Combination therapy with claudin-18.2 antagonists for cancer treatment

Abstract

The present invention relates to a combination therapy of a CLDN18.2 antagonist, chemotherapy, and optionally immunotherapy for treating CLDN18.2-expressing cancer.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to International Application No. PCT / CN2023 / 098182, filed on 3 June 2023, and International Application No. PCT / CN2024 / 093779, filed on 16 May 2024, the disclosures of which are incorporated herein by reference as a whole.

[0002] Sequence List The sequence listing contained in the 50,877-byte file named "063694-8016WO03_ST26," created on 28 May 2024, is submitted electronically with this specification and is incorporated herein by reference.

[0003] This disclosure relates to cancer therapy using a claudin 18.2 (CLDN18.2) antagonist in combination with a second-line therapy (e.g., chemotherapy and / or immunotherapy) for the treatment of cancers that express claudin 18.2. [Background technology]

[0004] The cell surface antigen Claudin 18 splice variant 2 (CLDN18.2) is considered an ideal target for the development of therapeutic agents for cancer treatment. In normal tissues, it is present only in the differentiated epithelial cells of the gastric mucosa and not in the gastric stem cell area or other normal tissues. However, abnormal ectopic expression of CLDN18.2 has been reported in gastric adenocarcinoma, pancreatic adenocarcinoma, ovarian adenocarcinoma, biliary adenocarcinoma, and lung adenocarcinoma (Sahin U et al., Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development, Clin Cancer Res. 2008 Dec 1;14(23):7624-34.), and has also been confirmed in other similar studies (Karanjawala ZE et al., New markers of pancreatic cancer identified through differential gene expression analyses: claudin 18 and annexin A8. Am J Surg Pathol. 2008 Feb;32(2):188-96., Micke P et al., Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer. Int J Cancer. 2014 Nov 1;135(9):2206-14., Keira Y et al., An immunohistochemical marker panel including claudin-18, maspin, and p53 improves diagnostic accuracy of bile duct neoplasms in surgical and presurgical biopsy specimens. Virchows Arch. 2015 Mar;466(3):265-77.). The currently available information indicates that CLDN18.2 is a promising therapeutic target for solid tumor treatment.

[0005] Currently available CLDN18.2-directed therapies have shown therapeutic efficacy in certain patients with CLDN18.2-expressing cancers, but this efficacy benefit is limited to patients with high CLDN18.2 expression. However, the medical needs of many patients with moderately or low CLDN18.2-expressing cancers remain unmet.

[0006] Therefore, there is a significant need for novel CLDN18.2-directed therapies for these many patients.

Summary of the Invention

[0007] The present disclosure particularly provides a method for identifying and treating a subject having Claudin 18.2 (CLDN18.2)-expressing cancer, which may benefit from treatment comprising a CLDN18.2 antagonist in combination with chemotherapy, the method comprising determining the level of CLDN18.2 protein in a cancer sample (e.g., in a tumor tissue sample), and selecting a subject having a moderate to high (e.g., moderate or high), or low level of the CLDN18.2 protein for said treatment.

[0008] In another aspect, the present disclosure provides a method for treating CLDN18.2-expressing cancer in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist in combination with chemotherapy, wherein the subject has been determined to have a moderate to high (e.g., moderate or high), or low CLDN18.2 expression level in a cancer sample (e.g., in a tumor tissue sample).

[0009] In some embodiments, the methods described herein correspond to a) a high CLDN18.2 protein level or CLDN18.2 expression level in which at least 70% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ as measured on a Leica Bond III LDT platform using an anti-CLDN18.2 diagnostic antibody, or an equivalent readout as measured on another test platform, and b) a moderate CLDN18.2 protein level or CLDN18.2 expression level in which at least 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ as measured on a Leica Bond III LDT platform using an anti-CLDN18.2 diagnostic antibody. c) The CLDN18.2 expression level is characterized by a membrane staining intensity of at least 2+ as measured on the LDT platform using an anti-CLDN18.2 diagnostic antibody, and less than 70% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ or corresponding to an equivalent readout measured on another test platform, and c) the low CLDN18.2 protein level or CLDN18.2 expression level is characterized by at least 1-10% (e.g., at least 1%, at least 5%, or at least 10%) of the cancer cells in the cancer sample (e.g., tumor tissue sample) being Leica Bond III The test results indicate that the CLDN18.2 expression level, as measured on the LDT platform using an anti-CLDN18.2 diagnostic antibody, is characterized by a membrane staining intensity of at least 1+, and that less than 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ or 3+, or an equivalent readout as measured on another test platform.

[0010] In some embodiments, the methods described herein correspond to a) a high CLDN18.2 protein level or CLDN18.2 expression level in which at least 70% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ as measured on a Leica Bond III LDT platform using 14G11, or an equivalent readout as measured on another test platform, and b) a moderate CLDN18.2 protein level or CLDN18.2 expression level in which at least 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ as measured on a Leica Bond III LDT platform using 14G11, or an equivalent readout as measured on another test platform, and c) The CLDN18.2 expression level is characterized by at least a membrane staining intensity of 2+ as measured on the LDT platform using 14G11, and less than 70% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by at least a membrane staining intensity of 2+, or correspond to an equivalent readout measured on another test platform, and c) the low CLDN18.2 protein level or CLDN18.2 expression level is due to at least 1-10% (e.g., at least 1%, at least 5%, or at least 10%) of the cancer cells in the cancer sample (e.g., tumor tissue sample) being Leica Bond III The CLDN18.2 expression level is characterized by at least a membrane staining intensity of 1+ as measured on the LDT platform using 14G11, and less than 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) have a CLDN18.2 expression level characterized by at least a membrane staining intensity of 2+ or 3+, or a corresponding equivalent threshold as measured on another test platform or with another anti-CLDN18.2 diagnostic antibody.

[0011] In another aspect, the present disclosure provides a method for treating a CLDN18.2-expressing cancer in a human subject of interest, comprising administering to the subject a therapeutically effective dose of a CLDN18.2 antagonist in combination with chemotherapy and a PD-1 / PD-L1 axis inhibitor, wherein the subject has been determined to have high or low PD-L1 expression levels in a cancer sample (e.g., in a tumor tissue sample).

[0012] In another aspect, the Disclosure provides the use of a CLDN18.2 antagonist in the manufacture of a pharmaceutical product for treating a CLDN18.2-expressing cancer in a subject of interest, wherein the treatment comprises administering the pharmaceutical product to the subject in combination with chemotherapy, and the subject is determined to have a moderate to high (e.g., moderate or high) or low CLDN18.2 expression level in a cancer sample (e.g., in a tumor tissue sample).

[0013] In another aspect, the Disclosure provides the use of a CLDN18.2 antagonist in the manufacture of a pharmaceutical product for treating a CLDN18.2-expressing cancer of a subject of interest, wherein the pharmaceutical product further comprises chemotherapy, and the subject is determined to have a moderate to high (e.g., moderate or high) or low CLDN18.2 expression level in a cancer sample (e.g., in a tumor tissue sample).

[0014] In another aspect, the Disclosure provides a kit for use in identifying and treating subjects with CLDN18.2-expressing cancer who may benefit from chemotherapy or a treatment comprising a CLDN18.2 antagonist in combination with both chemotherapy and a PD-1 / PD-L1 axis inhibitor, the kit comprising an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody) and a package insert containing instructions for using the treatment in subjects with moderate to high (e.g., moderate or high) or low levels of the CLDN18.2 protein. [Brief explanation of the drawing]

[0015] The drawings referred to herein form part of the specification. The features shown in the drawings illustrate only some embodiments of this application, and not all embodiments of this application, unless expressly indicated in the detailed description, and should not be construed otherwise when reading this specification.

[0016] [Figure 1] The study design for Cohort G from the Transtar102 trial (NCT04495296) is shown below.

[0017] [Figure 2] This shows progression-free survival in cohort G based on CLDN18.2 levels.

[0018] [Figure 3] This shows the percentage change in tumor size efficacy in cohort C of patients with low expression levels (1+ stained tumor cells ≥10%, and 2+ or 3+ stained tumor cells <40%). n: Efficacy population, subjects with measurable lesions at baseline and who underwent at least one post-baseline tumor assessment.

[0019] [Figure 4] This shows the percentage change in tumor size in cohort G of patients with low expression (staining 1+ tumor cells ≥10%, and staining 2+ or 3+ tumor cells <40%). n: Efficacy population, subjects with measurable lesions at baseline and who underwent at least one post-baseline tumor assessment.

[0020] [Figure 5] This shows the percentage change in tumor size in cohort G of patients with low expression (staining 1+ tumor cells ≥10%, and staining 2+ or 3+ tumor cells <40%). n: Efficacy population, subjects with measurable lesions at baseline and who underwent at least one post-baseline tumor assessment.

[0021] [Figure 6] This document outlines the expression levels of CLDN18.2 and PD-L1, as well as the overlap between GC / GEJ cancer tissues.

[0022] [Figure 7] The IHC staining results for CLDN18.2 under bright-field microscopy are shown.

[0023] [Figure 8] The results of IHC staining of PD-L1 under bright-field microscopy are shown.

[0024] [Figure 9A] The tumor growth curves (mean ± standard deviation, n=8) for GC PDX tumor growth inhibited by nivolumab and Hu18B10-HaLa in combination with oxaliplatin + 5-FU are shown.

[0025] [Figure 9B] This shows the tumor volume distribution (mean ± standard deviation, n=8) of GC PDX tumor growth inhibited by nivolumab and Hu18B10-HaLa in combination with oxaliplatin + 5-FU at day 22.

[0026] The same reference number is used throughout the drawing to refer to the same or similar parts. [Modes for carrying out the invention]

[0027] The following descriptions in this disclosure are intended to illustrate various embodiments of this disclosure. Therefore, any particular modifications considered should not be construed as limiting the scope of this disclosure. It will be obvious to those skilled in the art that various equivalents, variations, and modifications are possible without departing from the scope of this disclosure, and such equivalent embodiments are understood to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference as a whole.

[0028] I. Definition

[0029] As used herein, the terms “a, an,” “the,” and similar terms used in the context of the present invention (particularly in the context of the claims) are understood to cover both singular and plural forms unless otherwise indicated or explicitly contradicted in the context herein.

[0030] The term "CLDN18.2" refers to claudin 18 splice variant 2 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include the human CLDN18.2 protein (NCBI reference sequence number NP_001002026.1, or sequence number 29). Exemplary sequences of CLDN18.2 include the Mus musculus (mouse) CLDN18.2 protein (NCBI reference sequence number NP_001181852.1) and the Macaca fascicularis (cynomolgus monkey) CLDN18.2 protein (NCBI reference sequence number XP_015300615.1). CLDN18.2 is expressed in cancer cells. In one embodiment, the above CLDN18.2 is expressed on the surface of cancer cells.

[0031] As used herein, the term “antagonist” in relation to CLDN18.2 means any molecule that specifically binds to CLDN18.2 and / or partially or completely inhibits, blocks, or neutralizes the biological activity of CLDN18.2. Suitable CLDN18.2 antagonists may include, without limitation, antibodies, antisense oligonucleotides, peptides, and small organic molecules. In certain embodiments, the CLDN18.2 antagonist is an anti-CLDN18.2 antibody.

[0032] As used herein, "anti-CLDN18.2 antibody" refers to an antibody that can specifically bind to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity to provide, for example, diagnostic and / or therapeutic applications.

[0033] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific or bispecific antibody that binds to a specific antigen, or any polypeptide that mimics an antibody in terms of its ability to bind to a specific antigen. Naturally intact antibodies consist of two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3, respectively). Mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Antibodies are “Y” shaped, with the tail of the Y consisting of the second and third constant regions of two heavy chains joined together via a disulfide bond. Each arm of the Y includes the variable region and first constant region of a single heavy chain, which is joined to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, and LCDR3, while the heavy chain CDRs include HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibody and antigen-binding domains disclosed herein may be defined or identified by the provisions 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), NR Whitelegg et al, Protein Engineering, v13(12), 819-824 (2000), Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83). (1989), Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991), Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003), Marie-Paule Lefranc et al, Immunome Research, 1(3), (2005), Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015). These three CDRs are interposed between adjacent segments known as framework regions (FRs), which are more conserved than these CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of the constant region of their heavy chains. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively.Several major antibody classes are classified into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, the antibodies provided herein encompass any of their antigen-binding fragments.

[0034] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” refer to a fragment formed from an antibody fragment containing one or more CDRs (e.g., an antibody fragment), or any other moiety that binds to an antigen but does not contain an intact native antibody structure (e.g., an antibody moiety). Examples of antigen-binding fragments / moies include, without limitation, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments / moies are capable of binding to the same antigen as the parent antibody. In certain embodiments, an antigen-binding fragment / moiety may contain one or more CDRs from a particular parent antibody.

[0035] In relation to antibodies, "Fab" refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) attached to the variable and first constant regions of a single heavy chain by disulfide bonds. Fab can be obtained by papain digestion of the antibody at a residue near the N-terminus of the disulfide bond between the heavy chains in the hinge region.

[0036] "Fab'" refers to a Fab fragment that includes part of the hinge region, and can be obtained by pepsin digestion of the antibody at residues near the C-terminus of the disulfide bond between the heavy chains of the hinge region. Therefore, it differs from Fab in which only a few residues (including one or more cysteines) are present in the hinge region.

[0037] "F(ab')2" refers to a dimer of Fab' that contains two light chains and parts of two heavy chains.

[0038] In relation to antibodies, "Fv" refers to the smallest fragment of an antibody that has a complete antigen-binding site. The Fv fragment consists of a variable region of a single light chain that is bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable regions of a single light chain and a single heavy chain are linked via a disulfide bond.

[0039] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region that are directly linked to each other or linked via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain with a linker that contains two heavy chain variable regions and two light chain variable regions. In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) containing a dimerized (linked by a peptide linker) VH-VL portion with another VH-VL portion such that the VH portion of one portion coordinates with the VL portion of the other portion to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific diabody containing VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate, and each coordination pair has different antigen specificity.

[0040] A "single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of scFvs connected to the Fc region of the antibody.

[0041] "Camelized single-domain antibody," "heavy-chain antibody," "nanobody," or "HCAb" refers to an antibody that contains two VH domains and does not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999), Muyldermans S., J Biotechnol. Jun; 74(4):277-302 (2001), WO94 / 04678, WO94 / 25591, U.S. Patent No. 6,005,079). Heavy-chain antibodies were initially obtained from camelids (camels, dromedaries, and llamas). Although lacking a light chain, camelized antibodies possess a definitive antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8 (1993), Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr; 54(1):39-47 (2002), Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain (VHH domain) of heavy-chain antibodies represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)). A "diabody" contains a small antibody fragment having two antigen-binding sites, where these fragments contain a VH domain (VH-VL or VL-VH) connected to a VL domain in a single polypeptide chain (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8 (1993), EP404097, WO93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair; therefore, these domains are forced to pair with complementary domains on another chain, thereby generating two antigen-binding sites.Antigen-binding sites can target the same region of different antigens (or epitopes).

[0042] A "domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain embodiments, two or more VH domains are covalently linked to a peptide linker to form a bivalent or polyvalent domain antibody. The two VH domains of the bivalent domain antibody may target the same or different antigens.

[0043] In a particular embodiment, "(dsFv)2" comprises three peptide chains such that two VH portions are linked by a peptide linker and attached to two VL portions via disulfide crosslinks.

[0044] In certain embodiments, the "bispecific ds diabody" comprises VH1-VL2 (similarly linked by a peptide linker) linked to VL1-VH2 (linked by a peptide linker) via a disulfide crosslink between VH1 and VL1.

[0045] In certain embodiments, “bispecific dsFv” or “dsFv-dsFv'” comprises three peptide chains, such as VH1-VH2 moieties, where the heavy chain is linked by a peptide linker (e.g., a long, mobile linker) and paired with VL1 and VL2 moieties via disulfide crosslinks, respectively. Each disulfide-paired heavy and light chain has a different antigen specificity.

