WO2023196882A1 - Claudin 18.2 immunohistochemistry assay and use thereof - Google Patents

Abstract

This application describes an immunohistochemistry assay used to detect CLDN 18.2 in tissues from cancer patients with accuracy and precision, and the use of this assay. In embodiments, methods of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody and methods to treat a patient having cancer are described, where the methods comprise staining a sample from the patient with antibody EPR19202 in an IHC assay.

Description

CLAUDIN 18.2 IMMUNOHISTOCHEMISTRY ASSAY AND USE THEREOF

HELD

[0001] This application relates to methods which can be used for predicting effects of a claudin 18.2 antibody. More particularly, the present application relates to an immunohistochemistry (IHC) assay which may be useful for predicting the likelihood that a patient will respond therapeutically to treatment with a claudin 18.2 antibody.

BACKGROUND

[0002] Claudin- 18 (CLDN18) belongs to the claudin family of proteins which form important components of the tight cell junctions. The latter are apical cell-cell adhesion molecules in epithelial and endothelial cells that are involved in the regulation of paracellular permeability. The claudin family consists of 24 known transmembrane proteins that display a distinct tissue- and development-specific distribution pattern. A link has been established between the altered function of Claudin proteins and the formation of cancers. As such, changes in Claudin proteins can compromise the structure and function of the tight-j unctions and may impact cell signaling pathways. Claudin expression changes in a tissue specific manner (See Singh AB et al. Claudin family of proteins and cancer: an overview. Journal of Oncology (2010) 2010: 1- 11.)

[0003] Claudin- 18 (CLDN18) has two highly homologous isoforms, CLDN18.1 and CLDN18.2, which only differ from each other in the N-terminal 69 amino acids, including the first extracellular loop. Both isoforms have a tissue-specific expression: CLDN18.2 is highly expressed in the normal stomach and its expression is strictly confined to the differentiated cells of the gastric mucosa, while CLDN18.1, is specific for pulmonary tissue (see for example, Rohde C et al. Comparison of Claudin 18.2 expression in primary tumors and lymph node metastases in Japanese patients with gastric adenocarcinoma. Japanese Journal of Clinical Oncology (2019) 49(9): 870-876).

[0004] CLDN18.2 expression has the tendency to be conserved in gastric cancer and its metastases. More specifically, CLDN18.2 is observed in ± 30% of the primary cancers, and in ±34% of the metastases. There is also an association between CLDN18.2 expression and gastric tumor localization, whereby tumors localized in the gastric corpus have a high prevalence of CLDN18.2 (See Coati I et al. Claudin- 18 expression in oesophagogastric adenocarcinomas: a tissue microarray study of 523 molecularly profiled cases. British Journal of Cancer (2019) 121: 257-263). Ectopic activation of CLDN18.2 is also frequently observed, whereby CLDN18.2 is found in pancreatic, esophageal, ovarian and lung tumors (See Singh P et al. Anti-Claudin 18.2 antibody as new targeted therapy for advanced gastric cancer. Journal of Hematology and Oncology (2017) 10: 105).

[0005] In summary, CLDN18.2 is a very attractive pan-cancer target for antibody therapy of epithelial tumors, since this protein is absent from indispensable and vital normal organs, is expressed on the cell surface and belongs to a protein family that demonstrates tumor promoting cellular functions (See Tiireci O et al. A multicentre, phase Ila study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophages: the MONO study. Annals of Oncology (2019) 30(9): 1487-1495).

[0006] CLDN18.2 is highly expressed in gastric cancer (80%) and other cancer types (pancreatic cancer 60% positive), and in metastatic sites with different levels of CLDN 18.2 expression. CLDN18.2-targeting therapeutic antibodies have shown promising clinical efficacy in -30% of gastric cancers expressing high levels of CLDN18.2 and less pronounced activity in the majority of low expressing malignancies. Thus, it is desired to have a CLDN 18.2 antibody that shows promising activity against both high and low CLDN 18.2 expressing cancers. At the same time, a diagnostic assay is also needed to identify tumor cells not only with high but also with low CLDN 18.2 expression.

SUMMARY

[0007] This application discloses an IHC staining assay which can differentiate tumor cells with various levels of CLDN 18.2 expression from tumor cells with CLDN 18.1 expression. Through this assay, one can identify patient populations suitable for the treatment using CLDN 18.2 antibody. This application further discloses a CLDN 18.2 antibody that can be used together with the IHC assay disclosed herein for the treatment of cancer patients having tumor cells with various levels of CLDN 18.2 expression. The IHC assay described herein selectively targets CLDN 18.2 and allows for prospective selection of not only medium (+2) /high (+3) but also low (+1) CLDN 18.2 expression tumors. In other words, the IHC assay disclosed herein can expand the patient population that can be treated using the CLDN 18.2 antibody disclosed herein.

[0008] Provided is a method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, comprising: obtaining a sample from the patient, staining the sample with antibody EPR19202 in an IHC assay, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if the staining is positive. [0009] Provided is a method of treating a patient having cancer in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-claudin 18.2 antibody if a sample from the patient is stained positive with antibody EPR19202 in an IHC assay.

[0010] Provided is a method of treating a patient having cancer in need thereof, comprising: obtaining a sample from the patient, staining the sample with antibody EPR19202 in an IHC assay, determining the staining is positive, and administering to the patient a therapeutically effective amount of an anti-claudin 18.2 antibody.

[0011] In some embodiments, the method comprises obtaining a sample from the patient; staining the sample with antibody EPR19202 in an IHC assay; and determining presence or absence of CLDN18.2 in the sample. In the present application, CLDN 18.2 is determined to be present if the staining is positive and absent if the staining is negative. In some embodiments, if CLDN18.2 is present or the staining is positive, either additional testing of a sample from the patient can be conducted or the patient is administered a therapeutically effective amount of an anti-CLDN 18.2 antibody. In some embodiments, the additional testing can be a predictive biomarker assay which predicts the response of the patient to a therapy comprising the anti-CLDN 18.2 antibody. In some embodiments, the patient is administered a therapeutically effective amount of an anti-CLDN 18.2 antibody if the additional testing predicts the patient will respond to a therapy comprising the anti-CLDN 18.2 antibody. In some embodiments, additional testing of a sample is conducted on a new or fresh sample obtained from the patient, or on the obtained sample.

[0012] Provided is a method of treating a patient having cancer in need thereof with a therapy comprising a therapeutically effective amount of an anti-claudin 18.2 antibody, which method comprises, prior to treating the patient with the anti-claudin 18.2 antibody, obtaining a sample from the patient and staining the sample with antibody EPR19202 in an IHC assay.

[0013] Also provided is a use of antibody EPR 19202 in the manufacture of a kit for a method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody. [0014] Also provided is a use of antibody EPR 19202 in the manufacture of a kit for a method of deteimining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, wherein the method comprises obtaining a sample from the patient, staining the sample with antibody EPR19202 in an IHC assay, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if the staining is positive.

[0015] The sample from the patient for staining is in vitro sample of the patient having cancer. [0016] In some embodiments of the methods or use, staining is performed by following known IHC staining procedures. [0017] In some embodiments of the methods or use, the sample from the patient was preserved in the form of formalin fixed and paraffine embedded (FFPE) pellets before staining. In some embodiments, the sample is frozen tissue sample.

[0018] In some embodiments of the methods or use, the staining is membranous staining. In some embodiments, the staining is performed on an autostainer. In some embodiments, the autostainer is a Dako Link 48 autostainer or a LabVision Autostainer.

[0019] In some embodiments of the methods or use, the staining exhibits 0, 1+, 2+, or 3+ intensity, and the staining is positive if > 5% of the sample cells exhibit membranous staining at any intensity.

[0020] In some embodiments of the methods or use, the staining is positive if the H-score is > 5.

[0021] In some embodiments of the methods or use, the staining is positive if the H-score is > 15.

[0022] In some embodiments of the methods or use, the staining is positive if the H-score is > 25.

[0023] In some embodiments of the methods or use, the staining is positive if the H-score is > 35.

