, a targeted therapy, cell therapy, gene therapy, hormonal therapy, palliative care, surgery to treat cancer (e.g., tumorectomy), or one or more anti-emetic agents or other agents for the treatment of complications arising from chemotherapy.

[00247] A relapse or recurrence of a disease occurs when a person again suffers from a condition that affected them in the past. For example, if a patient suffered from a cancerous disease, received successful treatment for this disease, and then develops this disease again. This newly developed disease can be considered a relapse or recurrence. However, according to the present disclosure, a relapse or recurrence of a cancerous disease may, but does not necessarily, occur at the site of the original cancerous disease. Thus, for example, if a patient suffered from a stomach tumor and received successful treatment, a relapse or recurrence may be the occurrence of a stomach tumor or the occurrence of a tumor in a site other than the stomach. A relapse or recurrence of a tumor also includes situations where the tumor occurs in a site other than the site of the original tumor, as well as in the site of the original tumor. Preferably, the original tumor for which the patient received treatment is the primary tumor, and the tumor in a site other than the site of the original tumor is a secondary or metastatic tumor.

[00248] Sets

[00249] In some embodiments, the present invention provides kits comprising an isolated antibody or antigen-binding fragment thereof as provided herein. In some embodiments, a kit as provided herein is a diagnostic kit. The kits may be useful for detecting the presence or amount of CLDN18.2 in a biological sample, or may be useful in the diagnostic methods provided herein.

[00250] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof as provided herein, which may optionally be detectably labeled. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an indirectly detectable compound.

[00251] In some embodiments of the invention, the kit further comprises a reagent kit for detecting a complex of an antibody or antigen-binding fragment thereof bound to CLDN18.2, useful in various detection assays, including, for example, an immunoassay such as IHC, ICC, or ELISA (sandwich assay or competitive assay).

[00252] In another embodiment of the invention, reagents useful for performing immunohistochemistry on a FFPE tumor tissue site are provided in a kit along with instructions for performing the IHC analysis.

[00253] Any indirectly detectable labels or compounds provided herein can be used. In some embodiments, the indirectly detectable compound comprises biotin. In such embodiments, the reagent kit comprises detectably labeled avidin or stepavidin.

[00254] In some embodiments, a detectable label is conjugated to an antibody or antigen-binding fragment thereof as provided herein. In some embodiments, kits include an isolated antibody or antigen-binding fragment thereof as provided herein and a secondary antibody conjugated to a detectable label. In some embodiments, the secondary antibody includes an antibody that specifically binds to a primary antibody (e.g., an antibody or antigen-binding fragment thereof as provided herein). In some embodiments, the secondary antibody may be an anti-mouse antibody, an anti-rabbit antibody, or an anti-human antibody.

[00255] Prior to use, a detectable label may require combination with one or more components, such as buffering agents, antibody-enzyme conjugates, enzyme substrates, or the like, and such reagents may be included in kits. For example, when the detectable compound includes an enzyme, the kit will include substrates and factors required for the enzyme (e.g., a precursor substrate that provides a detectable chromophore or fluorophore). In addition, other reagents may be included, such as blocking reagents to reduce non-specific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The relative amounts of the various reagents may vary widely to provide concentrations of the reagents in solution that significantly optimize the sensitivity of the assay. In particular, the reagents may be supplied as dry powders, typically lyophilized, including excipients that, when dissolved, provide a reagent solution of the appropriate concentration. The kit may also include instructions, either in the form of inserts or a label, providing recommendations for detection and/or preparation of the assay system.

[00256] The components of the kit may be pre-attached to a solid support or may be applied to the surface of a solid support during use of the kit. The solid phase surface may be in the form of a tube, bead, microtiter plate, microsphere, or other material suitable for immobilizing proteins, peptides, or polypeptides.

[00257] Containers for use in such kits may typically include at least one vial, test tube, flask, bottle, syringe, or other suitable container into which one or more detecting compositions can be placed, and preferably a suitable aliquot. The kits provided herein also typically include means for containing the vial in a confined space for commercial sale, such as, for example, injection molded or blow molded plastic containers into which the desired vial is placed. If a radioactive, chromogenic, fluorogenic, or other type of detectable label or detecting agent is included in the kit, the detecting agent may be provided either in the same container as the detecting composition itself, or may be placed in a second separate container into which the second composition can be placed and appropriately aliquoted. Alternatively, the detection reagent may be prepared in a single container, and in most cases the kit also typically includes a means of containing the vial in a contained location for commercial sale and/or convenient packaging and shipping.