[0046] As used herein, a “bispecific” antibody is an artificial antibody having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be located on the same antigen or on two different antigens.

[0047] As used herein, the term "humanized" means that an antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human. In certain embodiments, the amino acid residues of the variable region framework of a humanized CLDN18.2 antibody are substituted for sequence optimization. In certain embodiments, the variable region framework sequence of a humanized CLDN18.2 antibody chain is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the corresponding human variable region framework sequence.

[0048] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy chain and / or light chain is derived from one species and the remaining portion of the heavy chain and / or light chain is derived from another species. In an exemplary example, a chimeric antibody can comprise a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0049] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0050] As used herein, the term "specific binding" or "binds specifically" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen, as used herein. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a binding affinity (KD) of ≤ 10 -6 M (e.g., ≤ 5 × 10 -7 M, ≤ 2 × 10 -7 M, ≤ 10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤ 10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, or ≤ 10 -9 M) and specifically binds to human and / or non-human CLDN18.2. As used herein, KD refers to the ratio of the dissociation rate to the association rate (koff / k on The KD value can be determined by conventional methods known in the art, not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (FACS, etc.). In certain embodiments, the KD value can be appropriately determined by flow cytometry. Various immunoassay formats can be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically react with a certain protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, specific or selective binding reactions produce a signal at least twice as loud as the background signal, and more typically at least 10 to 100 times louder than the background signal.

[0051] With respect to amino acid sequences (or nucleic acid sequences), "percent (%) sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after the sequences have been aligned to achieve maximum correspondence and gaps have been introduced as necessary. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be achieved using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI); see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990), Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Institute for Bioinformatics; see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996), Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by these tools or customize the parameters to suit the alignment by, for example, selecting a suitable algorithm. In certain embodiments, the positions of non-identical residues may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be up-adjusted to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference.

[0052] As used herein, “homologous sequence” means a polynucleotide sequence (or its complementary chain) or amino acid sequence that, when arbitrarily aligned, has at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with another sequence.

[0053] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-human mammals, e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0054] As used herein, "effector function" or "antibody effector function" refers to the biological activity resulting from the binding of the Fc region of an antibody to effectors such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody on the C1 complex with C1q, antibody-dependent cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to an Fc receptor on effector cells, and antibody-dependent cell-mediated phagocytosis (ADCP), in which nonspecific cytotoxic cells expressing FcγRs recognize antibodies bound to target cells and subsequently induce phagocytosis of the target cells. Effector functions include both functions that act after antigen binding and functions that act independently of antigen binding.

[0055] "Treating" or "treatment" of symptoms, as used herein, includes preventing or alleviating symptoms, delaying the onset or progression of symptoms, reducing the risk of developing symptoms, preventing or delaying the onset of symptoms associated with a symptom, reducing or terminating symptoms associated with a symptom, causing complete or partial regression of symptoms, curing symptoms, or any combination thereof.

[0056] "Cancer," as used herein, means any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, invasion or metastasis, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematological malignancies). "Solid tumor," as used herein, means a solid mass of neoplasm and / or malignant cells.

[0057] The term “therapeutic effective dose” or “effective dose” means the amount of a drug that produces any desired local or systemic therapeutic effect in a reasonable benefit / risk ratio applicable to any treatment, either alone or in combination with additional doses. In the case of treating a particular disease, the desired local or systemic therapeutic effect is preferably related to inhibiting disease progression. This includes delaying disease progression, in particular interrupting or reversing disease progression. When administered to prevent a disease, the dose is sufficient to avoid or delay the onset of the disease. The therapeutic effective dose or effective dose does not need to cure the disease or symptoms or prevent their onset. The effective dose of a drug described herein depends on the symptoms being treated, the severity of the disease, the patient’s age, physiological state, body size and weight, and other individual parameters, the duration of treatment, the type of combination therapy (if any), the specific route of administration, and similar factors. Therefore, the dosage of a drug described herein may depend on such various parameters. If the patient does not respond adequately to the initial dose, a higher dose (or an effective high dose achieved by a different, more localized route of administration) may be used. In certain embodiments, the therapeutically effective dose of a drug depends on its therapeutic index, solubility, and other factors.

[0058] The term "survival," as used herein, refers to the continued existence of a subject and can be expressed by progression-free survival (PFS) and / or overall survival (OS). Survival can be estimated by the Kaplan-Meier method. The difference in survival is calculated by the stratified log-rank test.

[0059] As used herein, the term “staining” refers to the process of conjugating a target (e.g., an antigen) in a biological sample (e.g., a cancer sample such as a tumor tissue sample) with a target-specific conjugate (e.g., a diagnostic antibody or nucleic acid), and then detecting the presence of the target-specific conjugate on the cells of the biological sample using a detectable label or chromogen. The detectable label may be directly conjugated to the target-specific conjugate (e.g., a primary antibody), or it may be conjugated to a secondary reagent (e.g., a secondary antibody) that specifically binds to an unlabeled target-specific reagent. In some cases, the target-specific reagent itself is detectable, and therefore no additional conjugation label is required.

[0060] As used herein, the term “target-specific conjugate” means an analyte of interest present in a tissue section (e.g., a tumor tissue section), such as an agent that specifically binds to a target of interest, e.g., a polypeptide or polynucleotide. Exemplary target-specific conjugates include antibodies, receptors, and ligands or their target-binding fragments, as well as polynucleotide probes.

[0061] As used herein, the term “chromogen” refers to a substance that can be converted into a coloring compound under specific conditions, for example, when subjected to the action of an enzyme or under specific chemical / reaction conditions.

[0062] As used herein, the terms “primary antibody” and “secondary antibody” refer to different antibodies, the primary antibody being a polyclonal or monoclonal antibody from one species (e.g., rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker such as CLDN18.2) in the biological sample under test (e.g., a cancer sample such as a tumor tissue sample), and the secondary antibody being an antibody from a different species (usually a polyclonal antibody) that specifically recognizes the primary antibody, for example, within its Fc region.

[0063] Please note that terms such as "comprises," "comprised," and "comprising," and "contains" are intended to be comprehensive or open-ended and do not exclude any additional elements or method steps that are not described.

[0064] In this specification, the expression “about” in reference to a value or parameter includes (describes) embodiments relating to the value or parameter itself. For example, a description referring to “about X” includes a description of “X.” Numerical ranges include the numerical values ​​that define the range. Generally, the term “about” refers to the indicated value of a variable and all values ​​of the variable within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater. When the term “about” is used in the context of a period (year, month, week, day, etc.), the term “about” means that period ± one next subperiod (e.g., about one year means 11 to 13 months, about six months means six months ± one week, and about one week means 6 to 8 days), or within 10% of the indicated value, whichever is greater.

[0065] II. Combination therapy for CLDN18.2-expressing cancers

[0066] This disclosure provides a method for treating CLDN18.2-expressing cancer in a human subject of interest. In certain embodiments, the subject is determined to have moderate to high (e.g., moderate or high) or low CLDN18.2 expression levels in a cancer sample (e.g., in a tumor tissue sample). In certain embodiments, the method comprises administering a therapeutically effective dose of a CLDN18.2 antagonist to the subject in combination with chemotherapy. In certain embodiments, the method further comprises administering immunotherapy.

[0067] In another embodiment, the Disclosure provides the use of a CLDN18.2 antagonist in the manufacture of a pharmaceutical product for treating CLDN18.2-expressing cancer in a subject of interest. In certain embodiments, the subject is determined to have moderate to high (e.g., moderate or high) or low CLDN18.2 expression levels in a cancer sample (e.g., in a tumor tissue sample). In certain embodiments, the treatment comprises administering the pharmaceutical product to the subject in combination with chemotherapy. In certain embodiments, the treatment further comprises administering immunotherapy. In certain embodiments, the pharmaceutical product further comprises chemotherapy and / or immunotherapy.

[0068] In another embodiment, the disclosure also provides a method for identifying and treating subjects with CLDN18.2-expressing cancer who may benefit from treatment including a CLDN18.2 antagonist in combination with chemotherapy. In certain embodiments, the treatment further includes immunotherapy. In certain embodiments, the method includes determining the level of CLDN18.2 protein in a cancer sample (e.g., in a tumor tissue sample). In certain embodiments, the method further includes selecting subjects having moderate to high (e.g., moderate or high) or low CLDN18.2 protein levels for the above treatment.

[0069] a. Subjects expressing CLDN18.2

[0070] In certain embodiments, the subject is determined to have moderate to high (e.g., moderate or high) CLDN18.2 expression levels in cancer samples. In certain embodiments, the subject is determined to have low CLDN18.2 expression levels in cancer samples. In certain embodiments, the subject is human.

[0071] As used herein, the term "low CLDN18.2 expression level," used interchangeably with the term "low CLDN18.2 protein level," is intended to mean a detectable CLDN18.2 expression level on the surface of the target cancer cells that is relatively low compared to a given threshold.

[0072] The predetermined threshold may be determined by those skilled in the art, for example, as the threshold for moderate or moderate CLDN18.2 protein expression levels in a particular type of cancer cell. Therefore, a level lower than the predetermined threshold for moderate or moderate expression levels means a low CLDN18.2 protein level.

[0073] The threshold for moderate or moderate CLDN18.2 protein expression levels can be determined by determining the actual expression level of CLDN18.2 protein in a sample, cell, tumor, or cancer population of a particular cancer type, and then ranking the population according to the actual expression level. For example, in a population with cancer (gastric adenocarcinoma and gastroesophageal adenocarcinoma), the threshold for moderate or moderate CLDN18.2 protein expression levels in that cancer type is 70% of the CLDN18.2 protein expression level in that population. th Percentile, 65 th Percentile, 60 th Percentile, 55 th Percentile, 50 th Percentile, 45 th Percentile, 40 th , 35 th , 30 th ,twenty five th , or 20 th These can be percentile levels. Such actual expression levels can be quantified in assays under specified assay conditions, such as immunohistochemistry (IHC) assays, hybridization assays, or amplification assays such as quantitative reverse transcription polymerase chain reaction (RT-PCR) assays.

[0074] In a particular embodiment, low CLDN18.2 protein levels in a particular cancer are associated with a 70% increase in a given population of that particular cancer. th Below the percentile, 65 th Percentile, 60 th Below the percentile, 55 th Below the percentile, 50 th Below the percentile, 45 th Below the percentile, 40 thBelow the percentile, 35 th Below the percentile, 30 th Below the percentile, 25 th Below the percentile, or 20th percentile th It's acceptable to be below the percentile.

[0075] As used herein, the term “moderate to high CLDN18.2 protein level” and the term “moderate to high CLDN18.2 expression level” are intended to mean a level of CLDN18.2 expression on the surface of the cancer cells in question that is relatively higher than or equivalent to a given threshold of moderate or moderate expression level.

[0076] The threshold for moderate or moderate CLDN18.2 protein expression levels can also be determined by the expression level measured by a suitable assay in cancer samples (e.g., tumor tissue samples). Various suitable methods are known in the art and include, without limitation, protein-based assays such as quantitative fluorescence cytometry, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA), or nucleic acid-based assays such as amplification assays, hybridization assays, or sequencing assays.

[0077] Those skilled in the art will understand that the absolute value representing the CLDN18.2 expression level in a particular sample can vary depending on the type of assay and the specific conditions of the assay. Therefore, a predetermined threshold for moderate or moderate CLDN18.2 protein expression levels in a particular cancer can also vary depending on the type of assay and the specific conditions of the assay, and can be determined by those skilled in the art by considering various factors, such as the type of sample, the type and conditions of the assay, the specific type of cancer or tumor, the CLDN18.2-diagnostic antibody used, and / or the type of treatment.

[0078] In certain embodiments, CLDN18.2 expression levels can be measured by an IHC assay using a CLDN18.2 diagnostic antibody and expressed as membrane staining intensity. In other words, CLDN18.2 protein levels or CLDN18.2 expression levels can be quantified by membrane staining intensity measured by IHC using an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody, e.g., 14G11 below). The range of membrane staining intensity may be, for example, 0 (no staining), 1+ (weak staining), 2+ (clear staining), 3+ (strong staining), and 4+ (very strong / saturated signal).

[0079] In certain embodiments, CLDN18.2 expression levels can be quantified based on the percentage of cells (e.g., cancer cells) exhibiting a specific membrane staining intensity level in the sample. In certain embodiments, CLDN18.2 expression levels can be quantified by the percentage of cancer cells whose membrane staining intensity, measured by IHC using an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody, e.g., 14G11 below), is a predetermined threshold. For example, to evaluate CLDN18.2 expression in a sample, an observer can examine the number of cells stained positive for CLDN18.2 in one or more selected fields under a microscope and calculate or estimate the percentage of cells (e.g., cancer cells) exhibiting a specific membrane staining intensity level for CLDN18.2. In certain embodiments, viable cells are stained and their claudin 18.2 expression levels are analyzed.

[0080] In certain embodiments, CLDN18.2 expression levels can also be quantified based on an intensity score calculated based on membrane staining intensity and the percentage of cells having a specific membrane staining intensity level. For example, an intensity score such as the 4-point HSCORE can be calculated by multiplying the percentage (0-100%) of cells stained at each intensity, based on staining intensities ranging from 0 (no staining), 1+ (weak staining), 2+ (clear staining), 3+ (strong staining), and 4+ (very strong / saturated signal) (see McCarty, KS Jr, et al, Cancer Res. 46(suppl 8):4244s-4248s (1986) for details). As another example, the Alfred score can be calculated based on a total score (TS, range 0-8) by adding a percentage score (PS) and an intensity score (IS). PS is the percentage of positive tumor cells ranging from 0 to 5 (0 = no positive cells, 1 = 1 / 100 cells positive, 2 = 1 / 10 cells positive, 3 = 1 / 3 cells positive, 4 = 2 / 3 cells positive, 5 = all tumor cells positive). IS represents the average staining intensity of positive tumor cells ranging from 0 to 3 (0 = negative, 1 = weak staining, 2 = moderate staining, 3 = strong staining) (see Alfred DC et al. Mod Pathol. 11: 155-168 (1998) for details).

[0081] In certain embodiments, CLDN18.2 expression levels are measured by an IHC assay. In short, cancer samples (e.g., tumor tissue samples, optionally formalin-fixed paraffin-embedded (FFPE) tissue samples) with a specific section thickness (e.g., 1–5 μm (e.g., 2–5 μm, 3–5 μm, or 4–5 μm)) are stained with a specific antigen retrieval solution (e.g., BOND Epitope Retrieval Solution 2 (Leica, AR9640), BOND Epitope Retrieval Solution 2 (Leica, AR9640), or CC1) that is compatible with a staining system (e.g., Leica Bond III LDT platform, Dako Autostainer Link 48 platform, or VENTANA BenchMark XT / ULTRA platform). The antigen is restored in Solution (Ventana, 760-700) at a specific temperature (e.g., 50°C-100°C, 60°C-100°C, 70°C-100°C, 80°C-100°C, 90°C-100°C, 92°C, 94°C, 96°C, 97°C, 98°C, or 100°C) for a set period of time (e.g., 15-120 min, 25-100 min, 35-90 min, 45-80 min, 55-90 min, 65-80 min, 70 min, 15-30 min, 20-30 min, or 64-104 min). After antigen recovery, the sample is exposed to (e.g., incubated with) anti-CLDN18.2 diagnostic antibody (e.g., 14G11 below) at a specific concentration (e.g., 0.05-20 ug / ml, 0.1-15 ug / ml, 0.15-10 ug / ml, 0.15-5 ug / ml, 0.2-10 ug / ml, 0.3-10 ug / ml, 0.4-10 ug / ml, 0.5-10 ug / ml, 0.6-10 ug / ml, 0.7-10 ug / ml, 0.8-10 ug / ml, 0.9-10 ug / ml, 1-10 ug / ml, or 0.15-5 ug / ml) for a certain period of time (e.g., 10-60 min, 15-50 min, 15-40 min, 15-35 min, 15-30 min, 16-32 min, or 20-30 min).Next, the sample may be brought into contact with a secondary antibody (e.g., BOND Polymer Detection (Leica, DS9800), FLEX / HRP reagent (Dako, K8002), or OptiView HRP Multimer reagent (Ventana, 760-700)) (e.g., incubated with it) and visualized using a staining system (e.g., Leica Bond III LDT platform, Dako Autostainer Link 48 platform, or VENTANA BenchMark XT / ULTRA platform). Those skilled in the art will understand that section thickness, time, specific temperature, and specific concentration of anti-CLDN18.2 diagnostic antibody may vary depending on the imaging system and the diagnostic antibody used.