[0024] In some embodiments of the methods or use, the staining is positive if the H-score is > 45.

[0025] In some embodiments of the methods or use, the staining is positive if the H-score is > 50.

[0026] In some embodiments of the methods or use, the staining is positive if the H-score is > 55.

[0027] In some embodiments of the methods or use, the staining is positive if the H-score is > 60.

[0028] In some embodiments of the methods or use, the staining is positive if the H-score is > 70.

[0029] In some embodiments of the methods or use, the staining is positive if the H-score is > 80.

[0030] In some embodiments of the methods or use, the staining is positive if the H-score is > 90.

[0031] In some embodiments of the methods or use, the staining is positive if the H-score is > 100. [0032] In some embodiments of the methods or use, the sample is a tissue sample comprising epithelial tumor cells.

[0033] In some embodiments of the methods or use, the sample is a tissue sample comprising cancer cells selected from gastric cancer, lung cancer (non-small cell lung cancer or small cell lung cancer), bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicle cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervix 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, vagina cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, cholangiocarcinoma, and adenocarcinoma.

[0034] In some embodiments of the methods or use, the sample is a tissue sample comprising cancer cells selected from gastric cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, and colorectal cancer.

[0035] In some embodiments of the methods or use, the sample comprises tumor cells that are primary. In some embodiments, the sample comprises tumor cells that are metastatic.

[0036] In some embodiments of the methods or use, the IHC assay further comprises staining the sample with a matched IgG isotype of antibody EPR19202 and detecting no staining thereof.

[0037] In some embodiments of the methods or use, the anti-claudin 18.2 antibody comprises a variable light chain region and a variable heavy chain region, wherein the variable light chain region comprises CDR1 having the sequence of SEQ ID NO: I, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3, the variable heavy chain region comprises CDR1 having the sequence of SEQ ID NO: 4, CDR2 having the sequence of SEQ ID NO: 5, and CDR3 having the sequence of SEQ ID NO: 6. In some embodiments, this anti-claudin 18.2 antibody is a humanized IgGl monoclonal antibody with mutations in the Fc domain to enhance ADCC, which is named herein after as Antibody 1.

[0038] In some embodiments of the methods or use, the anti-CLDN 18.2 antibody comprises the sequences of SEQ ID NOs: 7 and 8.

[0039] Also provided is a method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, comprising: obtaining a sample from the patient, dividing the sample into two or more sections, staining the samples on each section with antibody EPR19202, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if each staining is positive and the coefficient of variance meets the acceptance criteria. The aforementioned embodiments described for previous methods apply to this method.

[0040] Also provided is a use of antibody EPR 19202 in the manufacture of a kit for a method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody.

[0041] In some embodiments, the anti-claudin 18.2 antibody comprises a variable light chain region and a variable heavy chain region, wherein the variable light chain region comprises CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3, the variable heavy chain region comprises CDR1 having the sequence of SEQ ID NO: 4, CDR2 having the sequence of SEQ ID NO: 5, and CDR3 having the sequence of SEQ ID NO: 6. In some embodiments, this anti-claudin 18.2 antibody is a humanized IgGl monoclonal antibody with mutations in the Fc domain to enhance ADCC, which is named herein after as Antibody 1.

[0042] In some embodiments, the anti-CLDN 18.2 antibody comprises the sequences of SEQ ID NOs: 7 and 8.

[0043] Also provided is a use of antibody EPR 19202 in the manufacture of a kit for a method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, wherein the method comprises obtaining a sample from the patient, staining the sample with antibody EPR19202 in an IHC assay, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if the staining is positive. The aforementioned embodiments described for previous methods apply to this method.

[0044] Also provided is a kit, comprising antibody EPR19202 and a reagent for IHC assay. [0045] In some embodiments, the kit further comprises a matched IgG isotype of antibody EPR19202.

[0046] In some embodiments, antibody EPR19202 is used to stain samples from a patient having cancer in an IHC assay.

BRIEF DESCRIPTION OF THE FIGURES

[0047] FIG. 1A-C show representative images for CLDN18.2 staining obtained in parental HEK293 cells (FIG. 1A), custom generated HEK293 cells that overexpress CLDN18.1 (FIG. IB) and CLDN18.2 (FIG. 1C). Positive staining is only observed for HEK293 cells overexpressing CLDN18.2. Some non-specific background staining can be observed for HEK293 cells overexpressing CLDN18.1.

[0048] FIG. 1D-I show representative images for CLDN18.2 staining obtained in cancer cell lines showing different ranges in CLDN18.2 expression levels: MIAPACA2 (FIG. ID), PANCI (FIG. IE), BXPC3 (FIG. IF), HepG2 (FIG. 1G), PSN1 (FIG.1H), and KATOIII (FIG. II). Positive staining is only observed for KATOIII cells (FIG. II).

[0049] FIG. 2A-B show representative images for CLDN 18.2 expression in normal stomach (FIG. 2A) and gastric cancer cells (FIG. 2B). Samples were stained using antibody EPR19202.

[0050] FIG. 3A-B show representative images of the staining pattern obtained with the EPR19202 in noimal lung tissue (FIG. 3A) and normal gastric mucosa (FIG. 3B). A strong membranous staining is obtained in the gastric mucosa, while the lung epithelial cells remain completely negative.

[0051] FIG. 4A-B show representative images of the staining pattern obtained with EPR19202 in PDAC (FIG. 4A) and gastric cancer (FIG.4B) FFPE tissue samples.

[0052] FIG. 5 is a diagram illustrating the precision assessment strategy.

[0053] FIG. 6A-H show serial slides of PDAC stained in 3 runs (FIG. 6A-E for Run 1, FIG. 6F-G for Run 2, and FIG. 6H for Run 3) to verify intra-run and inter-run variability. In run 2 (FIG. 6F-G), samples were stained with the primary CLDN18.2 antibody or the IgG isotype control.

[0054] FIG. 7 is a chart showing CLDN18.2 expression levels in various MIA-PaCa2- hCLDN18.2 clones confirmed by flow cytometry.

[0055] FIG. 8A-E show various MIA-PaCa2-hCLDN18.2 clones in stained images obtained from the IHC staining assay described herein: FIG. 8A-E for parental clones #45, #42, #9, and #7, respectively.

[0056] FIG. 8F is a graph showing H-score for various MIA-PaCa2-hCLDN18.2 clones obtained from the IHC staining assay described herein.

[0057] FIG. 9A-B show ADCC activity of Antibody 1 and a Benchmark, respectively, with isolated human NK cells using MIA-PaCa2-hCLDN18.2 clones.

[0058] FIG. 9C show EC50 of Antibody 1 and a benchmark on MIA-PaCa2-hCLDN18.2 clones.

[0059] FIG. 10A show CLDN 18.1/18.2 expression level obtained from qPCR in normal lung, normal gastric, and gastric tumor cell lines (SNU 601, SNU620, SNU 5 and KATOIII).

[0060] FIG. 10B-E show CLDN 18.2 expression level obtained from CLDN18.2 IHC staining assay described herein in gastric tumor cell lines SNU 601, SNU620, KATOIII, and SNU5, respectively.

[0061] FIG. 10F is a graph showing H-score for gastric tumor cell lines SNU 601, SNU620, KATOIII, and SNU5 obtained from the IHC staining assay described herein. [0062] FIG. 11A-D show ADCC activity with PBMC of Antibody 1, a benchmark, and hlgGl on gastric tumor cell line SNU601, SNY 620, KATOIII, and SNU5, respectively.

[0063] FIG. 12A-D show ADCC activity with NK cells of Antibody 1 , a benchmark, and hlgGl on gastric tumor cell line SNU601, SNY 620, KATOIII, and SNU5, respectively.

[0064] FIG. 13A shows tumor volume in SNU620 xenograft models at days post treatment with Antibody 1, a benchmark CLDN 18.2 antibody and hlgGl.

[0065] FIG. 13B compares tumor volume in SNU620 xenograft models at day 42 between treatment with Antibody 1 and a benchmark CLDN 18.2 antibody.