[00258] A device or apparatus for implementing the detection or monitoring methods described herein is also provided. Such a device may include a chamber or tube into which a sample may be introduced, a fluid handling system optionally including valves or pumps for directing the flow of the sample through the device, optionally filters for separating plasma or serum from blood, mixing chambers for adding capture agents or detection reagents, and optionally a detection device for determining the amount of detectable label associated with the immunocomplex of the capture agents. The flow of the sample may be passive (e.g., due to capillary, hydrostatic, or other forces that do not require further manipulation of the device after the sample is introduced) or active (e.g., due to the application of force generated by mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or a combination of active and passive forces.

Examples

[00259] While the invention has been shown and described with reference to particular embodiments (some of which are preferred), it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present invention as presented herein.

[00260] Example 1: Preparation of antigen for immunization

[00261] 1. Construction of antigen peptide

[00262] In order to restrict the epitope of the antibody to CLDN18.2 rather than to CLDN18.1, a peptide unique to CLDN18.2 (Genbank accession number: NP_001002026) was developed by MabSpace Biosciences (Suzhou) Co., Limited. The coding sequence of this peptide (SEQ ID No. 19: DQWSTQDLYN) was located at amino acid residues Asp28-Asn37 (D28 - N37) of CLDN18.2.

[00263] 2. Peptide synthesis

[00264] The antigenic peptide was synthesized at SANGON BIOTECH (Shanghai) and used to immunize animals.

[00265] Example 2: Generation of antibodies

[00266] 1. Immunization and hybridoma fusion

[00267] 6-8 week-old mice of different strains were immunized with 40 μg/mouse peptide (conjugated with KLH) with CFA as an adjuvant, each mouse received 11 boosts (subcutaneous injection at several sites) 3 times a week for 4 weeks, and then boosts at a weekly interval 2 times, 3 days after the primary immunization. To determine the serum titer, 100 μl/well of serially diluted mouse serum was added to the plate coated with the antigen peptide and then incubated at 4°C for 30 min. The plate was washed 3 times with wash buffer, then 100 μl/well of goat anti-mIgG-HRP was added for repeated incubation at 4°C. After washing with wash buffer, TMB was added to react with HRP on the plate. The plate's OD450 nm was then read using a microplate reader, and the titer was analyzed using Graphpad Prism 6 software. Mice with higher titers were selected for subsequent fusion procedures.

[00268] 2. Mergers

[00269] Four days prior to fusion, each mouse was injected intraperitoneally with 20 μg of peptide. On the day of fusion, the spleens were aseptically removed and then processed into a single-cell suspension. Red blood cells were lysed to obtain splenocytes. Viable, log-phase myeloma cells (SP2/0) were mixed with mouse splenocytes at a 1:1 ratio in the fusion medium after fusion by electroshock for 1 min. The cells were resuspended and cultured in 96-well culture plates at 37°C, 5% CO2 incubator. After 7 days of culture, the growth medium was replaced with fresh one. Screening of hybridoma supernatants began 2-3 days after replacement of fresh growth medium.

[00270] Example 3: Binding screening, subcloning of positive hybridoma clones and small-scale production of antibodies

[00271] 1. Binding screening by ELISA

[00272] Hybridoma supernatants were collected for antigen binding screening. 100 μl/well of hybridoma supernatants were added to a plate coated with the antigen peptide and then incubated at 4°C for 1 hour. The plate was washed 3 times with wash buffer, then 100 μl/well of goat anti-mIgG-HRP was added for repeated incubation at 4°C. After washing with wash buffer, TMB was added to react with HRP on the plate. The OD490 nm of the plate was then read using a microplate reader. Positive clones binding by ELISA were selected and expanded. After 2 days, the binding of hybridoma supernatants of the selected clones was checked in the same manner, and the positive clones were proceeded to subcloning.

[00273] 2. Subcloning of positive hybridoma clones

[00274] Cells from positive hybridoma wells with the desired binding profile were selected for limiting dilution in 96-well plates. The diluted cells were allowed to grow for 7 days. After reaching a sufficient cell mass, the supernatant from each well was collected and retested using the same ELISA binding assay described above.

[00275] From each 96-well plate, the clone with the highest cell binding activity was expanded for a second round of limiting dilution into a 96-well plate with 200 μl of hybridoma growth medium per well. After 7 days, cell supernatants from the 96-well plates were analyzed by the same binding ELISA. Subcloning was performed more than 2 times until a positive binding signal appeared in more than 90/96 wells. Two subclones (i.e., 69H2 and 14G11) with the highest binding activity from each clone were identified, expanded, and cultured to obtain purified antibodies. Isotypes were determined by a standard method.