[0082] For example, different assay formats can be used, such as IHC assays using Leica automated staining systems (e.g., Leica Bond III LDT platform), IHC assays using Dako automated staining systems (e.g., Dako Autostainer Link 48 platform), or IHC assays using VENTANA automated staining systems (e.g., VENTANA BenchMark XT / ULTRA platform).

[0083] For Leica automated staining systems (e.g., Leica Bond III LDT platform), cancer samples (e.g., tumor tissue samples, or formalin-fixed paraffin-embedded (FFPE) tumor tissue samples) are sectioned to a thickness of 4-5 μm and allowed to recover antigens at 100°C for 15-30 minutes. Antigen recovery is performed using BOND Epitope Retrieval Solution 2 (Leica, AR9640) and detected with CLDN18.2 diagnostic reagent (e.g., 14G11 below) in a concentration range of 0.15-5 μg / mL, and the diagnostic reagent is allowed to come into contact with the cancer sample (e.g., tumor tissue sample) for 15-30 minutes. A more detailed description of the Leica Bond III LDT platform is provided in Example 1.

[0084] In certain embodiments, when a Leica automated staining system (e.g., Leica Bond III LDT platform) is used in an IHC assay, a predetermined threshold for moderate or moderate CLDN18.2 protein expression levels is 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of cancer cells in a cancer sample (e.g., tumor tissue sample) having a membrane staining intensity of at least 2+ or at least 3+. In such embodiments, a low CLDN18.2 protein level corresponds to less than 30%, or less than 31%, less than 32%, less than 33%, less than 34%, less than 35%, less than 36%, less than 37%, less than 38%, less than 39%, less than 40%, or less than 41%, less than 42%, less than 43%, less than 44%, less than 45%, less than 46%, less than 47%, less than 48%, less than 49%, or less than 50%. In certain embodiments, a low CLDN18.2 protein level corresponds to at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of cancer cells in a cancer sample (e.g., a tumor tissue sample) having at least 1+ membrane staining intensity.

[0085] In certain embodiments, a low CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 1% (e.g., at least 2%, at least 5%, or at least 10%) of cancer cells in a cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 1+, as optionally measured using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) on a Leica automated staining system (e.g., Leica Bond III LDT platform), and less than 40% of cancer cells in a cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ (e.g., 2+ or 3+). Low CLDN18.2 protein levels or CLDN18.2 expression levels may be characterized by equivalent readouts measured on a different test platform (e.g., on the Dako Autostainer Link 48 platform or the VENTANA BenchMark XT / ULTRA platform) using the same anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) or a different anti-CLDN18.2 diagnostic antibody.

[0086] In certain embodiments, moderate to high CLDN18.2 protein levels or CLDN18.2 expression levels correspond to at least 40% of cancer cells in a cancer sample (e.g., tumor tissue sample) having CLDN18.2 expression levels characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity, as optionally measured on a Leica automated staining system (e.g., Leica Bond III LDT platform) using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11). Moderate to high CLDN18.2 protein levels or CLDN18.2 expression levels may also be characterized by equivalent readouts measured on a different test platform using the same anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) or a different anti-CLDN18.2 diagnostic antibody.

[0087] In certain embodiments, the group of subjects having moderate to high CLDN18.2 protein levels or CLDN18.2 expression levels may be further divided into two subgroups: the group having moderate CLDN18.2 protein levels or CLDN18.2 expression levels, or the group having high CLDN18.2 protein levels or CLDN18.2 expression levels.

[0088] In certain embodiments, a high CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 70% of cancer cells in a cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity, as measured optionally on a Leica automated staining system (e.g., Leica Bond III LDT platform) using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11). A high CLDN18.2 protein level or CLDN18.2 expression level may also be characterized by equivalent readouts measured on a different test platform using the same anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) or a different anti-CLDN18.2 diagnostic antibody.

[0089] In certain embodiments, a moderate CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 40% of cancer cells in a cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity, as optionally measured on a Leica automated staining system (e.g., Leica Bond III LDT platform) using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11), and less than 70% of cancer cells in a cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity. A moderate CLDN18.2 protein level or CLDN18.2 expression level may also be characterized by equivalent readouts measured on a different test platform using the same anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) or a different anti-CLDN18.2 diagnostic antibody.

[0090] In addition to Leica automated staining systems (e.g., the Leica Bond III LDT platform), other suitable platforms can also be used to measure the expression level of the CLDN18.2 protein. Reference standards or calibration methods may be employed to establish equivalence between readouts obtained from different platforms. For example, a sample might be classified as having a membrane staining intensity of 1+ on the Leica Bond III LDT platform, while a different assay performed on a different platform might yield a different measurement of the expression level. Through appropriate calibration and comparative studies, these different readouts can be demonstrated to correspond to equivalent thresholds for the CLDN18.2 protein expression level. This ensures that, regardless of variability in readouts, different assays consistently provide equivalent measurements for the same sample.

[0091] For the Dako Autostainer Link 48 platform, cancer samples (e.g., tumor tissue samples) are sectioned to a thickness of 4 μm and antigen-recovered at 97–100°C for 20–30 minutes. Antigen recovery is performed using Target Retrieval Solution, High pH (Dako, K8002) and detected with CLDN18.2 diagnostic reagent in the concentration range of 0.5–10 μg / mL, with the diagnostic reagent in contact with the cancer sample (e.g., tumor tissue sample) for 20–30 minutes. In exemplary cases, IHC may be performed on slides of 4% neutral buffered formalin-fixed paraffin-embedded samples. Sections may be cut to a thickness of 4 μm and mounted on positively charged slides. With a preset PT Link pre-treatment module (Dako, PT100 / PT101 / PT200), deparaffinization, rehydration, and antigen recovery may be performed according to a 3-in-1 procedure by heating with Target Retrieval Solution, High pH (Dako, K8002) at 97-100°C for 20-30 minutes. After washing with a PT Link Rinse Station (Dako, PT109), the slides are placed on a Dako Autostainer Link 48 platform (Dako, AS480) using a pre-programmed staining protocol: quench and block with a peroxidase blocking reagent for 5 minutes, and incubate for 20-30 minutes with a diagnostic antibody in the dynamic concentration range (0.5-10 μg / mL) diluted with Antibody Diluent (Dako, S0809). Antibody binding was amplified with FLEX+Mouse (LINKER) and visualized with FLEX / HRP (Dako, K8002) by the formation of a brown precipitate due to reaction with the DAB substrate. Finally, sections were counterstained with hematoxylin and mounted with permanent mounting medium. IHC results may be scored under bright-field microscopy by a certified pathologist based on membrane staining intensity (negative (0), weak (1+), moderate (2+), strong (3+)) and the tumor percentage score (TPS) defined by the percentage of surviving invasive tumor cells showing a target staining intensity of ≥1+ in all surviving invasive tumor cells. Normal tissue and / or adjacent non-involved tissue are not included in the scoring evaluation.

[0092] In certain embodiments, cancer cells with low CLDN18.2 protein expression levels do not respond significantly to monotherapy with an anti-claudin 18.2 antibody, for example, monotherapy in which the anti-claudin 18.2 antibody is zolbetuximab. In certain embodiments, cancer cells with low CLDN18.2 protein expression levels do not respond significantly to combination therapy including an anti-claudin 18.2 antibody, for example, zolbetuximab. The combination therapy may include chemotherapy and / or immunotherapy.

[0093] ai). Anti-CLDN18.2 diagnostic reagent In some embodiments, the anti-CLDN18.2 diagnostic reagent is a nucleic acid or nucleic acid binder employed in, for example, an in situ hybridization (ISH, e.g., fluorescence in situ hybridization (FISH)) reaction. For example, the anti-CLDN18.2 diagnostic reagent may be RNA, DNA, DNA / RNA hybrid molecules, and / or peptide nucleic acids (PNA).

[0094] In certain embodiments, the anti-CLDN18.2 diagnostic reagent comprises an anti-CLDN18.2 diagnostic antibody. The anti-CLDN18.2 diagnostic antibody may be any anti-CLDN18.2 antibody capable of highly sensitively detecting CLDN18.2 expression on a cancer sample (e.g., a tumor tissue sample). The anti-CLDN18.2 diagnostic antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment thereof. For example, the anti-CLDN18.2 diagnostic antibody may be one described in PCT / CN2021 / 095411. In certain embodiments, the anti-CLDN18.2 diagnostic antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of 14G11, and / or the LCDR1, LCDR2, and LCDR3 sequences. As used herein, the term “14G11” refers to an anti-CLDN18.2 antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the anti-CLDN18.2 diagnostic antibody is a 14G11 comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the anti-CLDN18.2 diagnostic antibody is a 14G11 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 19 and a light chain containing the amino acid sequence of SEQ ID NO: 20. The anti-CLDN18.2 diagnostic antibody may also be other commercially available anti-CLDN18.2 antibodies such as [EPR19202](ab222512), 43-14A, SDT-102-24, MIL93, or 3B10.

[0095] The anti-CLDN18.2 diagnostic reagents described herein can be conjugated to any suitable detectable label or enzyme capable of producing a detectable label. Any convenient enzyme labeling / chromogenic deposition system can be employed (e.g., as used in standard IHC methods). In some embodiments, the detectable label is a fluorescent tag.

[0096] In certain embodiments, the label is indirectly conjugated to an anti-CLDN18.2 diagnostic antibody. Those skilled in the art will be aware of various techniques for achieving this. For example, the anti-CLDN18.2 diagnostic antibody may be conjugated with biotin, and the label may be conjugated with avidin, or vice versa. Since biotin selectively binds to avidin, the label can thus be indirectly conjugated to the antibody. Alternatively, to achieve indirect conjugation of the label and the antibody, the antibody may be conjugated with a small hapten, and the label may be conjugated with an anti-hapten antibody. In this way, indirect conjugation of the label and the antibody can be achieved. In certain embodiments, one or more secondary antibody molecules may be conjugated to a labeled conjugate polymer backbone so as to enable signal amplification.

[0097] Indirectly and directly labeled secondary antibodies are also commercially available, such as BOND Polymer Detection (Leica, DS9800), FLEX / HRP reagent (Dako, K8002), and OptiView HRP Multimer reagent (Ventana, 760-700).

[0098] a-ii) Sample In certain embodiments, the subject has or is determined to have a moderate to high or low CLDN18.2 expression level or a low CLDN18.2 protein level in cancer samples (e.g., tumor tissue samples) derived from the subject. In certain embodiments, the method provided herein further includes providing a biological sample from the subject. Any biological sample suitable for carrying out the method provided herein may be obtained from the subject. As used herein, “biological sample” means a biological specimen taken from the subject by sampling, which has undergone any additional processing. The collection of a sample from the subject is carried out, for example, by biopsy, in accordance with standard protocols commonly followed in hospitals or clinics.

[0099] In certain embodiments, the biological sample includes a cancer sample (e.g., a tumor tissue sample). In some embodiments, the biological sample is a fresh or stored sample obtained from tumor tissue, for example, by tumor biopsy or fine-needle aspiration. In some embodiments, the sample may be any biological fluid containing cancer cells or non-cancerous cells (e.g., peripheral blood mononuclear cells (PBMCs)).

[0100] Examples of biological samples include body fluids such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal lavage fluid, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, etc., and tissues such as biopsy tissue (e.g., biopsy bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, colon tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, colon tissue, kidney tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue), and paraffin-embedded tissue. In further embodiments, the biological sample includes cells, tissues, blood, plasma, serum, urine, mouthwash, feces, saliva, and any combination thereof.

[0101] In certain embodiments, the biological sample (e.g., cancer or tumor tissue sample) may be further processed to separate analytes such as nucleic acids or proteins. For example, the biological sample from the subject (e.g., cancer or tumor tissue sample) may be exposed to an anti-CLDN18.2 diagnostic reagent, which binds to translated CLDN18.2 mRNA or expressed CLDN18.2 protein and detects it.

[0102] In certain embodiments, the sample is a fixed tissue sample, optionally a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0103] FFPE tissue sections may be about 3–4 millimeters, preferably 4–40 micrometers, and are mounted on microscope slides and dried. Examples of paraffins include, but are not limited to, Paraplast, Broloid, and Tissue.ge. Fixed tissue samples, such as FFPE tissue samples, may be deparaffinized before contact with the anti-CLDN18.2 antibody or its antigen-binding fragment provided herein.

[0104] In some embodiments, the deparaffinized sample may be further processed to enable antigen recovery. Antigen recovery refers to any technique that demasks an epitope and restores epitope-antibody binding. The need for antigen recovery depends on several variables, not limited to the target antigen, the antibody used, the type of tissue, and the fixation method and duration. Antigen recovery techniques generally include protease-induced epitope recovery (PIER by enzymes such as proteinase K, trypsin, and / or pepsin) and heat-induced epitope recovery (HIER by microwave, pressure cooker, vegetable steamer, autoclave, or water bath).

[0105] In certain embodiments, the sample may be further processed in a manner preferred for determining the expression level of at least one biomarker, for example, CLDN18.2.

[0106] In certain embodiments, the CLDN18.2 expression level or CLDN18.2 protein level is determined from a cancer sample (e.g., a tumor tissue sample).

[0107] b.CLDN18.2 Antagonist

[0108] The CLDN18.2 antagonist used in the methods provided herein may include an anti-CLDN18.2 antibody or its antigen-binding fragment. The anti-CLDN18.2 antibody may be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotype antibody.

[0109] In certain embodiments, the anti-CLDN18.2 antibody comprises heavy chain HCDR1, HCDR2 and HCDR3 sequences and / or light chain LCDR1, LCDR2 and LCDR3 sequences. The HCDR1 sequence includes GYNMN (Sequence ID 1), or a homologous sequence having at least 80% sequence identity therewith. The HCDR2 sequence includes NIDPYYGGTSYNQKFKG (Sequence ID 2), or a homologous sequence having at least 80% sequence identity therewith. The HCDR3 sequence includes MYHGNAFDY (SEQ ID NO: 3), or a homologous sequence having at least 80% sequence identity therewith. The LCDR1 sequence includes KSSQSLLNSGNLKNYLT (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity therewith. The LCDR2 sequence includes WASTRKS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity therewith. The LCDR3 sequence includes QNDYSYPLT (sequence number 6) or a homologous sequence having at least 80% sequence identity thereto.

[0110] While CDRs are known to be involved in antigen binding, it has been discovered that not all six CDRs are necessarily essential or immutable. In other words, it is possible to replace, change, or modify one, two, or three CDRs in an anti-CLDN18.2 antibody while substantially maintaining specific binding affinity to CLDN18.2.

[0111] In certain embodiments, the anti-CLDN18.2 antibody contains the heavy chain CDR3 sequence MYHGNAFDY (SEQ ID NO: 3). Because the heavy chain CDR3 region is centrally located at the antigen-binding site, it is thought to have the most contact with the antigen and provide the most free energy for the antibody's affinity to the antigen. Furthermore, the heavy chain CDR3 is considered to be by far the most diverse CDR among antigen-binding sites in terms of length, amino acid composition, and conformation, due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81). The diversity of the heavy chain CDR3 is sufficient to provide most antibody specificity (Xu JL, Davis MM. Immunity. 13:37-45) and desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263:551-67).