[0066] FIG. 13C shows tumor volume in KATOIII xenograft models at days post treatment with Antibody 1, a benchmark CLDN 18.2 antibody and hlgGl.

[0067] FIG. 13D shows tumor volume in SNU5 xenograft models at days post treatment with Antibody 1, a benchmark CLDN 18.2 antibody and hlgGl.

[0068] FIG. 13E compares tumor volume in KATOIII xenograft models at day 40 between treatment with Antibody 1, hlgGl and a benchmark CLDN 18.2 antibody.

[0069] FIG. 13F compares tumor volume in SNU5 xenograft models at day 40 between treatment with Antibody 1, hlgGl and a benchmark CLDN 18.2 antibody.

[0070] FIG. 14A-14G show tumor volume in seven patient-derived xenografts (PDX) which according to DNAseq data expresses CLDN18.2 from human gastric tumors: FIG. 14A (PDX fragment GA0006), FIG. 14B (PDX fragment GA6831), FIG. 14C (PDX fragment GA2419), FIG. 14D (PDX fragment GA6208), FIG. 14E (PDX fragment GA0074), FIG. 14F (PDX fragment GA13765), and FIG. 14G (PDX fragment GA0060).

[0071] FIG. 15A-15G show CLDN 18.2 expression level in tested PDXs obtained from CLDN18.2 IHC staining assay described herein: FIG. 15A (PDX fragment GA0006), FIG. 15B (PDX fragment GA6831), FIG. 15C (PDX fragment GA2419), FIG. 15D (PDX fragment GA6208), FIG. 15E (PDX fragment GA0074), FIG. 15F (PDX fragment GA13765), and FIG. 15G (PDX fragment GA0060).

DETAILED DESCRIPTION

I. Definitions

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a, ” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” when applicable unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include” , “includes, ” and “included, ” is not limiting.

[0073] The term “antibody” as used herein refers to a molecule comprising at least a variable light chain region and a variable heavy chain region, wherein the molecule is capable of binding to antigen. Both “variable light chain region” and “variable heavy chain region” refer to a polypeptide comprising three CDRs. The term “antibody” also includes fragments that are capable of binding to antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, and (Fab’)2. The antibody can be chimeric antibody, humanized antibody, human antibody, and antibody of various species including mouse and cynomolgus monkey, etc.

[0074] The term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable light chain and heavy chain regions, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The system described by Kabat is used to describe the exact boundaries of the CDRs. The Kabat system, which is recognized in the art, refer to a system of numbering amino acid residues (See Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0075] The term “patient (s)” as used herein refer to animal (such as mammal) or human. [0076] The phrase “staining the sample with antibody EPR19202 in an IHC assay” or “a sample from the patient is stained positive with antibody EPR19202 in an IHC assay” means that antibody EPR19202 is the primary antibody in the IHC assay expected to bind an antigen to be detected.

[0077] As used herein, a “therapeutically effective amount” refers to the amount that, when administered to a subject for treatment of a disease, is sufficient to cause a desired treatment effect in the subject, including for example, alleviation of the symptoms or stop of the progression of the disease, such as reducing tumor volume.

[0078] The terms “treating”, “treatment”, or “treat” (of) a disease refers to slowing or arresting the development of a disease, providing relief from the symptoms or side-effects of the disease, and/or causing regression of the disease. The terms also refer to reduction of the occurrence of the disease in the subject when compared with a subject without the treatment. [0079] “Anti-CLDN18.2 antibody” or “an antibody against CLDN18.2” as used herein refers to an antibody that is capable of binding to CLDN18.2 (e.g. human or non-human CLDN18.2) with a sufficient affinity that can provide diagnostic and/or therapeutic use.

[0080] The term “affinity” as used herein refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e. antibody) or fragment thereof and an antigen. [0081] The term “coefficient of variance” (%CV) is calculated via the following formula: %CV =(H-Score STDEV/H-Score Average)* 100. The acceptance criteria for %CV is as follows:

H-score < 50: always concordant

H-score > 50 and < 100: maximum % CV = 50%

H-score > 100: maximum %CV = 20%

[0082] hmnunohistochemistry (IHC) refers to a process of detecting antigens (e.g., proteins) in cells of a tissue section, e.g. cells of the tissues mentioned herein, through a visualized antibody- antigen interaction (staining). IHC staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Staining can be accomplished by tagging the antibody with an enzyme or a fluorophore, which catalyze a color- or fluorophoreproducing reaction, respectively. Visualization of antigen can be accomplished through an indirect horseradish peroxidase (HRP)-based secondary detection method (e.g., Envision Flex Rabbit linker (K8009, Agilent) and EMM (K8009, Agilent)). Use of the EnVision Flex Rabbit linker can allow signal amplification (2-3 fold) of the primary rabbit antibody. The detection kit itself, for example, may comprise a dextran backbone to which a large number of HRP molecules and secondary antibody molecules have been coupled. The DAB (3,3'- diaminobenzidine) chromogen can be used for visualization.

II. Anti -CLDN 18.2 Antibody

[0083] Claudins (CLDNs) are central tight junction proteins that regulate epithelial-cell barrier function and polarity, thereby creating a boundary between the apical and basolateral plasma membrane domains. To date, 27 members of the CLDN family have been described with different organ-specific expression patterns. It has been shown that the expression levels of claudins are often abnormal in human neoplasias. One of the CLDN family members, CLDN- 18 isoform 2 (CLDN18.2) is a selective gastric lineage antigen, and its expression in normal tissues is confined to differentiated epithelial cells of the gastric mucosa.

[0084] The CLDN18.2 protein is highly conserved in mouse, rat, rabbit, dog, monkey, and human and comprises four transmembrane domains and two extracellular domains. About 8 of the 51 amino acid residues within the first extracellular domain differ from lung-tissue specific CLDN-18 isoform 1 (CLDN18.1), and may serve as an epitope for monoclonal antibody binding.

[0085] Under a cancer setting, CLDN18.2 has been shown to be involved in tumor development and progression. Indeed, CLDN18.2 has been shown to be displayed on the surface of human gastric cancer cells and its metastases (Sahin, et al, “Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development,” Clin Cancer Res 2008; 14(23): 7624-34) and its ectopic activation was observed in pancreatic cancer (Woll, et al., “Claudin 18.2 is a target for IMAB362 antibody in pancreatic neoplasms, ” Int J Cancer 2014; 134(3): 731-739 and Tanaka, et al., “Claudin-18 is an early-stage marker of pancreatic carcinogenesis,” J Histochem Cytochem 2011; 59(10): 942-952. Aberrant activation of CLDN18.2 was also observed in bile duct, esophageal, ovarian, and lung cancers, and was associated with poor overall survival and lymph node metastasis (Shinozaki, et al., “Claudin-18 in biliary neoplasms. Its significance in the classification of intrahepatic cholangiocarcinoma,” Virchows Arch 2011; 459: 73-80 and Micke, et al., “Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer,” Int J CNCER 2014; 135(9): 2206- 2214.

[0086] The anti-CLDN 18.2 antibody as used herein can be a monoclonal antibody, a genetically engineered antibody, a humanized antibody, a chimeric antibody, or a human antibody. The anti-CLDN 18.2 antibody can be selected from a Fab, a Fv, a scFv, a Fab’, or a (Fab’)2. In some embodiments, an anti-CLDN18.2 antibody described herein comprises a framework region selected from IgM, IgG (e.g., IgGl, IgG2, IgG3, or IgG4) , IgA, or IgE. In some cases, the anti-CLDN18.2 antibody comprises an IgM framework. In some cases, the anti-CLDN18.2 antibody comprises an IgG (e.g., IgGl, IgG2, IgG3, or IgG4) framework. In some cases, the anti-CLDN18.2 antibody comprises an IgGl framework. In some cases, the anti-CLDN 18.2 antibody comprises an IgG2 framework.

[0087] Exemplary anti-CLDN 18.2 antibody that can be used in the methods as disclosed herein can be any of the CLDN 18.2 antigen binding unit disclosed in PCT/CN2019/123588 (WO 2020/114480), which is incorporated herein by reference in its entirety and for all purposes.