[00276] 3. Small-scale production of antibodies

[00277] Hybridoma cells were inoculated and cultured for 14 days. Monoclonal antibodies (mAbs) were purified from the hybridoma cell culture using Protein A High Performance (Bio-Rad) affinity chromatography.

[00278] After chromatographic purification, these mAbs were expressed in PBS by dialysis followed by a filtration step.

[00279] Example 4: Immunocytochemical screening of antibodies on cell blocks

[00280] 1. Preparation of cell block sections

[00281] HEK293-human CLDN18.2 (hereinafter referred to as HEK293-CLDN18.2) and HEK293-human CLDN18.1 (hereinafter referred to as HEK293-CLDN18.1) cells were constructed by MabSpace Biosciences (Suzhou) Co.Limited. Briefly, HEK293 cells (Shanghai Institutes for Biological Sciences, Cat#GNhu43) were transfected with pcDNA3.1/hCLDN18.2 or pcDNA3.1/hCLDN18.1 plasmids and selected with G418 to generate a stable expression cell line of HEK293-CLDN18.2 or HEK293-CLDN 18.1. CLDN18.1 expression on HEK293-CLDN18.1 and CLDN18.2 expression on HEK293-CLDN18.2 were examined and confirmed by FACS using positive control antibodies, respectively. As shown in Figure 1, HEK293-CLDN18.1 cells showed a positive binding signal for the EPR19203 antibody (available from Abcam under the product name ab222513), which binds to CLDN18.1, and HEK293-CLDN18.2 cells showed a positive binding signal for the 18B10 antibody (see application PCT/CN2019/101563), which specifically binds to human CLDN18.2.

[00282] HEK293 cells and HEK293 cells with high levels of human CLDN18.2 (HEK293-CLDN18.2) or CLDN18.1 (HEK293-CLDN18.1) expression were harvested and fixed in 4% neutral buffered paraformaldehyde (PFA) for 30 min at room temperature. After centrifugation, the cells were resuspended in PBS, then dispersed in 200 μl of melted agar at 57°C and immediately solidified at 4°C. The agar-cell mixture was dehydrated in a gradient alcohol and cleared in xylene. After paraffin infiltration at 60°C, the agar-cell mixture was embedded in paraffin according to a standard procedure and cut into 3 μm thick sections, which were immediately placed on positively charged glass slides.

[00283] 2. Immunocytochemical screening of antibodies on cell block sections

[00284] To screen for specific and sensitive antibodies to CLDN18.2, immunocytochemistry (ICC) was performed using FFPE paraffin-embedded sections of HEK293, HEK293-CLDN18.2, and HEK293-CLDN18.1 cell blocks. After deparaffinization and rehydration, all sections were antigen-retrieved by boiling in En Vision™ FLEX Target Retrieval Solution (Dako, K8002) for 25 minutes at 97-99°C, then quenched, blocked with EnVision™ FLEX Peroxidase-Blocking Reagent (Dako, K8002), and incubated with appropriately diluted antibodies. Antibody binding was visualized using EnVision™ FLEX+, Mouse (LINKER), followed by EnVision™ FLEX /hRP and EnVision™ FLEX Substrate Working Solution (Dako, K8002). Sections were finally counterstained with hematoxylin and mounted using permanent mounting medium. No significant differences in staining were observed, and the staining intensity between antibodies varied from weak to strong.

[00285] As shown in Table 3 and Figure 2, antibody clones 69H2 and 14G11 strongly and specifically stained the surface of HEK293-CLDN18.2, but negatively stained HEK293 and HEK293-CLDN18.1 at a concentration of 1 nM and were further titrated to 0.5 nM to test the sensitivity. 69H2F7E6, 69H2D3B1, 69H2E1D3 are clones of antibody 69H2 and have the same heavy chain and light chain sequences with 69H2. 14G11G2D2, 14G11A4E1, and 14G11F5E1 are clones of antibody 14G11 and have the same heavy chain and light chain sequences with 14G11.

[00286] As a control, GC182 antibody staining against CLDN18.2 was used, which positively stained both HEK293-CLDN18.2 cells and HEK293-CLDN18.1 cells. The GC182 antibody has the heavy chain variable region sequence of SEQ ID NO: 22 and the light chain variable region sequence of SEQ ID NO: 23 and was produced by Mabspace Bioscience according to the sequences disclosed in WO2013167259. The results in Table 3 showed that the GC182 antibody can cross-react with CLDN18.1 and, therefore, is not specific for CLDN18.2.