[0112] In some embodiments, the anti-CLDN18.2 antibody comprises all or some heavy chain variable domains and / or all or some light chain variable domains. In one embodiment, the anti-CLDN18.2 antibody is a single-domain antibody comprising all or some of the heavy chain variable domains provided herein. Details of such single-domain antibodies are available in the art (see, for example, U.S. Patent No. 6,248,516).

[0113] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0114] In certain embodiments, the anti-CLDN18.2 antibody further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region. In certain embodiments, the anti-CLDN18.2 antibody further comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0115] In some embodiments, the immunoglobulin constant region includes heavy chain and / or light chain constant regions. The heavy chain constant region includes CH1, hinge, and / or CH2-CH3 regions. In certain embodiments, the heavy chain constant region includes an Fc region. In certain embodiments, the light chain constant region includes Cκ or Cλ.

[0116] In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein include a constant region of an IgG1 isotype. In certain embodiments, the constant region of human IgG1 includes SEQ ID NO: 9 or a homologous sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0117] The constant region of an IgG1 isotype can induce effector functions such as ADCC or CDC. The effector function of anti-CLDN18.2 antibodies can cause cytotoxicity in cells expressing CLDN18.2. Effector function can be evaluated by various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays, as well as any of the assays described above to determine ADCC or CDC.

[0118] In certain embodiments, the constant region includes one or more amino acid residue substitutions or modifications that result in increased CDC or ADCC compared to the wild-type constant region.

[0119] As used herein, “ADCC” or “antibody-dependent cytotoxicity” refers to a cell-mediated response in which nonspecific toxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a bound antibody on a target cell, subsequently causing lysis of the target cell. The lysis of the target cell is extracellular lysis, requiring direct intercellular contact and without complement involvement. ADCC may be considered a mechanism that directly induces varying degrees of immediate tumor destruction, leading to antigen presentation and induction of tumor-targeting T cell responses. In vivo induction of ADCC is thought to elicit tumor-targeting T cell responses and host-derived antibody responses. In certain embodiments, the constant region comprises one or more amino acid residue substitutions to SEQ ID NO: 9, selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO: 11, and optionally further comprises the sequence of SEQ ID NO: 10.

[0120] In certain embodiments, the anti-CLDN18.2 antibody is humanized. Humanized antibodies or antigen-binding fragments are desirable because they have reduced immunogenicity in humans. Humanized antibodies are chimeric in the variable region because non-human CDR sequences are transplanted into human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be essentially carried out by substituting non-human (e.g., mouse) CDR genes with corresponding human CDRs in human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-327, and Verhoeyen et al. (1988) Science 239:1534-1536). In certain embodiments, the humanized light and heavy chains of this disclosure are substantially non-immunogenic in humans and retain substantially the same or higher affinity to the parental antibody against CLDN18.2.

[0121] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein includes a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and homologous sequences thereof that have at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while maintaining specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0122] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein includes a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and homologous sequences thereof that have at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while maintaining specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0123] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, and SEQ ID NOs: 14, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and SEQ ID NOs: 16.

[0124] In certain embodiments, the humanized anti-CLDN18.2 antibody may include a heavy chain variable region fused to the constant region of a human IgG1 isotype and a light chain variable region fused to the constant region of a human kappa chain.

[0125] The humanized anti-CLDN18.2 antibodies provided herein retain specific binding affinity to biological samples expressing CLDN18.2, and in this respect are at least equivalent to or superior to the parent antibody. The humanized antibodies provided herein also retain their functionality, in that all antibodies can mediate cell death by ADCC and CDC, induction of apoptosis induced by cross-linking of targets on the surface of tumor cells, and direct inhibition of proliferation.

[0126] In certain embodiments, the anti-CLDN18.2 antibody is Hu18B10-HaLa. As used herein, the term "Hu18B10-HaLa" refers to a recombinant humanized IgG1 antibody that acts specifically on human CLDN18.2 by high affinity and enhanced NK cell FcR binding, and can induce strong antibody-dependent cytotoxicity (ADCC) activity. In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and the light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0127] The anti-CLDN18.2 antibody used in the methods provided herein may encompass various types of variants of the antibody sequences provided herein.

[0128] In certain embodiments, a variant includes one or more modifications or substitutions in the one, two, or three CDR sequences, one or more FR sequences, heavy or light chain variable region sequences provided herein, and / or constant regions (e.g., Fc regions) described above. Such antibody variants have one or more desirable properties brought about by the modifications or substitutions while retaining the specific binding affinity of their parent antibody to CLDN18.2. For example, an antibody variant may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or increased effector function, improved FcRn receptor binding, and increased pharmacokinetic half-life, pH sensitivity, and / or conjugation compatibility (e.g., one or more introduced cysteine ​​residues). In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein also includes glycosylated variants with improved effector function, such as ADCC or CDC.

[0129] In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein is afucosylated. The term “afucosylated” or “to be afucosylated” refers to the reduction or removal of core fucose on the N-glycan bound to the antibody. The majority of human IgG antibody glycans are known as G0, G1, and G2, and are complex branched molecules having core fucose residues with 0, 1, or 2 terminal galactoses.

[0130] Examples of afucosylated antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, and Okazaki et al. J. Mol. As described in Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004), it can be prepared using methods known in this field.

[0131] In certain embodiments, the antibody glycosylated variant is afcosylated at the Asn297 site of the CH2 region in the Fc region of the antibody. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues), however, due to slight sequence changes in the antibody, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0132] In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein also includes cysteine-operated variants, which include one or more introduced free cysteine ​​amino acid residues. The free cysteine ​​residues are residues that are not part of a disulfide crosslink. The cysteine-operated variants are useful for conjugating at the operated cysteine ​​site with, for example, maleimide or haloacetyl, cytotoxic compounds and / or imaging compounds, labels, or radioisotopes. Methods for manipulating antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art; see, for example, WO2006 / 034488.

[0133] In certain embodiments, the constant region of the anti-CLDN18.2 antibody or its antigen-binding fragment used in the method provided herein comprises one or more amino acid residue substitutions selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof, relative to SEQ ID NO: 9 (i.e., the wild-type sequence). In certain embodiments, the constant region comprises the sequence of SEQ ID NO: 11.

[0134] In certain embodiments, the anti-CLDN18.2 antibodies used in the methods provided herein also include anti-CLDN18.2 antigen-binding fragments such as diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0135] In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein is bivalent, tetravalent, hexavalent, or polyvalent. As used herein, “valency” refers to the presence of a specified number of antigen-binding sites in a given molecule. Therefore, the terms “bivalent,” “tetravalent,” and “hexavalent” indicate the presence of two, four, and six binding sites in the antigen-binding molecule, respectively. Any molecule with more than two valencies is considered polyvalent, including, for example, trivalent, tetravalent, and hexavalent antibodies.

[0136] A bivalent molecule can be monospecific if both binding sites specifically bind to the same antigen or epitope. This results in stronger binding to the antigen or epitope than its monovalent counterpart in certain embodiments. Similarly, a polyvalent molecule can also be monospecific. In certain embodiments, the primary and secondary binding sites in the bivalent or polyvalent antigen-binding moiety are either structurally identical (i.e., have the same sequence) or structurally different (i.e., have the same specificity but different sequences).

[0137] A bivalent molecule may be bispecific if its two binding sites are specific to different antigens or epitopes. The same applies to polyvalent molecules. For example, a trivalent molecule may be bispecific if two binding sites are monospecific to the first antigen (or epitope) and the third binding site is specific to the second antigen (or epitope). The bispecific antibodies that can be used in the methods provided herein can target both CLDN18.2 and checkpoint molecules such as PD-1, PD-L1, PD-L2, CLTA-4, SIRPα TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, or CD83. Exemplary bispecific antibodies that can be used in the methods provided herein include, without limitation, bispecific antibodies targeting CLDN18.2 and CD3, bispecific antibodies targeting CLDN18.2 and 4-1BB, bispecific antibodies targeting CLDN18.2 and TGFβ, bispecific antibodies targeting CLDN18.2 and SIRP, and bispecific antibodies targeting CLDN18.2 and IL-15.

[0138] The anti-CLDN18.2 antibody used in the methods provided herein may be an antibody-drug conjugate (ADC) comprising any of the above anti-CLDN18.2 antibodies conjugated to a cytotoxic agent.

[0139] In certain embodiments, the cytotoxic agent may be any agent that is harmful to cells or that can damage or kill cells. In certain embodiments, the cytotoxic agent may optionally be a toxin, a chemotherapeutic agent (DNA alkylating agent, topoisomerase inhibitor, tubulin binder, growth inhibitor, or other anticancer drug), or a radioisotope.

[0140] Examples of toxins include bacterial and plant toxins, such as diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine, abrin, modexin, α-sarcin, Aleurites fordi protein, dianthin protein, pokeweed protein (PARI, PAPII, and PAP-S), bitter melon inhibitors, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, restrictosin, phenomycin, enomycin, and trichothecenes (see, e.g., WO93 / 21232). Such macromolecular toxins can be conjugated to antibodies or antigen-binding fragments provided herein by methods known in the art, for example, as described in Vitetta et al (1987) Science, 238:1098.

[0141] Cytotoxic agents may be small molecule toxins and chemotherapeutic agents, for example, geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581, Mandler et al (2002) Bioconjugate Chem. 13:786-791), meitansine and meitansinoids (EP1391213, Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623, U.S. Patent No. 5,208,020), calicheamicin (Lode et al (1998) Cancer Res. 58:2928, Hinman et al (1993) Cancer Res.53:3336-3342), Taxol, Cytochalasin B, Gramicidin D, Ethidium Bromide, Emetine, Mitomycin, Etoposide, Tenoposide, Vincristine, Vinblastine, Vindesine, Colchicine, Doxorubicin, Daunorubicin, Dihydroxyanthracinedione, Mitoxanthrone, Mitramycin, Actinomycin D, 1-Dehydrotestosterone, Glucocorticoid, Procaine Tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites (e.g., methotrexate, 6-mercappurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromo These include mannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), calicheamicin, meitansinoids, dorastatin, auristatins such as MMAE and MMAF (U.S. Patent Nos. 5,635,483 and 5,780,588), drostatin, trichothecene, exatecan, berothecan, deruxtecan, MMAE, MMAF, DM1, DM4, SN-38, PBD, eribulin, and CC1065, and their cytotoxic derivatives.

[0142] Cytotoxic agents may be highly radioactive isotopes. Examples include the radioactive isotopes At211, I131, I125, Y90, Re186, Sm153, Bi212, P32, Pb212, and Lu. Methods for conjugating radioisotopes with antibodies are known in the art, for example, by suitable ligand reagents (see, e.g., WO94 / 11026, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. (1991)). Ligand reagents have a chelate ligand that can bind, chelate, or complex with the radioactive metal isotope, and also have a functional group that reacts with the cysteine ​​thiol of the antibody or antigen-binding fragment. Exemplary chelate ligands include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex.).

[0143] In certain embodiments, the ADC used in the methods provided herein is conjugated with an antibody (or its antigen-binding fragment) and one or more cytotoxic agents in an antibody-to-drug ratio of about 1 to about 20, about 1 to about 6, about 2 to about 6, about 3 to about 6, about 2 to about 5, about 2 to about 4, or about 3 to about 4.

[0144] c.Chemotherapy

[0145] In certain embodiments, the chemotherapy used in the methods provided herein includes a combination of chemotherapeutic agents. The term "chemotherapeutic agent" refers to a biocompound (molecular weight) or chemical compound (small molecule) that can be used in the treatment of cancer. Types of chemotherapeutic agents include, but are not limited to, histone deacetylase inhibitors (HDACIs), alkylating agents, antimetabolites, alkaloids, cytotoxic antibiotics / anticancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, kinase inhibitors, and the like. Examples of chemotherapy drugs include erlotinib, afatinib, docetaxel, adriamycin, 5-FU (5-fluorouracil), panobinostat, gemcitabine, cisplatin, pemetrexed, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, oteracil, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trametinib, tegafur, gimeracil, leucovorin calcium (folic acid) (LV), irinotecan hydrochloride (CPT-11), platinum (e.g., cisplatin), epirubicin, oxaliplatin, and capecitabine.

[0146] In certain embodiments, the chemotherapy comprises a chemotherapeutic agent selected from the group consisting of LV, 5-FU, CPT-11, epirubicin, oxaliplatin, capecitabine, platinum (e.g., cisplatin), tegafur, gimeracil, oteracil, docetaxel, and pemetrexed. In certain embodiments, the combination of chemotherapeutic agents comprises capecitabine and oxaliplatin. In certain embodiments, the combination of chemotherapeutic agents consists of capecitabine and oxaliplatin.

[0147] In certain embodiments, the chemotherapy regimen (or combination of chemotherapy agents) may be selected from the group consisting of FOLFIRI chemotherapy, EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, CAPOX chemotherapy, FOLFOX chemotherapy, DCF chemotherapy, SOX chemotherapy, and FLOT chemotherapy. The drug combination used in FOLFIRI chemotherapy includes or consists of LV, 5-FU, and CPT-11. The drug combination used in EOX chemotherapy includes or consists of epirubicin, oxaliplatin, and capecitabine. The drug combination used in ECF chemotherapy includes or consists of epirubicin, cisplatin, and 5-FU. The drug combination used in ECX chemotherapy includes or consists of epirubicin, cisplatin, and capecitabine. The drug combination used in EOF chemotherapy includes or consists of epirubicin, oxaliplatin, and 5-FU. The drug combination used in FLO chemotherapy includes or consists of 5-FU, LV, and oxaliplatin. The drug combination used in SOX chemotherapy includes or consists of tegafur, gimeracil, oteracil, and oxaliplatin.

[0148] In certain embodiments, the chemotherapy is 20 mg / m² 2 ~400mg / m 2 (For example, 40 mg / m²) 2 , 60 mg / m² 2 , 80 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 130 mg / m² 2 , 140 mg / m² 2 , 160 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 220 mg / m² 2 , 240 mg / m² 2 , 300 mg / m² 2 340 mg / m² 2 , 380 mg / m² 2 or 400 mg / m² 2It contains oxaliplatin. In certain embodiments, the chemotherapy is 500 mg / m². 2 ~2000mg / m 2 (For example, 600 mg / m²) 2 , 650 mg / m² 2 700 mg / m² 2 750 mg / m² 2 , 800 mg / m² 2 , 850 mg / m² 2 900 mg / m² 2 950 mg / m² 2 , 1000 mg / m² 2 , 1500 mg / m² 2 , 1700 mg / m² 2 or 2000 mg / m² 2 Contains capecitabine.

[0149] In certain embodiments, the chemotherapy used in the method provided herein is the CAPOX regimen. In certain embodiments, the CAPOX regimen is administered intravenously at a dose of 130 mg / m² on day 1. 2 This regimen consists of oxaliplatin (e.g., intravenous infusion over 2 hours or more, up to 8 cycles) and capecitabine (total daily dose of 1700 or 2000 mg / m2) administered orally twice daily at 850 mg / m2 or 1000 mg / m2 per dose over days 1 to 14. Several other CAPOX regimens exist that differ in dosage and administration method of both drugs.

[0150] Other chemotherapy regimens described above are described in PCT patent application PCT / JP2022 / 017017, which is incorporated herein by reference.

[0151] d. Immunotherapy

[0152] In certain embodiments, the immunotherapy used in the methods provided herein includes immunotherapeutic agents such as PD-1 / PD-L1 axis inhibitors.