[0088] In some embodiments, the anti-CLDN 18.2 antibody used in the method as disclosed herein comprises a variable light chain region and a variable heavy chain region, wherein the variable light chain region comprises CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3, the variable heavy chain region comprises CDR1 having the sequence of SEQ ID NO: 4, CDR2 having the sequence of SEQ ID NO: 5, and CDR3 having the sequence of SEQ ID NO: 6. In some embodiments, the anti-CLDN 18.2 antibody used in the method disclosed herein can be the 413H9F8-cp2-V2-DL antibody described in PCT/CN2019/123588 (“Antibody 1”).

[0089] In some embodiments, the anti-CLDN 18.2 antibody used in the method as disclosed herein comprises the sequences of SEQ ID NOs: 7 and 8. Various Fc engineering approaches, including S239D/I332E and F243L/R292P/Y300L/V305I/P396L mutations, have been developed to enhance effector functions of antibodies, such as to enhance ADCC. In some embodiments, the anti-CLDN 18.2 antibody comprise a Fc region which comprises S239D/1332E mutations. Anti-CLDN18.2 antibodies that are included in the studies have Fc variants with D356/L358 or E356/M358. A “DL” name suffix was added to indicate antibodies with D356/L358 in the Fc. In some embodiments, the anti-CLDN 18.2 antibody has Fc variant with D356/L358.

[0090] In some embodiments, the treatment comprises one or more additional therapeutic agent. The additional therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapeutic agent, a hormone-based therapeutic agent, a stem-cell based therapeutic agent, or radiation. In some embodiments, the additional therapeutic agent and the anti-CLDN18.2 antibody are administered simultaneously. In some embodiments, the additional therapeutic agent and the anti-CLDN18.2 antibody are administered sequentially. In some embodiments, the additional therapeutic agent is administered prior to the anti-CLDN 18.2 antibody. In some embodiments, the additional therapeutic agent is administered after the administration of the anti-CLDN 18.2 antibody. In some embodiments, the additional therapeutic agent and the anti-CLDN18.2 antibody are formulated as separate dosage.

III. IHC Staining and Analysis

[0091] Staining as used herein refers to IHC staining, which is calibrated IHC that can distinguish different levels of CLDN 18.2 expression in view of the staining intensity/pattems and percentages of stained cells.

[0092] The IHC staining assay may be performed using an automated pathology system, which may include automated staining (conventional stains, histochemical techniques, immunostainers); automated in situ hybridization systems; automatic slide preparation (coverslip, slide drying) and integrated slide and cassette labeling, as described in literature (See, for example, Roja et al., Review of imaging solutions for integrated, quantitative immunohistochemistry in the Pathology daily practice, Folia Histochemica et Cytobiologica, Vol. 47, No. 3, 349-354, 2009. [0093] Exemplary autostainers that can be used in an IHC staining include the commercially available Dako Link 48 autostainer and LabVisionmAutostainer.

[0094] The reagents can be used off the shelf for other autostainers or for manually-performed staining without using an autostainer.

[0095] The sample analyzed in an IHC can be in the form of formalin fixed and paraffine embedded (FFPE) pellets placed on a slide. Exemplary slides include 4 pm thick slides, SuperFrost UltraPlus yellow glass slides, baked for 2 hrs at 60°C, stored at 4°C.

[0096] After completing the staining process, the sample (e.g. a slide) is analyzed for antigen (such as CLDN 18.2) staining, either by a human, e.g., a pathologist, or by a computer programmed to distinguish between specific and non-specific staining results.

[0097] The staining is considered positive if the sample has an elevated level of membrane staining of any intensity relative to a negative control. The negative control can be the matched immunoglobulin isotype of the antibody used to stain the sample (antigen). Isotype control antibodies are used to estimate the non-specific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody (such as antibody EPR19202).

[0098] The level of membrane staining, which corresponds to the level of antigen expression (e.g., CLDN 18.2 expression), can be scored using a semi-quantitative scoring system H-score as described below.

[0099] The antibody used for CLDN 18.2 IHC staining as disclosed herein is EPR19202 (Clone number). This antibody is a rabbit monoclonal antibody binding to CLDN 18.2. It is developed by Abeam under its RabMAb® technology. It is also commercially available as Catalogue No.ab222512 from Abeam. The matched isotype is IgG. In other words, the negative control used in the IHC assay described herein is IgG.

[0100] Staining method used herein can be an indirect method wherein a secondary antibody is used. For example, for visualization of CLDN18.2 an indirect HRP (Horseradish peroxidase) - based secondary detection method is used (Envision Flex Rabbit linker (K8009, Agilent) and EMM (K8009, Agilent)). For example, the detection kit itself includes a dextran backbone to which a large number of HRP molecules and secondary antibody molecules have been coupled. The DAB chromogen can be used for visualization.

[0101] The staining intensity can be determined by a pathologist using a “white paper test.” The “white paper test” is a qualitative evaluation wherein the pathologists compares the staining intensity and staining pattern and assigns intensity score to cells. The slides that were stained are laid next to each other on a white paper or the white balanced digital images of the samples are tiled on a desktop display and are evaluated for staining consistency. There can be four (4) immunohistochemistry intensity categories assigned to the cells stained: negative (category 0), weak (category 1+), moderate (category 2+) and strongly (category 3+) stained membranes.

[0102] The H-Score (also known as histoscore) is a semi-quantitative analysis based on the staining intensity and percentages of stained cells. The pathologists assesses the intensity of the membranous CLDN18.2 staining that is observed for the cells. There are 4 immunohistochemistry categories: negative (category 0), weak (category 1+), moderate (category 2+) and strongly (category 3+) stained membranes. For each sample, a histoscore with a potential range of 0-300 is calculated according to following formula:

H-Score=((l x % weakly stained cells)+(2 x % moderately stained cells)+(3 x % strongly stained cells))

[0103] In some embodiments, the staining is positive if not less than 5% of the cells in a sample exhibit membranous staining at any intensity (1+, 2+, or 3+), alone or in combination. [0104] In some embodiments, the staining is scored according to H-score, and the staining is positive for CLDN18.2 if the H-score is equal to or above 5, 50, 100, 150 or 200. In certain embodiments, the anti-claudin 18.2 antibody is used to treat a cancer characterized by the expression of CLDN 18.2 at a level of 1+, 2+, or 3+ measured by IHC.

EXAMPLES

[0105] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure.

[0106] The benchmark anti-CLDN18.2 antibody used in Example 3 and 4 comprise a sequence set forth in SEQ ID NO: 9 and a sequence set forth in SEQ ID NO: 10.

[0107] Abbreviations

Ab antibody

AS autostainer

CCLE cancer cell line encyclopedia

CLDN 18 Claudin-18

CV coefficient of variance

FFPE formalin fixed and paraffin embedded FO form

IHC immunohistochemistry mRNA messenger ribonucleic acid

NAT normal tissue adjacent to the tumor

OE overexpressing

PDAC pancreatic ductal adenocarcinoma

RTqPCR reverse transcription polymerase chain reaction

Example 1

Validation of IHC Assay

[0108] This example describes the validation of the CLDN 18.2 IHC assay on the DAko Link 48 autosatiner to detect CLDN 18.2 in human cancer FFPE tissue samples.

[0109] Starting/ reference IHC staining conditions and representative staining patterns for CLDN 18 in FFPE tissue can be found for:

- clone 43-14A in Rohde C. et al 2019 and Tiireci O. et al 2018 for (manual staining procedures); the antibody is also available in a ready-to-use IVD kit from Ventana.

- clone EPR19202 on Leica Bond Rx in Dottermusch M. et al, Expression of potential therapeutic target claudin-18.2 is frequently decreased in gastric cancer: results from a large Caucasian cohort study. Virchows Arch. (2019) 475(5): 563-571.