[00287] GC182 heavy chain variable region sequence (SEQ ID NO:22):

[00288] GC182 light chain variable region sequence (SEQ ID NO:23):

[00290] Example 5: Cloning and recombinational production of selected antibodies

[00291] Production of recombinant antibodies

[00292] The variable region sequences of the light chain and heavy chain of the mouse anti-human antibodies CLDN18.2 14G11 and 69H2 were obtained by polymerase chain reaction (PCR) amplification from candidate hybridoma cell lines. After analysis and sequencing confirmation, the variable region genes, including the variable region sequence of the light chain (VL) fused to the mouse kappa constant region and the variable region sequence of the heavy chain (VH) fused to the mouse IgG1 constant region, were cloned into the recombinant expression vector pcDNA3.1(+) for antibody production and purification.

[00293] The heavy chain and light chain of antibodies 14G11 and 69H2 were fused to the constant region of the mouse IgG1 heavy chain and the constant region of the kappa light chain, respectively, as shown below:

[00294] Mouse IgG1 heavy chain constant region (SEQ ID NO: 17):

[00295] Mouse kappa light chain constant region (SEQ ID NO: 18):

[00296] Expression and purification of recombinant antibodies

[00297] ExpiCHO cells were transfected using the ExpiCHO transfection kit with equal amounts of DNA from the heavy chain vector and light chain vector. Transfected cells were cultured in a shaker at 125 rpm in an incubator at 37°C and 8% CO2. The cell culture was harvested on day 10, and the collected antibodies were purified by affinity chromatography. The resulting antibody was analyzed for purity by SDS-PAGE and size exclusion chromatography (TSK gel G3000SWXL, TOSOH).

[00298] Example 6: Evaluation of the selectivity of hCLDN18.2 binding to hCLDN 18.1 by ELISA and FACS

[00299] Specific binding to hCLDN18.2 peptide (aa28-37) by ELISA

[00300] 1 μg/mL of peptide (100 μL/well) was applied to high-binding clear polystyrene 96-well plates and blocked with blocking buffer. Serially diluted antibodies (150 to 0.0732 ng/mL) in blocking buffer were then added and incubated for 1.5 h at room temperature. The plate was washed 6 times with wash buffer, then 100 μL/well of goat pAb to Ms IgG (HRP) was added for incubation at room temperature for 1.5 h. After washing with wash buffer, TMB was added to react with HRP on the plate. The OD450 nm of the plate was then read using a SpectraMax i3x (Molecular Devices). 14G11G2D2 and 69H2F7E6 can specifically bind to the peptide with high affinity, while GC182 does not recognize the epitope in the linear peptide. (Figure 3)

[00301] Analysis of the selectivity of binding of recombinant antibodies against claudin 18.2 to recombinant protein hCLDN18.2 and hCLDN18.1 by ELISA

[00302] This assay utilized human recombinant CLDN18.2(N-6His) (obtained from Novoprotein under catalog number NC101) and human recombinant CLDN18.1(N-8His) (obtained from Novoprotein under catalog number CR54). Human recombinant CLDN18.2(N-6His) is a fusion polypeptide comprising the extracellular domain (residues A1a24-A1a81, SEQ ID NO:26) of human CLDN 18.2 flanked by sequences that can fold or associate, allowing the extracellular domain of human CLDN 18.2 to form a loop. Similarly, the recombinant human CLDN18.1(N-8His) variant is a fusion polypeptide comprising the extracellular domain of human CLDN 18.1 (residues Asp28-Leu76, SEQ ID NO:27) flanked by sequences that can fold or associate, allowing the extracellular domain of human CLDN 18.1 to form a loop.

[00303] 1 μg/ml of human recombinant CLDN 18.2(N-6His) or human recombinant CLDN 18.1(N-8His) (100 μl/well) was applied to high-binding clear polystyrene 96-well plates and blocked with blocking buffer. Serially diluted antibodies (100 to 0.0244 ng/ml) in blocking buffer were added and incubated for 1.5 h at room temperature. The plate was washed 6 times with wash buffer (PBS + 0.1% Tween), then 100 μl/well of goat pAb to Ms IgG (HRP) were added for incubation at room temperature for 1.5 h. After washing with wash buffer, TMB was added to react with HRP on the plate. The OD450 nm of the plate was then read using a SpectraMax i3x (Molecular Devices). Both 14G11G2D2 and 69H2F7E6 specifically bind to recombinant human CLDN 18.2(N-6His) with high affinity, with EC50 of 12.75 ng/mL and 13.87 ng/mL, respectively (Figure 4). Neither 14G11G2D2 nor 69H2F7E6 showed specific binding to human recombinant CLDN 18.1(N-8His) protein (Figure 5). As a control, GC182 showed no specific binding to recombinant CLDN 18.2(N-6His) or recombinant human CLDN 18.1(N-8His) (Figures 4, 5). This indicates that 14G11G2D2 and 69H2F7E6 bind to an epitope in the extracellular domain of CLDN18.2, but GC182 does not recognize such an epitope.