[0153] In certain embodiments, the PD-1 / PD-L1 axis inhibitors include nivolumab (OPDIVO, BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA, MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pidilizumab (CT-011), semiprimab (LIBTAYO, REGN2810), spartalizumab (PDR001), and cetrelimab (JNJ 63723283), Tripalimab (JS001), PF-06801591, Tislerizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lambrolizumab, Camrelizumab (SHR-1210), Syntilimab (Tyvyt, IBI308), Penplimab (AK105), Zin Bererimab, Letifanlimab, Selprulimab, Valstilimab, Geptanolimab, Prorugolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofadinlimab, Syndelizumab, MGA404, Sym021, BAT1306, HX008, Atezolizumab (TECENTRIQ, R055412) The following drugs are selected from the group consisting of 67, MPDL3280A, RG7446), BMS-936559, avelumab (Bavencio), rodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, emvafolimab (KN035), MDX-1105, STI-1040, CS1001, adebrelimab (SHR-1316), SHR-1701, TOB2450, vintrafusp, LP002, STI-3031, cosivelimab, pacumimab, NM01, LDP, AMP-224, galiblimab (BGB-A333), A167, SCD-135, opcolimab, and GR1405. In certain embodiments, the PD-1 / PD-L1 axis inhibitor is nivolumab.

[0154] In certain embodiments, the method includes administering a therapeutically effective dose of Hu18B10-HaLa to a subject in combination with chemotherapy (e.g., CAPOX) and nivolumab.

[0155] In certain embodiments, the subject is determined to have PD-L1 expression in cancer samples (e.g., tumor tissue samples).

[0156] In certain embodiments, PD-L1 expression levels are measured by immunohistochemistry (IHC) assays, hybridization assays, or amplification assays.

[0157] In certain embodiments, the PD-L1 expression level is quantified by membrane staining intensity measured by IHC using an anti-PD-L1 diagnostic reagent (e.g., an anti-PD-L1 diagnostic antibody).

[0158] In certain embodiments, PD-L1 expression is determined by IHC. PD-L1 staining by IHC requires appropriate patient samples, a highly sensitive primary anti-PD-L1 antibody, and a suitable staining protocol. Three scoring algorithms are primarily used for tumor type-specific PD-L1 staining and clinical judgment: tumor percentage score (TPS), immune percentage score (IPS), and composite positivity score (CPS) (see Schildhaus et al., Predictive value of PD-L1 diagnostics. Pathologe. 2018 Nov;39(6):498-519. doi: 10.1007 / s00292-018-0507-x).

[0159] TPS may also be defined as the percentage of PD-L1-positive tumor cells relative to all tumor cells in a biological sample. For more details, see, for example, Piper et al., Can PD-L1 tumor proportion score be used as the key to unlocking the KEYNOTE studies of pembrolizumab in advanced lung cancer? Transl Lung Cancer Res 2019;8(5):715-722.

[0160] iPS may also be defined as the percentage of PD-L1-positive tumor-associated immune cells relative to all tumor-associated immune cells. For more details, see, for example, Yang et al., PD-L1 expression on tumor cells and tumor infiltrating immune cells in Chinese colorectal cancer patients. Journal of Clinical Oncology, Volume 38, Issue 15_suppl.

[0161] In certain embodiments, PD-L1 expression levels are quantified by a composite positivity score (CPS), where CPS is determined by the ratio of PD-L1-stained immune cells and tumor cells to all viable tumor cells. CPS may also be defined as the number of PD-L1 stained cells (including tumor cells, lymphocytes, and macrophages without limitation) divided by the total number of surviving tumor cells and multiplied by 100. For details, see, for example, Yamashita et al., Prognostic impacts of the combined positive score and the tumor proportion score for programmed death ligand-1 expression by double immunohistochemical staining in patients with advanced gastric cancer. Gastric Cancer. 2020 Jan;23(1):95-104, and Dako et al., Development of the combined positive score (CPS) for the evaluation of PD-L1 in solid tumors with the immunohistochemistry assay PD-L1 IHC 22C3 pharmDx. Journal of Clinical Oncology, Volume 35, Issue 15_suppl, 2017.

[0162] For example, a PD-L1 expression level of 5% (or a CPS of 5) in a biological sample such as diseased tissue (e.g., cancerous or tumorous tissue) means that 5% of the cells (including tumor cells, lymphocytes, and macrophages without limitation) in the biological sample are PD-L1 stained positive.

[0163] The CPS of PD-L1 expression on a sample can be obtained using PD-L1 IHC 22C3 pharmDx or PD-L1 IHC 28-8 pharmDx. The PD-L1 IHC 28-8 pharmDx assay (Agilent Technologies, Santa Clara, California, USA), a kit for detecting PD-L1 expression, is approved as a complementary diagnostic for use with nivolumab in cancers such as non-squamous non-small cell lung cancer (NSQNSCL) and head and neck squamous cell carcinoma (SCCHN). The PD-L1 IHC 28-8 pharmDx includes optimized reagents and protocols necessary to complete IHC staining of formalin-fixed paraffin-embedded (FFPE) samples using the Autostainer Link 48 and Dako PT Link Pre-treatment Module (Phillips T, Simmons P, Inzunza HD, et al. Development of an automated PD-L1 mmunohistochemistry (IHC) assay for non-small cell lung cancer. Appl Immuno Molec Morph 2015; 23(8):541-9). In short, FFPE samples are first incubated with a primary monoclonal antibody against PD-L1 or a negative control reagent (NCR), then incubated with a host species-specific linker antibody of the primary antibody, and subsequently incubated with a readily available visualization reagent containing a secondary antibody molecule coupled to a dextran polymer backbone and a horseradish peroxidase molecule, before being subjected to optical microscopy to visualize PD-L1 staining.

[0164] PD-L1 IHC 28-8 pharmDx was used in CheckMate-649, a randomized, multicenter, open-label trial of previously untreated patients with advanced or metastatic gastric cancer, gastroesophageal junction cancer, and esophageal adenocarcinoma (n=1581). In this trial, patients were enrolled regardless of their PD-L1 expression level, and tumor samples from patients were evaluated in a central laboratory using the PD-L1 IHC 28-8 pharmDx assay. This clinical trial demonstrated a statistically significant improvement in overall survival (OS) and progression-free survival (PFS) in tumor patients with PD-L1 expression and a CPS of ≥5, which was not observed in tumor patients with PD-L1 expression and a CPS of <5 (information available from https: / / www.fda.gov / media / 124784 / download).

[0165] In certain embodiments, high PD-L1 expression levels correspond to CPS greater than 5 (>5) as measured optionally by an IHC assay using a PD-L1 diagnostic antibody (e.g., antibody 28-8) on a Dako automated scanning device (e.g., Dako Autostainer Link 48 platform). In certain embodiments, low PD-L1 expression levels correspond to CPS less than or equal to 5 (≤5) as measured optionally by an IHC assay using a PD-L1 diagnostic antibody (e.g., antibody 28-8) on a Dako Autostainer Link 48 platform.

[0166] In a particular embodiment, the anti-PD-L1 diagnostic antibody comprises an antibody selected from the group consisting of 28-8, SP263, 22c3, and SP142.

[0167] In a particular embodiment, the anti-PD-L1 diagnostic antibody 28-8 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 30 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 31.

[0168] In a particular embodiment, the anti-PD-L1 diagnostic antibody SP263 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 35.

[0169] In a particular embodiment, the anti-PD-L1 diagnostic antibody SP142 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 32 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 33.

[0170] In a particular embodiment, the anti-PD-L1 diagnostic antibody 22C3 comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 37.

[0171] In certain embodiments, the subject is determined to be a cancer sample (e.g., a tumor tissue sample) having either a) a high PD-L1 expression level or b) a low PD-L1 expression level.

[0172] In certain embodiments, the subject is a cancer sample determined to have a) a moderate to high CLDN18.2 expression level, or b) a low CLDN18.2 expression level. In certain embodiments, the subject is a cancer sample determined to have a) a high CLDN18.2 expression level, b) a moderate CLDN18.2 expression level, or c) a low CLDN18.2 expression level, as described herein.

[0173] In certain embodiments, subjects are determined to be cancer samples (e.g., tumor tissue samples) in which at least 1% (e.g., at least 2%, at least 5%, or at least 10%) of cancer cells have a CLDN18.2 expression level characterized by at least 1+ membrane staining intensity, as measured optionally on a Leica automated staining system (e.g., Leica Bond III LDT platform) using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11), and less than 40% of cancer cells have a CLDN18.2 expression level characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity. In such embodiments, subjects are determined to have a low PD-L1 expression level corresponding to a CPS of ≤5, or a high PD-L1 expression level corresponding to a CPS of >5, as measured optionally by an IHC assay using a PD-L1 diagnostic antibody (e.g., antibody 28-8).

[0174] In certain embodiments, the subject is determined to be a cancer sample (e.g., a tumor tissue sample) in which at least 40% of the cancer cells are cancer cells, having a CLDN18.2 expression level characterized by at least 2+ (e.g., 2+ or 3+) membrane staining intensity as optionally measured using an anti-CLDN18.2 diagnostic antibody (e.g., antibody 14G11) on a Leica automated staining system (e.g., Leica Bond III LDT platform). In such embodiments, the subject is determined to have a low PD-L1 expression level corresponding to a CPS of ≤5, or a high PD-L1 expression level corresponding to a CPS of >5, as optionally measured by an IHC assay using a PD-L1 diagnostic antibody (e.g., antibody 28-8).

[0175] e.CLDN18.2-expressing cancer

[0176] As used herein, "CLDN18.2-expressing cancer" refers to any cancer or tumor involving cancer cells that express CLDN18.2 (e.g., low, moderate, or high expression).

[0177] In certain embodiments, CLDN18.2-expressing cancers are selected from the group consisting of gastric cancer, esophageal adenocarcinoma, gastric / gastroesophageal junction (G / GEJ) cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, bile duct cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, malignant melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

[0178] In certain embodiments, CLDN18.2-expressing cancers include gastric cancer, esophageal adenocarcinoma, gastric / gastroesophageal junction (G / GEJ) cancer, ovarian cancer, pancreatic cancer, bile duct cancer (biliary tract cancer), colorectal cancer, or lung cancer.

[0179] In certain embodiments, CLDN18.2-expressing cancers are HER2-negative.

[0180] In certain embodiments, CLDN18.2-expressing cancers are gastric or gastroesophageal junction (mG / GEJ) adenocarcinoma, advanced gastric / gastroesophageal junction cancer, or esophageal adenocarcinoma.

[0181] In certain embodiments, CLDN18.2-expressing cancers are locally progressive.

[0182] In certain embodiments, CLDN18.2-expressing cancers are metastatic.

[0183] Over the past decade, targeted and immunotherapies have revolutionized the systemic treatment of various cancers, yet managing advanced and / or metastatic cancers remains a major challenge. Targeted therapy is a type of therapy that acts on specific molecules associated with cancer, such as certain proteins present in cancer cells but absent in normal cells, or present in greater quantities in cancer cells, or target molecules in the tumor microenvironment that contribute to cancer growth and survival. Targeted therapy reduces the impact of therapeutic agents on normal tissues by targeting the tumor. For example, many patients with advanced gastrointestinal cancers such as gastric cancer, pancreatic cancer, biliary tract cancer (including gallbladder and bile duct cancer), and lung cancer do not benefit significantly from current standard treatments. For most of these patients with advanced cancer, chemotherapy remains the dominant treatment, and the prognosis is still very poor.

[0184] In 2018, an estimated 55,440 people in the United States were diagnosed with pancreatic cancer, and approximately 43,330 died from the disease. Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. The prognosis for pancreatic cancer is extremely poor, with a median survival time of less than 6 months and a 5-year survival rate (including localized cancer) of 9% (SEER). Chemotherapy including gemcitabine plus nab-paclitaxel or FOLFIRINOX remains the first-line standard of treatment.

[0185] Biliary tract cancer (BTC) (including gallbladder cancer and bile duct cancer) is another type of tumor with a very poor prognosis. When metastatic bile duct cancer is treated with cisplatin + gemcitabine as first-line therapy, the median OS is 11.7 months and the median PFS is 8.0 months, which is still considered one of the preferred options in current standard treatment (Valle J, 2010).

[0186] Lung cancer was estimated to be the leading cause of both incidence and death worldwide in 2018 (Globocan, 2018). In recent years, significant advances in new targeted therapies (e.g., EGFR and ALK inhibitors) and immunotherapies have greatly improved overall survival in some subtypes, but many patients have not yet seen significant benefits, with the 5-year survival rate for patients with metastatic lung cancer (including NSCLC and SCLC) being only 5.2% (SEER).

[0187] According to GLOBOCAN's 2018 data, gastric cancer is the fifth most common cancer and the third leading cause of death worldwide. Esophageal cancer was the seventh most common cancer diagnosed in 2018 and the sixth leading cause of cancer-related death (Freddie et al., Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA CANCER J CLIN 2018;68:394-424). The overall survival rate for gastroesophageal adenocarcinoma is 20-25%. The five-year survival rate for patients with metastatic gastric or gastroesophageal adenocarcinoma is only 5.3% (SEER). Trastuzumab is only effective in patients with HER2-positive gastric or gastroesophageal cancer. Chemotherapy remains the dominant treatment for these patients.

[0188] In certain embodiments, the subjects are untreated. Untreated means the patient's condition or state of having not received any prior therapeutic intervention for the disease, in particular CLDN18.2-expressing cancer. In other words, the treatments provided herein are offered to the subjects as first-line treatments.

[0189] In certain embodiments, the subjects have not shown any resistance to CLDN18.2 antagonists, chemotherapy, or PD-1 / PD-L1 axis inhibitors. The term "resistance" refers to a decrease in the subject's response to a given treatment or a loss of its effectiveness over time. Resistance can be demonstrated by a reduced ability of the treatment to inhibit tumor growth, induce tumor cell death, or prevent tumor metastasis.

[0190] f. Pharmaceutical composition and route of administration

[0191] The CLDN18.2 antagonists, chemotherapeutic agents, and immunotherapeutic agents described above may each be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" refers to a formulation containing a therapeutically effective agent (e.g., the CLDN18.2 antagonists, chemotherapeutic agents, and immunotherapeutic agents described above) preferably together with pharmaceutically acceptable carriers, diluents, and / or excipients. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder when administered to a subject.

[0192] Pharmaceutical compositions are typically provided in a uniform dosage form and can be prepared in methods known in the art. Pharmaceutical compositions may be in liquid dosage forms, such as solutions or suspensions, or solid dosage forms, such as tablets and capsules. The pharmaceutical compositions described herein are generally applied in terms of “therapeutic dose” and “pharmaceutically acceptable preparation.” The term “pharmaceutically acceptable” means, as used herein, a non-toxic substance that does not interact with the action of the active ingredient of the pharmaceutical composition. For example, the term “pharmaceutically acceptable” may mean that a specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0193] The pharmaceutical compositions described herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of this disclosure comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0194] In certain embodiments, administration may be oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0195] In certain embodiments, the CLDN18.2 antagonist is administered in doses ranging from approximately 1 mg / kg to approximately 30 mg / kg (e.g., 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, or 30 mg / kg).

[0196] In certain embodiments, the CLDN18.2 antagonist is administered at a dose of approximately 4 mg / kg or 6 mg / kg.

[0197] In certain embodiments, the CLDN18.2 antagonist is administered once a week to once every 12 weeks (Q1W, Q2W, Q3W, Q4W, Q5W, Q6W, Q7W, Q8W, Q9W, Q10W, Q11W, or Q12W), or once a month to once every three months (Q1M, Q2M, or Q3M), or once a quarter.

[0198] In certain embodiments, the CLDN18.2 antagonist is administered at intervals of once every two weeks (Q2W) or once every three weeks (Q3W).

[0199] In certain embodiments, the CLDN18.2 antagonist is administered at a dose of 4 mg / kg every two weeks (Q2W) or at a dose of 6 mg / kg every three weeks (Q3W).