[0110] The staining performance was assessed on FFPE tissues and FFPE cell pellets covering the different levels of CLDN 18.2 expression. The final candidate antibody showing the best performance (specific, sensitive) was selected to be used for the optimization phase of the CLDN18.2 IHC assay. The preferred automated staining platform was also selected for better signal-to-noise ratio. During this phase, different staining parameters, including but not limited to pre-treatment conditions, blocking steps, detection method, were evaluated to discover the best antibody performance. The IHC staining was evaluated qualitatively by assay developers in order to get an optimal signal-to-noise ratio. The final interpretation of the staining would be done under supervision of a certified pathologist.

1.1 Materials and Instruments

A. Antibodies

[0111] The primary antibody and negative control are described in Table 1 as follows: Table 1: Antibody Specifications

B. Stainer

[0112] The LabVision Autostainer was used during the optimization process, which is solely used for optimizations due to its more open system (free programming), whereas the Dako Link 48 autostainers (used within the validation process of this assay) are locked and no free programming is allowed by lab technicians. A calibration certificate/data sheet has been received for each instrument that certifies that the autostainers have been tested and meet all specifications. All autostainers were cross validated twice a year.

C. Reference Sample

[0113] Following reference samples were used: a) a multi-cell pellet block consisting of duplicate cores for 3 FFPE cancer cell lines that have variable CLDN18.2 expression levels (negative for CLDN18.2, weakly positive for CLDNf 8.2 vs. strongly positive for CLDNf 8.2) and b) a multi-tissue block consisting of a normal lung FFPE tissue sample and a GC FFPE tissue sample containing normal adjacent tissue (NAT). The normal lung FFPE tissue sample should stain completely negative for CLDN18.2. For the GC sample, an intense membranous staining should be observed for CLDNf 8.2 in normal gastric mucosa, whereby a cytoplasmic component can also be present. Epithelial tumor cells can demonstrate a variety of CLDNf 8.2 expression levels, ranging from negative to strongly positive. 1.2 Accuracy & Specificity

A. Expected staining pattern in cell lines

[0114] The specificity of the IHC assay targeting CLDN18.2 was assessed on FFPE cell pellets. The cell pellets are selected on their origin of tissue and ideally demonstrate different CLDN18.2 expression levels (negative/low/medium/high). RNAseq data for the selected cell lines were retrieved from CCLE database and the flow cytometric data were provided by the Applicant (Table 2). A good correlation between the IHC data, the RNAseq and flow cytometric data strengthens the specificity of the IHC assay.

Table 2: RNAseq and flow cytometric data for CLDN18.2 expression levels in different cancer cell lines. The data were retrieved from the CCLE database¹ or provided by the Applicant.²

[0115] Given the low occurrence and availability of CLDN18.2 positive cells, custom generated CLDN18.1 and CLDN18.2 overexpressing HEK293 cells were also included in the specificity assessment. CLDN18 was not expected in parental HEK293 cells (CCLE database reports RNAseq values of -3.09). Therefore, positive staining is only expected for the CLDN18.2 overexpressing HEK293 cells.

[0116] The CLDN18.2 IHC assay performed according to expectations in the provided cell lines, namely a weakly positive (brown) staining in the KATOIII cells and an intense positive staining in the CLDN18.2 OE HEK293 cells. No immunoreactivity can be observed in the CLDN18.1 OE HEK293 cells, suggesting specificity of the antibody for the CLDN18.2 isoform (FIG. 1A-1I)

B. Tissue specific staining pattern

[0117] CLDN18 is normally expressed as two tissue-specific splice variants, whereby the expression of CLDN18.2 is restricted to normal gastric mucosa and CLDN18.1 is predominantly expressed in the lung. (Tiireci O et al.) The presence of staining of the epithelial cells in normal lung FFPE tissue would indicate cross-reactivity of the CLDN18.2 IHC assay with the CLDN18.1 isoform. In normal stomach FFPE tissue, a strong staining is expected for the stomach mucosa. In cancer FFPE tissue, CLDN18.2 expression can be detected in a diverse variety of epithelial tumor types. (Dottermusch M et al. Expression of potential therapeutic target claudin-18.2 is frequently decreased in gastric cancer: results from a large Caucasian cohort study (2019) 475: 563-571). FIG. 2A-B, taken from Dottermusch M et al., depicts representative stained images for CLDN18.2 expression in normal stomach (a) and gastric cancer cells (b) using clone EPR19202. While CLDN18.2 shows a membranous staining pattern, but a weak cytoplasmic staining can also be present.

[0118] The primary CLDN18.2 antibody (clone EPR19202) displayed a strong immunoreactivity in normal stomach tissue, whilst no immunoreactivity was observed in the epithelial cells of normal lung tissue (FIG. 3A-B). These results indicated that the assay using primary CLDN18.2 antibody (EPR19202 clone), is specific for the CLDN18.2 isoform and that the antibody does not show cross-reactivity with the CLDN18.1 isoform.

[0119] The CLDN18.2 IHC assay using the primary CLDN18.2 antibody (clone EPR19202), displayed immunoreactivity in epithelial tumor types (for example, PDAC and gastric cancer), where a moderate to strong membranous staining was observed for the epithelial tumor cells. Occasionally, a weak cytoplasmic staining can also be observed. Immunoreactivity was absent in the stromal tissue. (FIG. 4A-B).

C. Comparison IHC signal to alternative method: qPCR

[0120] The quantitative measurement of transcript abundance can be used to support the validation of protein expression for a panel of FFPE human tumors of target cancer indication/segment. The mRNA levels for CLDN18.1 and CLDN18.2 were measured in selected cell lines and FFPE tissue samples via TaqMan based RTqPCR experiments in cells and tissue samples. A good correlation between the IHC data and mRNA data using the validated TaqMan probes strengthens the specificity of the IHC assay. The correlation then was qualitatively assessed by sorting the samples from low to high expression.

[0121] Commercially available TaqMan probes were used for targeting human CLDN18.1 and CLDN18.2 (CLDN18.1: Hs00981422_ml (#4351372); CLDN18.2: Hs00981430_m2 (#4351372)). A linearity and precision experiment should be conducted to validate the selected TaqMan probes before using them for validation purposes. The primer efficiencies should lie in the range of 1.8 and 2.2 and the coefficient of determination (R2) between the Ct value and the log concentration should be > 0.95. Furthermore, precision testing is performed to assess the intra- and inter-assay variability. The difference in Ct between duplicate measurements of the same sample in the same run should not exceed 0.5 to be considered valid. Intra-assay variation and inter-assay variation per assay should be <2%. [0122] In addition, given the high degree of homology (91.9%) between the CLDN18.1 and CLDN18.2 isoforms, the specificity of the TaqMan probes will be assessed on parental and custom HEK293 cells and FFPE tissue of normal lung, normal stomach and normal liver. The expected results for these samples are indicated in Table 3.

Table 3: Expected mRNA levels for CLDN18.1 and CLDN18.1

[0123] The specificity of the TaqMan probes was first assessed in parental and custom HEK293 cells. In parallel, the CLDN18.1 and CLDN18.2 mRNA levels were determined for different cancer cell lines as listed in Table 2 that display variable levels of CLDN18.2 expression. The results indicated that (1) a good correlation was observed between the IHC data and the RTqPCR data. Indeed, normal lung and liver tissue lack both CLDN18.2 protein expression and CLDN18.2 mRNA, (2) there is a good correlation between the CLDN18.2 mRNA levels and the intensity of the CLDN18.2 IHC signal. Indeed, high CLDN18.2 mRNA levels were detected for normal stomach tissue and a PDAC sample, which also show very intense CLDN18.2 staining, and (3) lower CLDN18.2 mRNA levels are detected in the samples that show a weaker CLDN18.2 protein expression.

D. Comparison staining pattern of different antibodies

[0124] For further accuracy, the IHC staining pattern using the EPR19202 clone (selected clone) was compared with the staining profile from clone 43-14A. Assuming that both antibodies are target specific, immunostaining with both clones should result in comparable staining patterns. The antibody specifications of each clone are summarized in Table 4.