[00304] FACS analysis on HEK293-hCLDN18.2 and HEK293-hCLDN 18.1 cell lines

[00305] Log-phase HEK293-hCLDN18.2 or HEK293-hCLDN18.1 cells were harvested, washed, and resuspended. Diluted antibodies (20 μg/mL) in blocking buffer were added and incubated at 4°C for 1 hour. The cells were then washed twice with blocking buffer, after which a second antibody (goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488, ThermoFisher) was added in blocking buffer. The cells were incubated at 4°C for 1 hour, then washed twice with blocking buffer, and resuspended in blocking buffer. The cells were then transferred to a FACS tube, and antibody binding to the cells was determined by flow cytometry (BD Accuri C6). Antibodies 18B10 and EPR19203 were used as positive controls for the analysis on HEK-hCLDN 18.2 and HEK-hCLDN 18.1 cells. As shown in Figure 1, neither antibodies 14G11G2D2 nor 69H2F7E6 could bind human claudin 18.2 or claudin 18.1 expressed on the cell surface with the native conformation.

[00306] Example 7: Determination of the binding affinity of recombinant anti-claudin 18.2 to recombinant hCLDN18.2 protein using ForteBio

[00307] 100 nM human recombinant CLDN18.2(N-6His) protein (Novoprotein, NC101) in 1× kinetic buffer ((PBS+0.1%BSA+0.02% Tween20)) was loaded onto pre-wetted Ni-NTA biosensors (PALL, 18-5101) for 200 sec. After equilibration in 1× kinetic buffer as a baseline (60 sec), the sensor was immersed in serially diluted antibodies (50 nM, 25 nM) with 1× kinetic buffer, respectively, and incubated for 200 sec to determine association (koff) and then dissociation in 1× kinetic buffer for 200 sec (koff). The biosensors were regenerated for 5 s in regeneration buffer and then neutralized for 5 s in neutralization buffer three times. All procedures were performed at 30°C using Octet RED 96 (PALL). The binding affinity KD was calculated and analyzed using the koff to kon ratio (Figure 6). 69H2F7E6 showed a KD value of 1.48 nM, and 14G11G2D2 showed a KD value of 0.213 nM.

[00308] Example 8: Testing the specificity of an antibody using normal tissues

[00309] CLDN18.2 is a highly selective gastric lineage antigen, the expression of which is restricted to short-lived differentiated epithelial cells of the gastric mucosa, whereas CLDN18.1 expression is restricted to the lungs. Based on this, selected antibodies were analyzed on various relevant normal tissues to confirm specific binding to CLDN18.2, but not to CLDN18.1. Immunohistochemistry (IHC) was performed on normal intestine, kidney, tonsil, thyroid, skeletal muscle, stomach, lung, and breast sections fixed in 4% neutral buffered formalin and embedded in paraffin (FFPE). After deparaffinization and rehydration, all sections were antigen-retrieved by boiling in EnVision™ FLEX Target Retrieval Solution (Dako, K8002) for 25 minutes at 97-99°C, then quenched, blocked with EnVision™ FLEX Peroxidase-Blocking Reagent (Dako, K8002), and incubated with appropriately diluted antibodies. Antibody binding was visualized with EnVision™ FLEX+, Mouse (LINKER), then EnVision™ FLEX /hRP and EnVision™ FLEX Substrate Working Solution (Dako, K8002). Sections were finally counterstained with hematoxylin and mounted with permanent mounting medium.

[00310] Clones 14G11G2D2 and 69H2F7E6 were selected for further specificity analysis on normal FFPE tissues. Both 69H2F7E6 and 14G11G2D2 showed good results in terms of staining intensity, pattern, and selectivity. Both 69H2F7E6 and 14G11G2D2 showed strong staining intensity on FFPE stomach sections, but little staining on lung, intestine, kidney, skeletal muscle, tonsil, thyroid, and breast sections (see Table 4 and Figures 7A and 7B). All stained cells were epithelial cells, but no other cell types, including lymphocytes, vessels, fibrocytes, or smooth muscle cells. In contrast, the GC182 antibody against CLDN 18.2 showed strong and moderate staining intensity in FFPE sections of the stomach and lungs, respectively, confirming that it binds to both CLDN18.2 and CLDN18.1. Compared with 69H2F7E6, 14G11G2D2 showed superior results and was selected as a candidate for further IHC analysis. The IHC staining results are presented in Table 4 and Figures 7A and 7B.