[0200] In certain embodiments, the CLDN18.2 antagonist is administered before, concurrently with, or after chemotherapy and / or immunotherapy.

[0201] III. Kit

[0202] In another embodiment, the Disclosure also provides a kit useful for identifying and treating individuals with CLDN18.2-expressing cancer who are likely to benefit from treatment, wherein the treatment comprises a CLDN18.2 antagonist and chemotherapy. In certain embodiments, the treatment further comprises immunotherapy. In certain embodiments, the kit comprises the above-mentioned anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody) and a package insert containing instructions for using the treatment in individuals with moderate to high or low levels of CLDN18.2 expression.

[0203] As used herein, the term “packaging instructions” means instructions contained in the packaging of a commercially available medicine, which include, for example, information on indications, dosage, usage, administration, contraindications, other medicines to be used in combination with the packaged product, and / or warnings regarding the use of such medicines. In certain embodiments, the instructions include selecting a subpopulation having low CLDN18.2 protein levels for therapeutic purposes.

[0204] The kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes. [Examples]

[0205] While this disclosure has been specifically illustrated and described with reference to certain embodiments (some of which are preferred embodiments), those skilled in the art will understand that various modifications can be made to the form and details without departing from the spirit and scope of this disclosure as disclosed herein.

[0206] Example 1: Detection of claudin 18.2 expression in gastric / gastroesophageal junction cancer using the Leica Bond III LDT assay.

[0207] Claudin 18.2 expression was evaluated by indirect immunohistochemistry (IHC) using clone 14G11, a mouse anti-human claudin 18.2 monoclonal antibody developed in-house and validated in the central laboratory, on a Leica automated staining platform (Leica Bond III Automated IHC Stainer, Leica, Germany). In short, immunohistochemistry (IHC) was performed on slides of 4% neutral buffered formalin-fixed, paraffin-embedded specimens. Sections were cut to a thickness of 4 μm and mounted on positively charged slides. After deparaffinization and rehydration, all slides were restored to antigen by heating in BOND® Epitope Retrieval Solution 2 (Leica, AR9640) at 100°C for 30 minutes. The slides were then quenched and blocked with a peroxidase inhibitor and incubated for 30 minutes with mouse anti-CLDN18.2 monoclonal antibody (clone 14G11) at a dynamic concentration range (0.45 μg / mL) diluted in primary antibody buffer (Leica Biosystem, AR9352). Antibody binding was amplified by a post-primary IgG linker and visualized by the formation of a brown precipitate using BOND® Polymer Detection (Leica, DS9800). Finally, sections were counterstained with hematoxylin and mounted with permanent mounting medium.

[0208] IHC results were scored by a certified pathologist under bright-field microscopy based on membrane staining intensity (negative (0), weak (1+), moderate (2+), strong (3+)) and the percentage of surviving invasive tumor cells showing a Claudin 18.2 staining intensity of ≥1+ in all surviving invasive tumor cells, as well as a defined tumor percentage score (TPS). Normal tissue and / or adjacent non-involved tissue were not included in the scoring evaluation.

[0209] The following are the definitions of claudin 18.2 (CLDN18.2) in H / M expression, low expression, and residual expression.

[0210] H / M (High / Moderate Expression): Patient samples showing ≥40% of tumor cells stained ≥2+ are classified as the high / moderate expression group. Of these, ≥70% of tumor cells stained ≥2+ are classified as the high-expression subgroup, or ≥40% of tumor cells stained ≥2+ and <70% of tumor cells stained ≥2+ are classified as the moderate-expression subgroup.

[0211] L (low expression): Patient samples showing ≥10% tumor cells stained ≥1+ and <40% tumor cells stained ≥2+ or ≥3+ are classified as the low expression group.

[0212] Patient samples showing <10% of tumor cells with R (remaining expression): staining ≥1+ are classified as the remaining expression group.

[0213] Example 2: Efficacy of Hu18B10-HaLa+CAPOX or Hu18B10-HaLa+CAPOX+nivolumab in Claudin 18.2-expressing G / GEJ cancer

[0214] Hu18B10-HaLa+CAPOX (Cohort C):

[0215] A Phase I / IIa clinical trial was designed to evaluate the safety, tolerability, pharmacokinetics, and preliminary efficacy of the claudin 18.2 monoclonal antibody Hu18b10-HaLa in the treatment of locally advanced, unresectable, or metastatic solid tumors.

[0216] Administration: A combination of Hu18B10-HaLa (6 mg / kg IV on day 1 in Q3W) and CAPOX (1000 mg / m2 capecitabine twice daily on days 1-14 of each 3-week cycle + 130 mg / m2 IV oxaliplatin on day 1 in Q3W) for the treatment of gastric / gastroesophageal junction (G / GEJ) cancer. Results are shown in Table 1, with a cutoff date of April 18, 2024. CLDN18.2 expression levels were required (1+ stained tumor cells ≥10%), however, some patients treated at the same dose levels early in the trial did not have their CLDN18.2 expression tested (reported as unknown). The expression status of claudin 18.2 was determined by indirect immunohistochemistry (IHC) using the in-house developed and centrally validated mouse anti-human claudin 18.2 monoclonal antibody clone 14G11 on a Leica automated staining platform (Leica Bond III Automated IHC Stainer, Leica, Germany). See Example 1 for details.

[0217] Hu18B10-HaLa + CAPOX + nivolumab (Cohort G):

[0218] A Phase I / IIa trial was designed to evaluate the combination of Hu18B10-HaLa (same as osemitamab in Figure 1), nivolumab, and chemotherapy as a first-line treatment for patients with claudin 18.2-positive locally advanced or metastatic gastric or gastroesophageal junction (G / GEJ) adenocarcinoma. Eligible patients included those with HER2-negative or unknown, unresectable locally advanced or metastatic G / GEJ cancer, and those who had not previously received systemic anticancer therapy for advanced disease.

[0219] Administration: Hu18B10-HaLa (6 mg / kg or 3 mg / kg administered intravenously on day 1 of Q3W) was combined with CAPOX (1000 mg / m2 capecitabine twice daily on days 1-14 of each 3-week cycle + 130 mg / m2 intravenously administered oxaliplatin on day 1 of Q3W) and nivolumab (360 mg administered intravenously on day 1 of Q3W for less than 2 years). The study design is shown in Figure 1. The results are shown in Table 1, with April 18, 2024, as the cutoff date. The expression status of claudin 18.2 was determined by indirect immunohistochemistry (IHC) using the in-house developed and centrally validated mouse anti-human claudin 18.2 monoclonal antibody clone 14G11 on a Leica automated staining platform (Leica Bond III Automated IHC Stainer, Leica, Germany). See Example 1 for details.

[0220] The results will be compared with other clinical trials (see the comparative trials below for details): 1) A randomized phase II trial comparing zolbetuximab + EOX (epirubicin, capecitabine, and oxaliplatin) with EOX monotherapy as the first-line treatment for FAST-positive, advanced CLDN18.2-positive gastric and gastroesophageal adenocarcinoma. 2) Phase III trial of zolbetuximab + CAPOX in GLOW, 1L claudin 18.2+ (CLDN 18.2+) / HER2- locally advanced (LA) or metastatic gastric / gastroesophageal junction (mG / GEJ) adenocarcinoma. 3) CHECKMATE649, a trial comparing the efficacy of nivolumab plus chemotherapy versus chemotherapy alone in gastric cancer or gastroesophageal junction cancer.

[0221] Table 1 and Figure 2 show that, in all PDL1, CPS-positive populations, Hu18B10-hala in combination with nivolumab and oxaliplatin + capecitabine, as first-line treatment for advanced or metastatic gastric / gastroesophageal junction (GAJ) adenocarcinoma, showed better mPFS outcomes for CLDN18.2 expression in the H / M or L groups compared to the Checkmate649 trial, with a cutoff date of April 18, 2024. In particular, the mPFS in the H / M group was significantly longer than that of the nivolumab + chemotherapy group from the Checkmate649 trial, regardless of PD-L1 CPS status. The H / M group showed a better tumor response compared to the PDL1 CPS <5 population in the Checkmate649 trial. Both the H / M and L groups showed better ORR than the PDL1 CPS <5 population in the Checkmate649 trial.

[0222] In the efficacy analysis of cohort G of patients (with known PD-L1 CPS and CLDN18.2 levels), a clear trend was observed between antitumor efficacy and CLDN18.2 expression: mPFS was 12.6 months for patients with H / M expression, 8.5 months for patients with L expression, and 6.7 months for patients with R expression. Patients with H / M CLDN18.2 and PD-L1 CPS < 5 also had an mPFS of 12.6 months. This trend was even more pronounced in the subgroup with PD-L1 CPS < 5. In addition, as an alternative approach, the inventors compared subsets based on CLDN18.2 expression levels. There is a clear trend between antitumor efficacy and CLDN18.2 expression, with hazard ratios (HR) for PFS being 0.56 (H / M / L vs R) and 0.443 (H / M vs R) in patients with known PD-L1 CPS and CLDN18.2, and 0.602 (H / M / L vs R) and 0.388 (H / M vs R) in patients with PD-L1 CPS < 5.

[0223] As is clear from Tables 2A and 2B below, Hu18B10-HaLa+CAPOX exhibits better efficacy outcomes, ORR, mPFS, or mDOR than the FAST trial in all patients (tumor cells stained 2+ or 3+ for CLDN18.2 ≥40%) or only low-expressing tumor cells (tumor cells stained 2+ or 3+ for CLDN18.2 ≥40-69%), or the GLOW trial in patients (tumor cells stained 2+ or 3+ for CLDN18.2 ≥75%).

[0224] As is evident from Tables 2A and 2B below, Hu18B10-HaLa+CAPOX+nivolumab exhibits better efficacy outcomes, ORR, or mPFS than Glow, Fast, or Checkmate649, particularly in CLDN18.2 low-expression regimens, demonstrating superior tumor response.

[0225] As shown in Tables 1A and 1B, Figure 3 (Cohort C), and Figures 4 and 5 (Cohort G), Hu18B10-HaLa+CAPOX (and further +nivolumab) showed significant therapeutic efficacy in low-expression tumors (1+ stained tumor cells ≥10%, and 2+ or 3+ stained tumor cells <40%).

[0226] [Table 1]

[0227] [Table 2A]

[0228] [Table 2B]

[0229] Comparative trial: FAST vs. GLOW: Zolbetuximab + chemotherapy

[0230] Zolbetuximab is a first-in-class chimeric monoclonal antibody specific to the CLDN18.2 epitope. Early-stage clinical trials (NCT00909025, NCT01197885, and NCT01671774) (Sahin U, 2015, Sahin U, 2018, Tuereci O, 2019) have shown evidence of antitumor activity and good tolerability of zolbetuximab in patients with CLDN18.2-expressing solid tumors. In a Trial IIa in patients with advanced GC / GEJ cancer treated with zolbetuximab monotherapy, the overall response rate (ORR) was 14% in a subgroup of patients with strong CLDN18.2 expression, defined as ≥70% of tumor cells with immunohistochemistry (IHC) staining intensity ≥2+ (4 / 29) (Tuereci O, 2019, Ann Oncol. 2019 Sep 1;30(9):1487-1495. doi: 10.1093 / annonc / mdz199.)

[0231] FAST: Zolbetuximab + EOX (epirubicin, capecitabine, and oxaliplatin): A randomized phase II trial comparing zolbetuximab + EOX with EOX monotherapy as the first-line treatment for advanced CLDN18.2-positive gastric and esophageal adenocarcinoma.

[0232] FAST was a randomized phase 2 trial (NCT01630083) that enrolled patients (≥18 years old) with advanced gastric / gastroesophageal junction adenocarcinoma and esophageal adenocarcinoma with moderate to strong CLDN18.2 expression in ≥40% of tumor cells. Patients received either epirubicin + oxaliplatin + capecitabine (EOX, Group 1, n=84) every 3 weeks (Q3W) or zolbetuximab (800 mg / m2 at Q3W, then 600 mg / m2) + EOX (Group 2, n=77) as first-line treatment. The primary endpoint was progression-free survival (PFS), and overall survival (OS) was a secondary endpoint.

[0233] Zolbetuximab combined with EOX chemotherapy extended overall survival and progression-free survival compared to EOX monotherapy in patients with advanced GC / GEJ cancer exhibiting moderate to strong CLDN18.2 expression (defined as membrane staining intensity ≥2+ by IHC in ≥40% of tumor cells). Median PFS was extended in the zolbetuximab + EOX group compared to the EOX monotherapy group (7.5 months vs. 5.3 months, p<0.0005, HR 0.44, 95% CI 0.29, 0.67). Median OS (13.0 months vs. 8.3 months, p<0.0005, HR 0.55, 95% CI 0.39, 0.77) was also significantly higher in the zolbetuximab + EOX group compared to the EOX monotherapy group. The increase in efficacy was more pronounced in patients with strong CLDN18.2 expression (staining intensity ≥2+ in ≥70% of tumor cells), while there was no clinically significant increase in PFS (4.3 months vs. 4.1 months, HR 0.71, 95% CI 0.32, 1.57, p=0.497) and OS (8.3 months vs. 7.4 months, HR 0.78, 95% CI 0.40, 1.49, p=0.401) in the low-expression group (40%–69% of tumor cells with moderate to strong CLDN18.2 staining). Due to the lack of efficacy improvement in the low-expression subgroup, Astellas underwent an important Phase 3 trial in the "high-expression" group, defined as "moderate to strong membranous CLDN18 staining in ≥75% of tumor cells."

[0234] Astellas conducted two phase 3 trials, SPOTLIGHT (using mFOLFOX6 as the chemotherapy backbone) and GLOW (using CAPOX as the CAPOX backbone), in patients with 1L claudin 18.2+ (CLDN18.2+) / HER2- locally advanced (LA) or metastatic gastric / gastroesophageal junction (mG / GEJ) adenocarcinoma, to evaluate the addition of zolbetuximab to chemotherapy. Both trials were conducted using the same claudin 18.2 selection criteria (moderate to strong membranous CLDN18 staining in ≥75% of tumor cells) and reached similar conclusions that the addition of zolbetuximab improved efficacy. In particular, in the GLOW trial III, the ORR (95% CI) was 53.8% (46.58–60.99) in the zolbetuximab + CAPOX group, and the median PFS was 8.21 months (95% CI, 7.46–8.84).

[0235] GLOW: 1L Claudine 18.2+ (CLDN18.2+) / HER2 - Zolbetuximab + CAPOX (GLOW Phase III trial) in locally advanced (LA) or metastatic gastric / gastroesophageal junction (mG / GEJ) adenocarcinoma.

[0236] Patients with CLDN18.2+ (moderate to severe membranous CLDN18 staining by IHC in ≥75% of tumor cells) / HER2-LA unresectable or mG / GEJ adenocarcinoma were randomized in a 1:1 ratio to receive either 8 cycles of 800 mg / m2 IV zolbetuximab + CAPOX (capecitabine 1000 mg / m2 orally administered via BID from D1 to D14 of each cycle, and oxaliplatin 130 mg / m2 IV administered on D1 of each cycle) or placebo (PBO) + CAPOX. Patients were continued for more than 8 cycles with either zolbetuximab or placebo + capecitabine until disease progression (PD) or termination criteria were met (at the discretion of the investigator). The primary endpoint (EP) was progression-free survival (PFS) based on RECIST v1.1 as determined by the IRC. Overall survival (OS) was a major secondary endpoint, and other secondary endpoints included objective response rate (ORR) and safety. Differences in PFS and OS between treatment groups were examined using stratified log-rank tests, and OS was examined only if PFS was statistically significant.