Table 4. Overview of 2 anti-CLDN18 antibodies tested

[0125] The IHC staining profiles obtained for clone EPR 19202 and clone 43-14A were compared for 14 different samples. A similar staining pattern can be appreciated in a PDAC sample and two gastric cancer sample. The staining intensity, however, slightly differed between both clones. In contrast to clone EPR19202, clone 43-14A however showed crossreactivity with the CLDN18.1 isoform. Indeed, clone 43-14A showed immunoreactivity in normal lung tissue and in CLDN18.1 overexpressing HEK293 cells. This observation can be explained by the fact that clone 43-14A targets a C-terminal epitope of CLDN18, which is shared by both isoforms, whereas clone EPR19202 recognizes a N-terminal epitope that is unique for the CLDN18.2 isoform.

E. Negative Control

[0126] Use of an isotype control instead of the CLDN18.2 primary antibody (clone EPR19202) at the same concentration should omit the IHC signal. The isotype control serves to ensure that the staining that is observed is not the result of immunoglobulins binding non-specifically to Fc receptors present on the cell surface. In the second run of the precision experiment described later, an isotype control Ab was included at the same concentration as the primary CLDN18.2 antibody. No immuno-reactive staining was detected in any of the tissue slides.

F. Linearity

[0127] The linearity of the method is its ability to elicit results that are proportional to the concentration of the analyte in the sample. In here, the primary antibody will be serially diluted (on average five dilutions) and used to label tissue samples of different indications covering the spectrum of expression levels (low, moderate, high expression). Each staining pattern will be qualitatively evaluated. Dilution of the primary antibody should result in a gradual signal loss and if possible with complete omission of the IHC signal at low concentration.

[0128] The optimal antibody concentration will be selected. This is the concentration for which the best signal to noise ratio is observed. Background non-specific staining patterns should become absent or should be clearly distinguishable from specific staining in solid tumor samples.

[0129] A linearity experiment was performed as follows. A dilution series with 5 different dilutions of the primary antibody (1/25, 1/50, 1/100, 1/400, and 1/800) was performed on serial sections of 5 samples showing variable levels of CLDN18.2 expression. The dilution was set in such a way that the background unspecific staining patterns can be clearly distinguishable from the specific staining pattern on cells. The 5 samples included 4 gastric cancer tissue samples and 1 PDAC tissue sample. [0130] Based on the qualitative evaluation, a signal proportional to the antibody concentration was discerned for all samples. A dilution 1/50 was determined as the optimal dilution because it resulted in the best signal to noise ratio.

G. Precision

[0131] Precision is the ability of the assay to consistently reproduce a result using serial sections from the same specimen processed within one run and over different runs. Intra-assay and inter-assay precision are two distinct measures, which is part of the assay validation procedure. The experimental design of the precision study is given in FIG. 5.

[0132] In summary, eight serial slides of five different FFPE blocks with different expression levels will be processed over three different staining runs. In the first run, 5 serial sections of each block will be included to assess intra-run agreement. In the second run, 2 serial slides (1 positive and 1 IgG control) will be processed and in the third run, 1 serial slide will be included. For each indication a minimum of 1 block should be included. The precision runs will be executed by minimum 2 different lab techs and on minimum 2 different staining instruments.

[0133] Qualitative evaluation: assessment will be performed in a qualitative manner by a pathologist via a side by side comparison of the serial slides following white paper test. For the precision of CLDN18.2 IHC to be valid, at least 80% of the samples should display a good concordance. This means that the signal intensity must be concordant in all serial slides whereby also the same staining pattern must be present.

[0134] Semi-quantitative evaluation by H-score: samples stained during the precision experiment will be scored by a pathologist, who will assess the % of tumor cells that exhibit membranous staining at 0, 1+, 2+ and 3+ intensity. A global H-score will be calculated according to following formula:

H-score = [(1* % of cells displaying a 1+ positivity) + (2* % of cells displaying a 2+ positivity) + (3* % of cells displaying a 3+ positivity)]

[0135] The coefficient of variance (%CV) will be calculated to evaluate intra-run variability and inter-run variability:

- H-score < 50: always concordant

- H-score > 50 and < 100: maximum % CV = 50%

- H-score > 100: maximum %CV = 20%

(%CV intra = (STDEVintra/AVGintra)* 100; % CV inter = STDEVi_nter/AVGinter)*100) Results must be at least 80% concordant at sample level.

[0136] If >5% of the tumor cells exhibit membranous staining for CLDN18.2 at any intensity, a sample is considered to be positive for CLDN18.2. Precision will also be evaluated based on this cut-off for positivity, whereby the percentage of positive agreement and percentage of negative agreement will be established. Results must be at least 90% concordant on slide level. [0137] Precision was performed on 4 gastric cancer samples and 1 PDAC sample to evaluate intra- and inter-run variability. The qualitative assessment of all samples included in precision, was performed by a certified pathologist following ‘white paper test’. Analysis demonstrated a good concordance within runs and between different runs (> 80% of the serial slides were concordant). A minimal variation in the staining intensity /DAB color tone was observed for the samples stained in the 3rd run, but these variations are within the limits of acceptability. For illustration, FIG. 6A-H shows the PDAC/negative control sample used in the precision study of CLDN18.2 IHC.

[0138] Samples were also evaluated at a semi-quantitative level to allow for a more standardized and objective assessment of the precision performance. A pathologist assessed the % of tumor cells that exhibit membranous staining at 0, 1+, 2+ and 3+ intensity. A global H- score was also calculated according to following formula: H-score = [(! * % of cells with 1% positivity)+(2 * % of cells with 2+ positivity)+(3* % of cells with 3+ positivity)].

Table 5: Semi-quantitative analysis of precision testing for CLDN18.2 IHC assay performed on gastric cancer sample and PDAC

((%CV intra = (STDEVintra/AVGintra)*100; % CV inter = STDEVinter/AVGinter)*100)

0139] For the intra-run variability and inter-run variability, a concordance of 100% was obtained on sample level.

[0140] Samples for which >5% of the tumor cells exhibit membranous staining for CLDN18.2 at any intensity were considered to be positive for CLDN18.2. The cut-off for positivity was used to evaluate precision and results are shown in Table 6.

Table 6: Semi-quantitative analysis of precision testing for CLDN18.2 IHC assay performed on gastric cancer sample and PDAC.

Semi-quantitative analysis of precision testing for CLDN18.2 IHC assay was performed on gastric cancer sample and PDAC. The % of TCs that demonstrate membranous staining for CLDN 18.2 (at any intensity) were quantified by a pathologist. Based on the >5 % cut-off, all samples were CLDN18.2 positive.

H. Normal Tissue Screening

[0141] Using a tissue micro-array (TMA) that contained tissue cores of 32 types of normal human organs, expression of CLDN18.2 was evaluated in a range of normal human tissues. All cores were reviewed and scored by a pathologist. A read-out based on an H-score was applied, whereby membranous CLDN 18.2 expression was evaluated for the normal epithelial cells. A sample is considered to be positive for CLDN18.2 in case > 5% of the epithelial cells show membranous CLDN18.2 expression (of any intensity).

[0142] Normal tissue screening was performed on a normal tissue TMA, containing 32 types of normal human organs, each type taken from 3 different individuals. One section of the TMA was stained with the CLDN 18.2 antibody on the DAKO autostainer. A gastric cancer FFPE sample was included as run control. The run control was stained with the CLDN18.2 antibody and the IgG isotype control.

[0143] Membranous CLDN18.2 expression was evaluated for the normal epithelial cells. The cores were scored by a pathologist according to the histoscore. This means that the percentage of epithelial cells stained according to 0, 1+, 2+ and 3+ intensity were determined. Only membranous CLDN18.2 staining was considered to be positive. The H-score was calculated according to following formula:

H-score = [(1 * % cells 1+) + (2 * % cells 2+) + (3 * % cells 3+)]

[0144] A sample is considered to be positive for CLDN18.2 in case > 5% of the epithelial cells show membranous CLDN18.2 expression. Based on these criteria, 0.04 % were considered to be positive for CLDN18.2. CLDN18.2 positivity was only observed for normal stomach tissue. The other normal human organs, including lung, are negative for CLDN18.2.