[00311] Furthermore, the specificity and sensitivity of the selected antibodies were also compared with the existing EPR19202 antibody (Abcam, ab222512) against CLDN18.2 on normal tissues. IHC was performed as described above. As shown in Figure 8, the EPR19202 antibody at a concentration of 0.374 μg/mL showed weaker staining intensity on gastric tissue than the 14G11G2D2 antibody at a lower concentration, i.e., 0.15 μg/mL, indicating that ERP19202 is less sensitive than 14G11G2D2. In particular, the EPP19202 antibody showed non-specific staining in skeletal muscle (Figure 8), while the 14G11G2D2 antibody retained specificity for CLDN18.2 and did not cross-react with any of the normal tissues lacking CLDN18.2.

[00312] Example 9: Use of identified antibodies to assess CLDN18.2 expression using IHC based on tumor tissue samples from various tumor types

[00313] It is reported that the expression level of CLDN18.2 in Japanese patients with gastric cancer is detected in 87% of tumor samples, and moderate or strong expression of CLDN18.2 is observed in 52% of tumor samples (Rohde et al, Jpn J Clin Oncol. 2019 Sep 1; 49(9):870-876). Furthermore, aberrant ectopic expression of CLDN18.2 has been reported in other cancer cells, including pancreatic, ovarian, biliary, and lung adenocarcinomas (Sahin U et al. Clinical Cancer Research, 2008, 14(23): 7624–7634; Karanjawala ZE et al, Am J Surg Pathol. 2008 Feb; 32(2): 188–96; Micke P et al, Int J Cancer. 2014 Nov 1; 135(9):2206–14; Keira Y et al, Virchows Arch. 2015 Mar; 466(3):265–77).

[00314] 14G11G2D2 was further analyzed on various relevant cancer tissues to confirm staining intensity, pattern, and positivity. Immunohistochemistry (IHC) was performed on sections of gastric, pancreatic, cholangiocarcinoma, and non-small cell lung cancer (NSCLC) tumors fixed in 4% neutral buffered formalin and embedded in paraffin (FFPE). After deparaffinization and rehydration, all sections were subjected to antigen retrieval by boiling in EnVision™ FLEX Target Retrieval Solution (Dako, K8002) for 25 minutes at 97-99°C, then quenched, blocked with EnVision™ FLEX Peroxidase-Blocking Reagent (Dako, K8002), and incubated with appropriately diluted 14G11G2D2 antibodies. Antibody binding was visualized with EnVision™ FLEX+, Mouse (LINKER), then EnVision™ FLEX /hRP and EnVision™ FLEX Substrate Working Solution (Dako, K8002). Sections were finally counterstained with hematoxylin and mounted with permanent mounting medium. All samples were analyzed for the relative proportion of positively stained tumor cells to all visible tumor cells with membranous staining of varying intensities (negative (-), weak (+), moderate (++), strong (+++)). Only membranous staining was considered positive, and normal human stomach served as a positive control for each staining. Weak or strong membrane signals were generated by 14G11G2D2 in gastric, pancreatic, cholangiocarcinoma, and NSCLC tissues, respectively (see Fig. 9), and the percentage of positive tumor cells varied individually for different tumor types (see Table 5-1 for gastric cancer, Table 5-2 for pancreatic cancer, Table 5-3 for cholangiocarcinoma, and Table 5-4 for NSCLC tissues). The positivity and prevalence of CLDN18.2 expression in gastric, pancreatic, cholangiocarcinoma, and NSCLC cancer tissues were summarized in Table 6.

[00320] Example 10: Verification of CLDN18.2 staining by IHC in the stomach using biotinylated 14G11G2D2

[00321] Biotinylated 14G11G2D2 (14G11G2D2-Biotin) was prepared. Briefly, biotinamidocaproate ester NHS (Sigma, B2643-10MG) was prepared in anhydrous DMF at a concentration of 20 mg/mL as a stock solution. For each 1 mg of 14G11G2D2 antibody to be labeled, 10 μL of the stock solution was added and gently mixed for 1 hour at room temperature. Low molecular weight reaction products were removed by desalting the product on Zeba™ Spin Desalting Columns (ThermoFisher, 89890) according to the manufacturer's instructions.