[0237] The results showed that 507 patients were randomized in a 1:1 ratio to receive either zolbetuximab + CAPOX (N=254) or PBO + CAPOX (N=253). Both PFS (median 8.21 months vs. 6.80 months, HR 0.687, P=0.0007) and OS (median 14.39 months vs. 12.16 months, HR 0.771, P=0.0118) were significantly extended with zolbetuximab + CAPOX (Table 1). In patients with measurable disease, the ORR (95% CI) was 53.8% (46.58~60.99) vs. 48.8% (41.76~55.84) in the comparison between the zolbetuximab group and the placebo group. The most common TEAEs with zolbetuximab + CAPOX were nausea (68.5% vs 50.2% in the zolbetuximab group vs placebo group), vomiting (66.1% vs 30.9%), and decreased appetite (41.3% vs 33.7%). Both groups had similar rates of serious TEAEs (47.2% vs 49.8%), grade ≥3 TEAEs (72.8% vs 69.9%), and drug-related TEAEs resulting in death (2.4% vs 2.8%) (Source: https: / / meetings.asco.org / abstracts-presentations / 217906).

[0238] Therefore, according to the data, zolbetuximab is effective in high-claudin 18.2 cancer cells (defined as ≥70% of tumor cells with immunohistochemistry (IHC) staining intensity ≥2+) or ≥75% of tumor cells with CLDN18 staining, but not in moderately and lowly claudin 18.2 expressing cells. There is still a significant unmet medical need for moderately and lowly claudin 18.2 expressing tumor cells that are not covered by zolbetuximab. By default, the standard treatment for these patients remains chemotherapy or chemotherapy plus checkpoint inhibitors such as nivolumab.

[0239] Comparative study: Checkmate-649

[0240] In recent years, immunotherapy has become a new treatment option for gastric cancer patients. Nivolumab, a PD-1 checkpoint inhibitor, has demonstrated clinical benefit in many late-stage clinical trials. In a placebo-controlled phase 3 trial (ATTRACTION-2) in patients with unresectable advanced or recurrent gastric cancer that had progressed after two or more chemotherapy regimens, nivolumab significantly improved overall survival (OS) (5.26 months vs. 4.14 months, HR=0.63, P<0.001). The 12-month OS was 26.2% in the nivolumab group (n=330) compared to 10.9% in the placebo group (n=163). The ORR in the nivolumab group was 11% (Kang et al., Nivolumab in patients with advanced gastric or gastro-oesophageal junction cancer refractory to, or intolerant of, at least two previous chemotherapy regimens (ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. The Lancet, 390(10111), 2461-2471).

[0241] Checkmate-649 was another phase 3 trial comparing nivolumab plus chemotherapy with chemotherapy alone in patients with untreated advanced G / GEJ cancer. More than 2,200 patients were randomized into three groups, with 1,581 patients randomized to receive either nivolumab (240 mg Q3W or 360 mg Q3W) plus chemotherapy (XELOX Q3W or FOLFOX Q2W) (n=789) or chemotherapy alone (n=792). Nivolumab plus chemotherapy significantly extended overall survival (OS) compared to chemotherapy alone in the PD-L1 CPS ≥ 5 group (14.4 months vs 11.1 months, HR=0.71, p<0.0001), and median progression-free survival (PFS) was 7.7 and 6.1 months, respectively, in the PD-L1 CPS ≥ 5 group (HR=0.68, P<0.0001). However, patients with PD-L1 CPS < 5 showed only minimal benefit from nivolumab. The overall survival (OS) with nivolumab plus chemotherapy was 12.4 months vs. 12.3 months, with an HR of 0.94, and the progression-free survival (PFS) was 7.5 months vs. 8.2 months, with an HR of 0.93.

[0242] Based on the results of CheckMate-649, nivolumab in combination with chemotherapy was approved by the FDA as a first-line treatment for metastatic gastric cancer (regardless of PD-L1 expression), and EMEA approved this combination regimen only for patients with PD-L1 CPS ≥ 5. The NCCN and CSCO guidelines recommend nivolumab in combination with chemotherapy for patients with advanced metastatic gastric cancer with PD-L1 CPS ≥ 5 (Category 1 evidence). For patients with advanced metastatic gastric cancer with PD-L1 CPS < 5, nivolumab in combination with chemotherapy may be used, but only as a Category 2 recommendation.

[0243] CheckMate649: Nivolumab + Chemotherapy: CheckMate649: Efficacy study comparing nivolumab + chemotherapy with chemotherapy in gastric cancer or gastroesophageal junction cancer.

[0244] This multicenter, randomized, open-label, phase 3 trial (CheckMate649) enrolled previously untreated adults (≥18 years) with unresectable non-HER2-positive gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, or esophageal adenocarcinoma from 175 hospitals and cancer centers in 29 countries, regardless of PD-ligand 1 (PD-L1) expression. Patients were randomly assigned using interactive web-response technology (block size 6) to receive nivolumab (360 mg every 3 weeks or 240 mg every 2 weeks) plus chemotherapy (capecitabine and oxaliplatin every 3 weeks, or leucovorin, fluorouracil, and oxaliplatin every 2 weeks), or chemotherapy alone (all three groups were open-label in a 1:1:1 ratio). The primary endpoint comparing nivolumab plus chemotherapy with chemotherapy alone was blinded, independent central review-based overall survival (OS) or progression-free survival (PFS) in patients whose tumors had a PD-L1 composite score (CPS). Safety was evaluated in all patients who received at least one dose of the assigned treatment.

[0245] Example 3: Determination of CLDN18.2 and PD-L1 co-expression status in patient tumor samples

[0246] To investigate the expression levels and overlap status of CLDN18.2 and PD-L1 in GC / GEJ cancer, immunohistochemistry (IHC) was performed on 4% neutral buffered formalin-fixed paraffin-embedded (FFPE) tumor sections using our proprietary and validated recombinant anti-CLDN18.2 antibody (14G11) and a commercially available anti-PD-L1(28-8) monoclonal antibody (e.g., Abcam, catalog number ab205921). The sequences of the heavy and light chain variable regions of the 28-8 monoclonal antibody are shown in SEQ ID NOs. 30 and 31, respectively (see US9212224B2). After deparaffinization and rehydration, all sections were restored to antigen by boiling in EnVision® FLEX Target Retrieval Solution (Dako, K8002) at 97-99°C for 25 minutes. Subsequently, they were quenched and blocked with EnVision® FLEX Peroxidase-Blocking Reagent (Dako, K8002), and incubated with appropriately diluted 14G11 (0.6 ug / mL) and 28-8 (1 ug / mL) antibodies, respectively. Antibody binding was visualized using EnVision® FLEX+, Mouse (LINKER), and then EnVision® FLEX / HRP and EnVision® FLEX Substrate Working Solution (Dako, K8002). Finally, the sections were counterstained with hematoxylin and mounted with permanent mounting medium.

[0247] All 300 participants were examined, and the expression levels and overlaps of CLDN18.2 and PD-L1 between GC / GEJ cancer tissues are summarized in Figure 6. The results showed that most samples with CLDN18.2 expression in GC / GEJ cancers had CPS < 5 and low PDL1 expression.

[0248] Example 4: Evaluation of CLDN18.2 and PD-L1 expression in sections of the GC-02-0007 PDX tumor model by IHC assay.

[0249] A. CLDN18.2 expression status in the PDX tumor model GC-02-0007

[0250] Immunohistochemistry (IHC) was performed on slides of 4% neutral buffered formalin-fixed, paraffin-embedded PDX samples. Sections were cut to a thickness of 4–5 μm and mounted on positively charged slides. After deparaffinization and rehydration, all slides were restored to antigen by heating in BOND Epitope Retrieval Solution 2 (Leica, AR9640) at 100°C for 30 minutes. Subsequently, the slides were quenched and blocked with a peroxidase inhibitor and incubated for 30 minutes with 0.15 μg / mL mouse anti-claudin 18.2 monoclonal antibody (clone 14G11) diluted in primary antibody buffer. Antibody binding was amplified by a post-primary IgG linker and visualized by the formation of a brown precipitate using BOND Polymer Detection (Leica, DS9800). Finally, the sections were counterstained with hematoxylin and mounted with permanent mounting medium.

[0251] All surviving tumor cells on the entire slide were evaluated and included in the scoring method. In general, at least 100 surviving tumor cells were recommended for the percentage score. IHC results were scored based on membrane staining intensity (negative (0), weak (1+), moderate (2+), strong (3+)) under bright-field microscopy, and the percentage of surviving invasive tumor cells showing ≥1+ claudin 18.2 staining intensity in all surviving tumor cells, as well as a defined tumor percentage score (TPS). Normal tissue and / or adjacent non-involved tissue were not included in the scoring evaluation. CLDN18.2 positivity was observed in the GC-02-0007 PDX model with moderate to strong intensity in ≥75% of tumor cells across all groups before and after treatment (see Figure 7).

[0252] B.GC-02-0007 Regulation of PD-L1 expression after Hu18B10-HaLa monotherapy or combination therapy with nivolumab / chemotherapy in a PDX tumor model

[0253] In order to better understand the tumor regulation before and after Hu18B10-HaLa treatment in GC-02-0007 PDX tumor samples, the PD-L1 expression status was also evaluated. Immunohistochemistry (IHC) was performed on these 4% neutral buffered formalin-fixed paraffin-embedded (FFPE) tumor sections using a commercially available rabbit anti-human PD-L1 monoclonal antibody (SP263). After deparaffinization and rehydration, all slides were subjected to antigen retrieval at 97 - 99 °C for 30 minutes with BOND Epitope Retrieval Solution 2 (Leica, AR9640). Subsequently, quenched and blocked with a peroxidase inhibitor, and incubated at room temperature (RT) for 30 minutes with appropriately diluted SP263 (0.2 μg / mL) antibody. Antibody binding was visualized for 8 minutes with BOND Polymer Detection (Leica, DS9800) on an automated staining Leica BOND III. Finally, the sections were counterstained with hematoxylin and mounted with a permanent mounting medium.

[0254] For PD-L1 with different membrane staining intensities (negative (0), weak (1+), moderate (2+), strong (3+)), all samples were scored by the composite positive score (CPS) based on the ratio of all stained immune cells and all viable tumor cells to all viable tumor cells. Interestingly, the staining results showed that treatment with Hu18B10-HaLa alone or in combination with nivolumab / chemotherapy could upregulate the expression level of PD-L1 compared to the isotype control, as shown in Figure 8 and Table 3.

[0255]

Table 3

[0256] Example 5 Efficacy of Hu18B10-HaLa in combination with nivolumab and oxaliplatin + 5-FU against the GC-02-0007 PDX tumor model in PBMC humanized B-NDG-hIL-15 mice

[0257] ​GC-02-0007 gastric cancer PDX was obtained from Beijing Cancer Hospital, passaged in NODSCID mice, and a PDX bank was constructed. As shown in Example 4, claudin 18.2 expression was high, exceeding 75% of tumor cells in this mode, while PD-L1 expression was low, with CPS < 5 in this mode. Each B-NDG-hIL-15 mouse was subcutaneously inoculated with a small tumor tissue block approximately 2-3 mm in diameter, excised from tumor dissection of a mouse with a tumor. Seventeen days after inoculation, animals with a tumor size of approximately 50-100 mm³ were selected and randomly divided into six groups of eight mice each. The animals were intravenously inoculated with 0.1 mL of 5*10^6 human PBMCs. Animals were selected and administered the drug several hours after human PBMC infusion. Animals from groups 1 to 6 were each administered 15 mg / kg of hIgG1 and vehicle, 10 mg / kg of Hu18B10-HaLa, 1 mg / kg of oxaliplatin + 10 mg / kg of 5-FU, 10 mg / kg of Hu18B10-HaLa combined with 1 mg / kg of oxaliplatin + 10 mg / kg of 5-FU, 5 mg / kg of nivolumab combined with 1 mg / kg of oxaliplatin + 10 mg / kg of 5-FU, and 10 mg / kg of Hu18B10-HaLa combined with 5 mg / kg of nivolumab and 1 mg / kg of oxaliplatin + 10 mg / kg of 5-FU. hIgG1, Hu18B10-HaLa, and nivolumab were administered intraperitoneally twice a week for 3 weeks, while vehicle, oxaliplatin, and 5-FU were administered intravenously once. The animals were euthanized by CO2 inhalation at the end of the experiment. Tumor size was measured two-dimensionally twice a week using calipers (INSIZE), and the volume was recorded in mm². 3 The coefficient of the tumor was expressed by the formula V = 0.5a*b^2, where a and b are the longest and shortest diameters of the tumor, respectively. The results were analyzed using Prism GraphPad, and the mean value was expressed as SEM. A t-test was performed to compare the two groups. A statistically significant difference was considered to exist when p < 0.05 and ** < 0.01.

[0258] As shown in Figures 9A and 9B, in a human gastric cancer GC-02-0007 PDX tumor model of B-NDG-hIL15 PBMC humanized mice with high human CLDN18.2 expression and negative for human PD-L1, Hu18B10-HaLa combined with nivolumab and oxaliplatin + 5-FU was found to be superior to oxaliplatin + 5-FU alone, Hu18B10-HaLa combined with oxaliplatin + 5-FU, and nivolumab combined with oxaliplatin + 5-FU in terms of tumor growth inhibition.

[0259] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

Claims

1. A method for treating CLDN18.2-expressing cancer in humans that requires it, The aforementioned subjects are administered a therapeutically effective dose of a CLDN18.2 antagonist in combination with chemotherapy. The subject is a method that has been determined to have moderate to high or low CLDN18.2 expression levels in cancer samples (e.g., tumor tissue samples).

2. A method for identifying and treating subjects with claudin 18.2 (CLDN18.2)-expressing cancer who are likely to benefit from treatment including a CLDN18.2 antagonist in combination with chemotherapy, (a) Determining the level of CLDN18.2 protein in cancer samples (e.g., in tumor tissue samples), (b) A method comprising selecting a subject having moderate to high or low levels of the CLDN18.2 protein for the treatment.

3. The method according to claim 1 or 2, wherein the CLDN18.2 protein level or the CLDN18.2 expression level is measured by an immunohistochemistry (IHC) assay, a hybridization assay, or an amplification assay.

4. The method according to any one of the claims, wherein the CLDN18.2 protein level or the CLDN18.2 expression level is quantified by membrane staining intensity measured by IHC using an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody).

5. The method according to any one of the claims, wherein the CLDN18.2 protein level or the CLDN18.2 expression level is quantified by the percentage of cancer cells whose membrane staining intensity, measured by IHC using an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody), is a predetermined threshold.

6. a) The moderate to high CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ (e.g., 2+ or 3+) as measured on the Leica Bond III LDT platform using an anti-CLDN18.2 diagnostic antibody, or to an equivalent readout as measured on another test platform, and / or b) The low CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 1% (e.g., at least 2%, at least 5%, or at least 10%) of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 1+ as measured on the Leica Bond III LDT platform using an anti-CLDN18.2 diagnostic antibody, and less than 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ or 3+, or to equivalent readouts measured on another test platform. The method according to any one of the above claims.

7. The anti-CLDN18.2 diagnostic antibody comprises HCDR1, HCDR2, and HCDR3 sequences, and LCDR1, LCDR2, and LCDR3 sequences. The HCDR1 sequence includes RNYFH (SEQ ID NO: 23), or a homologous sequence having at least 80% sequence identity therewith. The HCDR2 sequence includes WIYPGGFDIEYSEKFKG (Sequence ID 24), or a homologous sequence having at least 80% sequence identity therewith. The HCDR3 sequence includes NYGSTFGY (SEQ ID NO: 25), or a homologous sequence having at least 80% sequence identity therewith. The LCDR1 sequence includes RSSQNIVHSNGNTYLE (SEQ ID NO: 26) or a homologous sequence having at least 80% sequence identity therewith. The LCDR2 sequence includes KVSNRFS (Sequence ID 27) or a homologous sequence having at least 80% sequence identity therewith, The method according to any one of claims 4 to 6, wherein the LCDR3 sequence includes FQGSHVPFT (SEQ ID NO: 28) or a homologous sequence having at least 80% sequence identity thereof.