I. Prevalence

[0145] Using tissue micro-arrays (TMA) that contain normal and malignant tissue cores, expression of CLDN18.2 will be evaluated in PDAC and GC. All cores will be reviewed and scored by a pathologist. A read-out based on an H-score will be applied, whereby membranous CLDN18.2 expression will be evaluated for the tumor cells. A sample is considered to be positive for CLDN18.2 in case > 5% of the tumor cells show membranous CLDN18.2 expression (of any intensity).

[0146] The objective of this study was to determine the target expression profile in tumor tissues of the indications of interest (ie. pancreas cancer and gastric cancer) using the CLDN18.2 (clone EPR19202) IHC assay.

[0147] For the prevalence study 2 tumor TMAs (pancreas cancer and gastric cancer) were stained for CLDN18.2 on the DAKO autostainer. In each staining run, a gastric cancer sample was included as run control.

[0148] In total, 400 cores were stained for CLDN18.2 of which 75 cores were excluded from the analysis due to tissue detachment, loss of antigenicity (QC failure of specific cores on TMA slides) or the absence of tumor.

[0149] The cores were scored by a pathologist according to the histoscore for tumor cells. This means that the percentage of tumor cells stained according to 0, 1+, 2+ and 3+ intensity were determined. Only membranous CLDN18.2 staining was considered to be positive. The H-score was calculated according to following formula:

H-score = [(1 * % cells 1+) + (2 * % cells 2+) + (3 * % cells 3+)]

[0150] A sample is considered to be positive for CLDN18.2 in case > 5% of the tumor cells show membranous CLDN 18.2 expression.

[0151] Pancreas carcinoma: CLDN18.2 expression was assessed on 1 advanced stage pancreatic carcinoma TMA, containing 86 cases of adenocarcinoma, 3 undifferentiated carcinoma, 2 adenosquamous carcinoma, 1 squamous cell carcinoma, 2 carcinoid, 1 solid pseudopapillary carcinoma and 1 neuroendocrine carcinoma. The TMA contains duplicate cores per case, divided into two identical 96 core arrays. In total 38 cores were excluded from analysis: 33 because of failed QC, 4 due to the absence of tumor cells and 1 due to tissue detachment.

[0152] Membranous CLDN18.2 expression was evaluated for the tumor compartment. Based on the criteria above, 33/154 cores (21.43%) were considered to be positive for CLDN18.2 (tumor positivity >5%). CLDN18.2 positivity was only observed for the adenocarcinomas, which represented the majority of the cases within this TMA. Most samples show a rather weak (1+) to moderate (2+) CLDN18.2 expression, whereby a substantial proportion of the tumor cells also lack membranous CLDN18.2 staining.

[0153] Gastric cancer: CLDN18.2 expression was assessed on 1 stomach cancer tissue array with adjacent normal tissue, containing 156 cases of stomach adenocarcinoma, 40 stomach metastatic adenocarcinoma, 8 stomach adjacent normal tissue and 4 stomach normal tissue (208 cores and 208 cases). In total 30 cores were excluded from analysis: 15 because of failed QC, 9 due to the absence of tumor cells and 6 due to tissue detachment.

[0154] Membranous CLDN18.2 expression was evaluated for the tumor compartment. Based on the criteria defined above, 44 out of 178 tumor cases (24.72 %) were considered to be positive for CLDN18.2. More specifically, 40 out of 143 adenocarcinomas (27.97%) and 4 out of 35 (11.43%) metastatic adenocarcinoma cases were positive for CLDN18.2. In 30 out of 44 positive cases, > 50% of the tumor cells lacked CLDN18.2 expression (0+), whereas the remainder tumor cells displayed CLDN18.2 expression at variable intensities. In 6 out of 44 cases, all tumor cells stained positive for CLDN18.2 whereby the majority of the tumor cells showed a moderate to strong expression of the target.

J. Epitope stability testing

[0155] Comparing the staining performance of serial sections stained at 4 different time points (eg. time point 0, 1 month, 3 months and 6 months) - sectioned at the same time and stored at the same temperature, allows studying the stability of the epitope. The stability of the CLDN18.2 epitope will be tested on 4 tissue samples with different expression levels of the target. In each staining run, freshly sectioned slides of a run control will be included. The run control will be stained according to the positive protocol and the negative protocol. The staining pattern and intensity will be qualitatively evaluated for the different timepoints for each sample.

[0156] The objective of this study was to determine the target expression profile in tumor tissues of the indications of interest (ie. pancreas cancer and gastric cancer) using the CLDN18.2 (clone EPR19202) IHC assay. [0157] For the prevalence study 2 tumor TMAs (pancreas cancer and gastric cancer) were stained for CLDN18.2 on the DAKO autostainer. In each staining run, a gastric cancer sample was included as run control.

[0158] The epitope stability testing was performed on 3 gastric cancer samples and 1 PDAC sample at the timepoints described above. The selected blocks displayed a range of CLDN18.2 expression. Some fluctuations in CLDN18.2 staining intensity were observed for timepoints t = 3 months and t = 6 months. The fluctuations were most obvious for weak CLDN18.2 positive cells. To assess whether these fluctuations were reproducible, these specific timepoints were retested. In each run, except for timepoint 0, fresh sectioned slides of a gastric cancer sample containing NAT were included as a run control. The run control stained whereby a strong positive staining could be observed for the normal stomach mucosa and the tumor cells demonstrated a variety of CLDN18.2 expression levels, ranging from negative to strongly positive. Non-specific staining could not be observed for the sections stained with the IgG isotype control.

[0159] Results: for timepoints up to 3 months, the observed staining intensity on aged slides fell within the range of variability expected from the assay. At the subsequent 6 month timepoint staining intensity on aged slides decreased to a level outside of this range, whereby the decreased staining intensity was most prominent for the weak CLDN18.2 positive tumor cells. A more subtle decrease of the staining intensity could, however, also be observed for the strongly CLDN18.2 positive tumor cells. The staining intensity is thus stable up to the 3 month timepoint, and the epitope loses its stability at the 3 month timepoint.

Example 2

Antibody 1 exhibits potent ADCC activity not only for CLDN18.2-high but also -low tumor cells

[0160] CLDN 18.2-negative MIA-PaCa2 was transduced with retrovirus carrying human CLDN18.2 gene and the clones were isolated. CLDN18.2 expression was confirmed by flow cytometry (FIG. 7) and CLDN18.2 IHC assay disclosed herein (FIG. 8A-E). FIG. 8F showed the H-score for various MIA-PaCa2-hCLDN18.2 clones. CLDN18.2 expression levels represented by flow cytometry and IHC correspond well with each other.

[0161] ADCC with isolated human NK cells (E:T = 5:1) was performed using the MIA- PaCa2-hCLDN18.2 clones (FIG. 9A-B). ECso for each of the clones were compared (FIG. 9C). The results indicated that Antibody 1 exhibited robust ADCC activity in not only CLDN18.2- high/med but also CLDN18.2-low MIA-PaCa2 clones. In contrast, a benchmark CLDN 18.2 antibody has limited/no activity on low-expressing clones. Example 3

Antibody 1 induces robust ADCC for gastric tumor cell lines expressing endogenous CLDN18.2 compared to benchmark antibody

[0162] CLDN18.2 expression in gastric tumor cell lines SNU601, 620, KATOIII, and SNU5 was confirmed by qPCR (FIG. 10A) and CLDN18.2 IHC disclosed herein (FIG. 10B-E). FIG. 10F showed the H-score from the CLDN 18.2 IHC for the various gastric tumor cell lines. CLDN 18.2 expression level in these cells obtained from qPCR and CLDN 18.2 IHC correspond well with each other.

[0163] ADCC with human PBMC (E:T = 20: 1) (FIG. 11A-D) and with purified human NK cells (E:T = 1:1) was performed for the indicated tumor cell lines (FIG. 12A-D).