[00322] Immunohistochemistry (IHC) was performed on gastric slides fixed in 4% neutral buffered formalin and embedded in paraffin. After deparaffinization and rehydration, all slides were antigen retrieved by boiling in EnVision™ FLEX Target Retrieval Solution (Dako, K8002) for 25 minutes at 97-99°C, then quenched, blocked with the IHC Biotin Block Kit (MaiXin, BLK-0001) according to the instructions, and incubated with 3 μg/ml of in-house biotinylated mouse monoclonal antibody against claudin 18.2 (14G11G2D2-Biotin) for 30 minutes at 37°C. Antibody binding was visualized using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision™ FLEX working substrate solution (Dako, K8002). Sections were finally counterstained with hematoxylin and mounted using permanent mounting medium.

[00323] As shown in Figure 10, 14G11G2D2-6hotiih showed identical staining to 14G11G2D2 in terms of intensity and specificity.

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SEQUENCE LIST

<110> MABSPACE BIOSCIENCES (SUZHOU) CO., LIMITED

<120> ANTIBODIES AGAINST CLAUDINU 18.2 AND THEIR DIAGNOSTIC APPLICATIONS

<130> 063694-8004WO02

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<---

Claims (65)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a) CDR1 of the heavy chain with the amino acid sequence X ₁ X ₂ YX ₃ H (SEQ ID NO: 8), CDR2 of the heavy chain with the amino acid sequence WIYPX ₄ GX ₅ X ₆ X ₇ X ₈ YX ₉ EKFKG (SEQ ID NO: 12), and CDR3 of the heavy chain with the amino acid sequence NYX ₁₀ STFGY (SEQ ID NO: 24); and b) CDR1 of the light chain with the amino acid sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 2), CDR2 of the light chain with the amino acid sequence KX ₁₁ SNRFS (SEQ ID NO: 25), and CDR3 of the light chain with the amino acid sequence FQGSHVPFT (SEQ ID NO: 6); where X ₁ represents R or T, X ₂ represents N or Y, X ₃ represents F or I, X ₄ represents G or R, X ₅ represents F or G, X ₆ represents D or N, X ₇ represents I or T, X ₈ represents E or V, X ₉ represents S or N, X ₁₀ represents G or R, and X ₁₁ represents V or I; and wherein said antibody or antigen-binding fragment thereof comprises: a) CDR1 of the heavy chain with the amino acid sequence presented in SEQ ID NO: 1, CDR2 of the heavy chain with the amino acid sequence shown in SEQ ID NO: 3, CDR3 of the heavy chain with the amino acid sequence shown in SEQ ID NO: 5, CDR1 of the light chain with the amino acid sequence shown in SEQ ID NO: 2, CDR2 of the light chain with the amino acid sequence shown in SEQ ID NO: 4, CDR3 of the light chain with the amino acid sequence shown in SEQ ID NO: 6; or b) CDR1 of the heavy chain with the amino acid sequence presented in SEQ ID NO: 7, CDR2 of the heavy chain with the amino acid sequence shown in SEQ ID NO: 9, CDR3 of the heavy chain with the amino acid sequence shown in SEQ ID NO: 11, CDR1 of the light chain with the amino acid sequence shown in SEQ ID NO: 2, CDR2 of the light chain with the amino acid sequence shown in SEQ ID NO: 10, CDR3 of the light chain with the amino acid sequence shown in SEQ ID NO: 6. 2. An antibody or antigen-binding fragment thereof according to claim 1, comprising: a) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO: 13 and a light chain variable region with the amino acid sequence presented in SEQ ID NO: 14; or b) a heavy chain variable region with the amino acid sequence presented in SEQ ID NO: 15 and a light chain variable region with the amino acid sequence presented in SEQ ID NO: 16. 3. An antibody or antigen-binding fragment thereof according to any one of claims 1, 2, comprising an immunoglobulin constant region, optionally comprising an IgG heavy chain constant region, and/or a light chain constant region. 4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the constant region of the heavy chain comprises the amino acid sequence presented in SEQ ID NO: 17, or a sequence with a degree of identity of at least 80%, and/or the constant region of the light chain comprises the amino acid sequence presented in SEQ ID NO: 18, or a sequence with a degree of identity of at least 80%. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a monoclonal antibody or a recombinant antibody. 6. A conjugate that specifically binds to CLDN18.2, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-5, linked to one or more molecules, wherein the molecule comprises a radioactive isotope, a lanthanide, a chemiluminescent label, a chromophore molecule, colloidal gold particles, a fluorescent label, an enzyme-substrate label, a digoxigenin label, biotin/avidin, a hapten, a DNA molecule for detection or labeling of particles. 7. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-5. 8. An expression vector comprising an isolated polynucleotide according to claim 7. 9. A host cell expressing the antibody or antigen-binding fragment thereof according to claim 1, comprising the vector according to claim 8. 10. A method for expressing an antibody or antigen-binding fragment thereof according to claim 1, comprising culturing a host cell comprising a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1, under conditions under which said vector is expressed. 