8. The aforementioned anti-CLDN18.2 diagnostic antibody comprises VH and VL. a) The VH comprises the amino acid sequence of SEQ ID NO: 21 or a homologous sequence having at least 80% sequence identity therewith, b) The method according to claim 7, wherein the VL includes the amino acid sequence of SEQ ID NO: 22 or a homologous sequence having at least 80% sequence identity therewith.

9. The method according to any one of claims 4 to 6, wherein the anti-CLDN18.2 diagnostic antibody comprises an antibody selected from the group consisting of [EPR19202] (ab222512), 43-14A, SDT-102-24, MIL93, and 3B10.

10. a) The moderate to high CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by a membrane staining intensity of at least 2+ (e.g., 2+ or 3+) as measured on the Leica Bond III LDT platform, or to an equivalent readout as measured on another test platform, and / or b) The low CLDN18.2 protein level or CLDN18.2 expression level corresponds to at least 1% (e.g., at least 2%, at least 5%, or at least 10%) of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by at least 1+ membrane staining intensity, as measured optionally on the Leica Bond III LDT platform using 14G11, and less than 40% of the cancer cells in the cancer sample (e.g., tumor tissue sample) having a CLDN18.2 expression level characterized by at least 2+ or 3+ membrane staining intensity, or to equivalent readouts measured on another test platform. The method according to any one of claims 4 to 8.

11. The method according to any one of the claims, wherein the concentration of the anti-CLDN18.2 diagnostic antibody is about 0.05 to 20 μg / mL (for example, about 0.05 to 20 μg / mL, about 0.1 to 15 μg / mL, about 0.15 to 10 μg / mL, about 0.15 to 5 μg / mL, about 0.2 to 10 μg / mL, about 0.3 to 10 μg / mL, about 0.4 to 10 μg / mL, about 0.5 to 10 μg / mL, about 0.6 to 10 μg / mL, about 0.7 to 10 μg / mL, about 0.8 to 10 μg / mL, about 0.9 to 10 μg / mL, about 1 to 10 μg / mL, or about 0.15 to 5 μg / mL).

12. The method according to any one of the claims, wherein the anti-CLDN18.2 diagnostic antibody is diluted in dilution buffer for about 10 to 60 minutes (for example, about 10 to 60 minutes, about 15 to 50 minutes, about 15 to 40 minutes, about 15 to 35 minutes, about 15 to 30 minutes, about 16 to 32 minutes, or about 20 to 30 minutes).

13. The method according to claim 12, wherein the dilution buffer is selected from the group consisting of Leica (AR9352) and PBS.

14. The method according to any one of the claims, wherein the IHC assay includes antigen recovery for about 15 to 120 min (for example, about 15 to 120 min, about 25 to 100 min, about 35 to 90 min, about 45 to 80 min, about 55 to 90 min, about 65 to 80 min, about 70 min, about 15 to 30 min, about 20 to 30 min, or about 64 to 104 min).

15. The method according to claim 14, wherein the antigen recovery is performed at a temperature of about 50 to 100°C (for example, about 50°C to 100°C, about 60°C to 100°C, about 70°C to 100°C, about 80°C to 100°C, about 90°C to 100°C, about 92°C, about 94°C, about 96°C, about 97°C, about 98°C, or about 100°C).

16. The method according to any one of the claims, wherein the chemotherapy comprises a chemotherapeutic agent selected from the group consisting of erlotinib, afatinib, docetaxel, adriamycin, 5-fluorouracil (5-FU), panobinostat, gemcitabine, cisplatin, pemetrexed, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, oteracil, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trametinib, tegafur, gimeracil, leucovorin calcium (folic acid) (LV), irinotecan hydrochloride (CPT-11), platinum (e.g., cisplatin), epirubicin, oxaliplatin, and capecitabine.

17. The method according to any one of the claims, wherein the chemotherapy includes a combination of chemotherapeutic agents.

18. The method according to claim 17, wherein the chemotherapy comprises 1) capecitabine and oxaliplatin (CAPOX), 2) LV, 5-FU and CPT-11 (FOLFIRI), 3) epirubicin, oxaliplatin and capecitabine (EOX), 4) epirubicin, cisplatin and 5-FU (ECF), 5) epirubicin, cisplatin and capecitabine (ECX), 6) epirubicin, oxaliplatin and 5-FU (EOF), 7) 5-FU, LV and oxaliplatin (FLO), or 8) tegafur, gimeracil, oteracil and oxaliplatin (SOX).

19. The method according to any one of the claims, wherein the treatment further comprises immunotherapy, or the method further comprises administering immunotherapy to the subject in a therapeutically effective dose.

20. The method according to any one of the claims, wherein the immunotherapy comprises an immunotherapy agent such as a PD-1 / PD-L1 axis inhibitor.

21. The method according to any one of the claims, wherein the subject is a cancer sample (for example, a tumor tissue sample) that has been determined to have a high PD-L1 expression level or b) a low PD-L1 expression level.

22. The method according to claim 21, wherein the PD-L1 expression level is quantified by membrane staining intensity measured by IHC using an anti-PD-L1 diagnostic reagent (e.g., an anti-PD-L1 diagnostic antibody).

23. The method according to claim 22, wherein the PD-L1 expression level is quantified by a composite positive score (CPS), the CPS being determined by the ratio of PD-L1-stained immune cells and tumor cells to all viable tumor cells.

24. The method according to claim 23, wherein the high PD-L1 expression level corresponds to a CPS greater than 5 (>5) measured by IHC using antibody 28-8, and / or the low PD-L1 expression level corresponds to a CPS of 5 or less (≤5) measured by IHC using antibody 28-8.

25. A method for treating CLDN18.2-expressing cancer in humans that requires it, The aforementioned subjects are administered a therapeutically effective dose of a CLDN18.2 antagonist in combination with chemotherapy and a PD-1 / PD-L1 axis inhibitor. The subject is a method that has been determined to have a low PD-L1 expression level in cancer samples (for example, tumor tissue samples).

26. The method according to claim 25, wherein the PD-L1 expression level is measured by an immunohistochemistry (IHC) assay, a hybridization assay, or an amplification assay.

27. The method according to claim 26, wherein the PD-L1 expression level is quantified by membrane staining intensity measured by IHC using an anti-PD-L1 diagnostic reagent (e.g., an anti-PD-L1 diagnostic antibody).

28. The method according to claim 27, wherein the PD-L1 expression level is quantified by a composite positive score (CPS), the CPS being determined by the ratio of PD-L1-stained immune cells and tumor cells to all viable tumor cells.

29. The method according to claim 28, wherein the low PD-L1 expression level is measured by IHC using antibody 28-8 and optionally corresponds to a CPS of 5 or less (≤5) measured by an IHC assay on a Dako Autostainer Link 48 platform.

30. The method according to claim 27, wherein the anti-PD-L1 diagnostic antibody comprises an antibody selected from the group consisting of 28-8, SP263, 22c3, and SP142.

31. The method according to any one of claims 25 to 30, wherein the subject is determined to be the cancer sample having a) a moderate to high CLDN18.2 expression level, or b) a low CLDN18.2 expression level.

32. The method according to any one of claims 25 to 31, wherein the CLDN18.2 expression level is quantified as defined in any one of claims 3 to 15.

33. The method according to any one of claims 25 to 32, wherein the chemotherapy is as defined in any one of claims 16 to 18.

34. The aforementioned PD-1 / PD-L1 axis inhibitors are nivolumab (OPDIVO, BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA, MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pidilizumab (CT-011), semiprimab (LIBTAYO, REGN2810), spartalizumab (PDR001), and cetrelimab (JNJ 63723283), Tripalimab (JS001), PF-06801591, Tislerizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lambrolizumab, Camrelizumab (SHR-1210), Syntilimab (Tyvyt, IBI308), Penplimab (AK105), Zimberelimab Letifanlimab, Selprulimab, Valstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofadinelimab, Syndelizumab, MGA404, Sym021, BAT1306, HX008, Atezolizumab (TECENTRIQ, R05541267, MPDL3280) A, RG7446), BMS-936559, avelumab (Bavencio), rhodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, emvafolimab (KN035), MDX-1105, STI-1040, CS1001, adebrelimab (SHR-1 The method according to any one of claims 25 to 33, selected from the group consisting of 316), SHR-1701, TOB2450, vintrafusp, LP002, STI-3031, cosivelimab, pacumimab, NM01, LDP, AMP-224, galiblumab (BGB-A333), A167, SCD-135, opcolimab, and GR1405.

35. The method according to any one of the claims, wherein the CLDN18.2 antagonist comprises an anti-CLDN18.2 antibody, for example, a monoclonal anti-CLDN18.2 antibody, a bispecific antibody targeting CLDN18.2, and a second antigen containing an anti-CLDN18.2 antigen-binding domain.

36. The CLDN18.2 antagonist comprises an anti-CLDN18.2 antigen-binding domain containing HCDR1, HCDR2, and HCDR3 sequences and / or LCDR1, LCDR2, and LCDR3 sequences. The HCDR1 sequence includes GENMN (SEQ ID NO: 1), or a homologous sequence having at least 80% sequence identity therewith. The HCDR2 sequence includes NIDPYYGGTSYNQKFKG (Sequence ID 2), or a homologous sequence having at least 80% sequence identity therewith. The HCDR3 sequence includes MYHGNAFDY (Sequence ID 3), or a homologous sequence having at least 80% sequence identity therewith. The LCDR1 sequence includes KSSQSLLNSGNLKNYLT (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity thereof. The LCDR2 sequence includes WASTRKS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity therewith, The method according to any one of the claims, wherein the LCDR3 sequence includes QNDYSYPLT (Sequence ID 6) or a homologous sequence having at least 80% sequence identity thereof.

37. The CLDN18.2 antagonist comprises an anti-CLDN18.2 antigen-binding domain including VH and VL. The aforementioned VH includes the amino acid sequence of SEQ ID NO: 7 or a homologous sequence having at least 80% sequence identity therewith, The method according to claim 36, wherein the VL includes the amino acid sequence of SEQ ID NO: 8 or a homologous sequence having at least 80% sequence identity therewith.

38. The method according to any one of the claims, wherein the anti-CLDN18.2 antagonist further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

39. The method according to claim 38, wherein the immunoglobulin constant region is the constant region of human IgG1, IgG2, IgG3, or IgG4.

40. The method according to claim 39, wherein the immunoglobulin constant region is a constant region of human IgG1 containing sequence number 9 or a homologous sequence having at least 80% sequence identity thereto.

41. The method according to any one of claims 38 to 40, wherein the immunoglobulin constant region comprises one or more amino acid residue substitutions or modifications resulting in increased CDC or ADCC compared to the wild-type constant region.

42. The method according to claim 41, wherein the immunoglobulin constant region comprises one or more amino acid residue substitutions selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof, relative to SEQ ID NO:

9.

43. The method according to any one of claims 38 to 42, wherein the immunoglobulin constant region includes the sequence of SEQ ID NO:

11.

44. The method according to any one of claims 38 to 43, wherein the immunoglobulin constant region further comprises the sequence of SEQ ID NO:

10.

45. The method according to any one of the claims, wherein the CLDN18.2 antagonist is a humanized anti-CLDN18.2 antibody.

46. The aforementioned anti-CLDN18.2 antibody comprises VH and VL. The aforementioned VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, and 14, and The method according to claim 45, wherein the VL includes an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO:

16.

47. The anti-CLDN18.2 antibody comprises a heavy chain and a light chain. The heavy chain includes the amino acid sequence of SEQ ID NO: 17 or a homologous sequence having at least 80% sequence identity therewith, The method according to claim 46, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 18 or a homologous sequence having at least 80% sequence identity thereof.

48. The method according to any one of the claims, wherein the CLDN18.2 antagonist is further connected to one or more conjugate portions.

49. The method according to claim 48, wherein the conjugate portion comprises a clearance regulator, a chemotherapeutic agent, a toxin (e.g., exatecan, berotecan, deruxtecan, MMAE, MMAF, DM1, DM4, SN-38, PBD, eribulin), a radioisotope, a lantanide, a luminescence label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a cytokine (e.g., IL-15, IL-2, IL-7), or another anticancer drug.

50. The method according to any one of the claims, wherein the subject is a human.

51. The method according to any one of the claims, wherein the administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

52. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered in a dose of about 1 mg / kg to about 30 mg / kg (for example, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, or 30 mg / kg).

53. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered in a dose of approximately 4 mg / kg or 6 mg / kg.

54. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered at intervals of once a week to once every 12 weeks (Q1W, Q2W, Q3W, Q4W, Q5W, Q6W, Q7W, Q8W, Q9W, Q10W, Q11W, or Q12W), or once a month to once every three months (Q1M, Q2M, or Q3M), or once a quarter.

55. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered at intervals of once every two weeks (Q2W) or once every three weeks (Q3W).

56. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered at a dose of 4 mg / kg at intervals of two weeks (Q2W), or at a dose of 6 mg / kg at intervals of three weeks (Q3W).

57. The method according to any one of the claims, wherein the CLDN18.2 antagonist is administered before, simultaneously with, or after the administration of the chemotherapy.

58. The method according to any one of the claims, wherein the CLDN18.2-expressing cancer is selected from the group consisting of gastric cancer, esophageal adenocarcinoma, gastric / gastroesophageal junction (G / GEJ) cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, bile duct cancer (biliary tract cancer), head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, malignant melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

59. The method according to claim 58, wherein the CLDN18.2-expressing cancer is gastric cancer, esophageal adenocarcinoma, gastric / gastroesophageal junction (G / GEJ) cancer, ovarian cancer, pancreatic cancer, bile duct cancer (biliary tract cancer), colorectal cancer, or lung cancer.

60. The method according to claim 59, wherein the CLDN18.2-expressing cancer is gastric or gastroesophageal junction (mG / GEJ) adenocarcinoma, advanced gastric / gastroesophageal junction cancer, or esophageal adenocarcinoma.

61. The method according to any one of the claims, wherein the CLDN18.2-expressing cancer is HER2-negative.

62. The method according to any one of the claims, wherein the CLDN18.2-expressing cancer is locally progressive.

63. The method according to any one of the claims, wherein the CLDN18.2-expressing cancer is metastatic.

64. The method according to any one of the claims, wherein the subject is untreated.

65. The method according to any one of the claims, wherein the subject has not shown any resistance to a CLDN18.2 antagonist, chemotherapy, or PD-1 / PD-L1 axis inhibitor.

66. The method according to any one of the claims, wherein the subject has a longer survival time compared to a subject treated with a reference antibody (e.g., nivolumab or zolbetuximab) in combination with chemotherapy.

67. The method according to claim 66, wherein the survival period of the subject is at least five months (for example, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, or at least fifteen months).

68. Use of a CLDN18.2 antagonist in the manufacture of a pharmaceutical product for treating a CLDN18.2-expressing cancer of a subject requiring treatment, wherein the treatment comprises administering the pharmaceutical product to the subject in combination with chemotherapy, and the subject is determined to have a moderate to high or low CLDN18.2 expression level in a cancer sample (e.g., in a tumor tissue sample).

69. The use of a CLDN18.2 antagonist in the manufacture of a pharmaceutical product for treating a CLDN18.2-expressing cancer of a subject requiring such use, wherein the pharmaceutical product further comprises chemotherapy, and the subject is determined to have a moderate to high or low CLDN18.2 expression level in a cancer sample (e.g., in a tumor tissue sample).

70. A kit for use in identifying and treating subjects with CLDN18.2-expressing cancer who may benefit from chemotherapy-containing therapy including a CLDN18.2 antagonist, comprising: an anti-CLDN18.2 diagnostic reagent (e.g., an anti-CLDN18.2 diagnostic antibody); and a package insert containing instructions for using the therapy in subjects with moderate to high or low levels of CLDN18.2 expression.

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