[0164] Efficacy of Antibody 1 in comparison to a benchmark in vivo was also evaluated. 5 x 10⁶ cells of SNU620, KATOIII, or SNU5 were subcutaneously inoculated into Balb/c nude mouse at the right hind flank with 0.1 ml of 1 : 1 mixture of PBS and Matrigel (354263 , Coming). After randomization, 10 mg/kg of control antibody (hlgGl) or the indicated concentration of Antibody 1 or benchmark antibody was injected by IP route once weekly (QW) and tumor volume was measured twice a week. Antibody 1 could suppress tumor growth even at the concentration of 0.1 and 1 mg/kg, but benchmark required 10 mg/kg to achieve inhibition of tumor growth although exposure of benchmark was about twice as high as Antibody 1. Antibody 1 demonstrated greater tumor growth inhibition effect than a benchmark CLDN 18.2 antibody (FIG. 13A). At day 42, the tumor volume was compared (FIG. 13B). ****, ***, or ns indicates p < 0.0001, p < 0.001, or not significant, respectively. [0165] KATOIII minimally expressing CLDN18.2 and CLDN 18.2-negative SNU5 were subcutaneously inoculated into Balb/c nude mice (n = 10). 10 mg/kg of the indicated antibodies was injected by IP route once weekly (QW). The results showed that Antibody 1 suppressed KATOIII but not SNU5 growth in vivo (FIG. 13C and 13D). KATOIII tumor growth was also inhibited by Antibody 1 more potently than benchmark (FIG. 13C) while both antibodies failed to inhibit CLDN18.2- negative SNU5 growth (Fig. 13D). Antibody 1 showed better efficacy for KATOIII than benchmark at day 40 (FIG. 13E). Both antibodies failed to inhibit SNU5 growth at day 40 (FIG.13F). ****, *, or ns indicates p < 0.0001, p < 0.05, or not significant, respectively. These results indicate that Antibody 1 can target not only CLDN 18.2 -high gastric tumors but also CLDN18.2-low gastric tumors and exhibit better in vivo efficacy than benchmark.

[0166] Additional studies also showed that Antibody 1 demonstrated greater efficacy than a benchmark in vivo in engineered xenograft models. Specifically, Antibody 1 treatment resulted in greater anti-tumor activity and longer duration of response than the benchmark in CLDN18.2 high-expressing MIA PaCa xenograft models. Antibody 1 also exhibited stronger efficacy than the benchmark in CLDN18.2 high-expressing patient derived GC tumor model.

Example 4

Antibody 1 in vivo efficacy in gastric patient-derived xenograft (PDX) models [0167] Seven gastric PDXs were selected for Antibody 1 treatment based on RNAseq data for CLDN18.2. The PDX fragments (2-3 mm in diameter) were subcutaneously inoculated into Balb/c nude mouse (n=10) and the PDX-bearing mice were injected with Antibody 1 or hlgGl (50 mg/kg) by IP route three times a week (T1W) and tumor volume was measured twice a week. Although RNAseq data showed that all the PDX models are CLDN 18.2-positive, only three PDXs (GA0006, 6831, 2419) responded to Antibody 1 treatment (FIG. 14A-14C) while four PXDs (GA6208, 0074, 13765, and 0060) did not respond to Antibody 1 treatment (FIG. 14D-14G). * or ns in FIG. 14A-14G indicates p < 0.05 or not significant, respectively. CLDN18.2 protein levels in the PDXs were confirmed by CLDN18.2 IHC (FIG. 15A-15G). Strikingly, all three responders showed membrane staining of CLDN 18.2 but non-responders (GA6208, 0074, 13765, 0060) were CLDN18.2 IHC-negative. These data indicated that Antibody 1 can target only CLDN18.2-expressing tumors. In addition, the CLDN18.2-staining intensity correlated with tumor growth inhibition (TGI) since GA2419 (TGI = 40.3%) that showed weak CLDN18.2-positivity was less sensitive to Antibody 1 treatment than GA0006 (TGI = 56.6%) and GA6831 (TGI = 56.0%) that showed high and medium CLDN18.2-staining intensity, respectively (Fig. 6A, B). This study also demonstrated that the CLDN18,2 IHC assay described here can be more accurate than other CLDN18.2 detecting method (e.g., RNAseq data) in predicting the responsiveness to anti-CLDN 18.2 antibody treatment in cancer patients.

Claims

We Claim:

1. A method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, comprising: obtaining a sample from the patient, staining the sample with antibody EPR19202 in an 1HC assay, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if the staining is positive.

2. A method of treating a patient having cancer in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-claudin 18.2 antibody if a sample from the patient is stained positive with antibody EPR19202 in an IHC assay.

3. A method of treating a patient having cancer in need thereof, comprising: obtaining a sample from the patient, staining the sample with antibody EPR19202 in an IHC assay, determining the staining is positive, and administering to the patient a therapeutically effective amount of an anti-claudin 18.2 antibody.

4. A method of treating a patient having cancer in need thereof with a therapy comprising a therapeutically effective amount of an anti-claudin 18.2 antibody, which method comprises, prior to treating the patient with the anti-claudin 18.2 antibody, obtaining a sample from the patient and staining the sample with antibody EPR19202 in an IHC assay.

5. The method of any of claims 1-4, wherein staining is performed by following known IHC staining procedures.

6. The method of any of claims 1-5, wherein the sample from the patient was preserved in the form of formalin fixed and paraffine embedded (PEPE) pellets before staining.

7. The method of any of claims 1-6, wherein the staining is membranous staining.

8. The method of any of claims 1-7, wherein the staining is performed on an autostainer.

9. The method of claim 8, wherein the autostainer is a Dako Link 48 autostainer or a LabVision Autostainer.

10. The method of any of claims 1-3 and 5-9, wherein the staining exhibits 1+, 2+, or 3+ intensity, and wherein the staining is positive if > 5% of the sample cells exhibit membranous staining at any intensity.

11. The method of any of claims 1-3 and 5-9, wherein the staining is positive if (1) the sample has an elevated level of membrane staining of any intensity relative to a negative control or (2) the H-score is > 5, wherein the negative control is IgG isotype of antibody EPR19202.

12. The method of any of claims 1-11, wherein the sample is a tissue sample comprising epithelial tumor cells.

13. The method of any of claims 1-12, wherein the sample is a tissue sample comprising cancer cells selected from gastric cancer, lung cancer (non-small cell lung cancer or small cell lung cancer), bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicle cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervix 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, vagina cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, cholangiocarcinoma, and adenocarcinoma.

14. The method of claim 13, wherein the sample is a tissue sample comprising cancer cells selected from gastric cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, and colorectal cancer.

15. The method of any of claims 1-14, wherein the sample comprises tumor cells that are primary.

16. The method of any of claims 1-14, wherein the sample comprises tumor cells that are metastatic.

17. The method of any of claims 1-16, wherein the method further comprises staining the sample with a matched IgG isotype of antibody EPR19202.

18. The method of any of claims 1-17, wherein the anti-claudin 18.2 antibody comprises a variable light chain region and a variable heavy chain region, wherein the variable light chain region comprises CDR1 having the sequence of SEQ ID NO: 1, CDR2 having the sequence of SEQ ID NO: 2, and CDR3 having the sequence of SEQ ID NO: 3, the variable heavy chain region comprises CDR1 having the sequence of SEQ ID NO: 4, CDR2 having the sequence of SEQ ID NO: 5, and CDR3 having the sequence of SEQ ID NO: 6.

19. The method of claim 18, wherein the anti-CLDN 18.2 antibody comprises the sequences of SEQ ID NOs: 7 and 8.

20. The method of claim 18 or 19, wherein the anti-CLDN 18.2 antibody is a humanized TgGl monoclonal antibody with mutations in the Fc domain to enhance ADCC.

21. A method of determining whether to treat a patient having cancer with an anti-claudin 18.2 antibody, comprising: obtaining a sample from the patient, dividing the sample into two or more sections, staining the sample on the two or more sections with antibody EPR19202, and determining to treat the patient with a therapeutically effective amount of the anti-claudin 18.2 antibody if each staining is positive and the coefficient of variance meets the acceptance criteria.

22. A kit, comprising antibody EPR19202 and a reagent for IHC assay.

23. The kit of claim 22, further comprising a matched IgG isotype of antibody EPR19202.