11. A method for determining the presence of CLDN18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-5 or a conjugate according to claim 6 under conditions that ensure specific binding of the antibody or antigen-binding fragment thereof to human CLDN18.2, and determining the presence of CLDN18.2 in the sample, wherein said antibody or antigen-binding fragment thereof in said conjugate is linked to a detectable label, which allows for direct visualization without the need for further interaction of the antibodies; or said antibody or antigen-binding fragment thereof is not labeled, but can react with a second molecule that is detectably labeled. 12. A method for determining the level of expression of CLDN18.2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-5 or a conjugate according to claim 6 under conditions that ensure specific binding of the antibody or antigen-binding fragment to human CLDN18.2, and determining the level of expression of CLDN18.2 in the sample, wherein said antibody or antigen-binding fragment thereof in said conjugate is linked to a detectable label, which allows for direct visualization without the need for further interaction of the antibodies; or said antibody or antigen-binding fragment thereof is not labeled, but can react with a second molecule that is detectably labeled. 13. A method for diagnosing a CLDN18.2-associated disease or condition in a subject, comprising: a) contacting a sample obtained from a subject with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a conjugate according to claim 6 under conditions that ensure specific binding of the antibody or antigen-binding fragment thereof to CLDN18.2; and b) determining the presence or expression level of CLDN18.2 in a sample; wherein said antibody or antigen-binding fragment thereof in said conjugate is linked to a detectable label, allowing direct visualization without the need for further interaction of the antibodies; or said antibody or antigen-binding fragment thereof is not labeled but can react with a second molecule that is detectably labeled, wherein a subject is diagnosed with a CLDN18.2-associated disease or condition when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 reaches a threshold level. 14. A method for treating or preventing a CLDN18.2-associated disease or condition in a subject, comprising: a) selection of the subject suitable for treatment, including: i) contacting a sample obtained from a subject with an antibody or antigen-binding fragment thereof according to any one of claims 1-5 under conditions that ensure specific binding of the antibody or antigen-binding fragment to CLDN18.2; ii) determining the presence or expression level of human CLDN18.2 in the sample; iii) selecting a subject as suitable for treatment of a CLDN18.2-associated disease or condition when the presence of CLDN18.2 is detected or when the expression level of CLDN18.2 in a sample reaches a threshold level; b) administering a therapeutically effective amount of a CLDN18.2-targeting agent to the selected subject. 15. The method according to any one of claims 11-14, wherein the sample is a cell sample or a tissue sample, possibly wherein the sample comprises tumor tissue or circulating tumor cells. 16. The method of any one of claims 11-15, wherein CLDN18.2 is cell surface-associated or membrane-associated CLDN18.2, and wherein the presence or expression level of CLDN18.2 is determined using immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), or immunoblotting, and wherein the expression level is determined based on the percentage of positively stained cells in the sample or the staining intensity for CLDN18.2 in the sample. 17. The method according to any one of claims 13-16, wherein the CLDN18.2-associated disease or condition is cancer. 18. The method of claim 17, wherein the cancer is a primary cancer or a metastatic cancer. 19. The method according to claim 17 or 18, wherein the cancer is stomach 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, spine 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, cholangiocarcinoma and/or adenocarcinoma, possibly wherein the esophageal cancer is esophageal adenocarcinoma, gastric/gastroesophageal junction (G/GEJ) cancer. 20. The method according to any one of claims 14-19, wherein the CLDN18.2-targeting agent is capable of causing cytotoxicity to CLDN18.2-expressing cells, and wherein the CLDN18.2-targeting agent is a therapeutic antibody against CLDN18.2 or a CLDN18.2-binding molecule, a CLDN18.2-targeted cell therapy, a chemical compound targeting CLDN18.2, or a therapeutic nucleic acid targeting CLDN18.2. 21. The method according to any one of claims 13-20, wherein the subject is receiving or has received anti-cancer therapy or is suffering from a recurrence of cancer. 22. A kit for diagnosing a CLDN18.2-associated disease or condition, comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-5 or a conjugate according to claim 6, and instructions indicating recommendations for detection and/or preparation of the assay system. 23. The kit of claim 22, comprising a reagent kit for detecting a complex of an antibody or its antigen-binding fragment bound to CLDN18.2, and wherein the reagent kit comprises a secondary antibody that binds to the antibody or its antigen-binding fragment, optionally the secondary antibody is detectably labeled. 24. The kit of claim 22, wherein the antibody or antigen-binding fragment thereof in said conjugate is detectably labeled, or the antibody or antigen-binding fragment thereof is conjugated to an indirectly detectable molecule.