Combination therapy involving antibodies against claudin 18.2 for treatment of cancer

Abstract

To provide a combination therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2.SOLUTION: The present invention provides a combination therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head and neck cancer, and cancer of the gallbladder and metastases thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Gastric and esophageal (gastroesophageal; GE) cancers are among the malignant tumors with the highest unmet medical need. Gastric cancer is the second leading cause of cancer death worldwide. The incidence of esophageal cancer has been increasing in the last decade in line with the shift in histotype and primary tumor location. In the United States and Western Europe, adenocarcinoma of the esophagus is now more prevalent than squamous cell carcinoma, and most tumors are located in the lower esophagus. The 5-year overall survival rate for GE cancer is 20 - 25%, despite the aggressiveness of established standard treatments with substantial side effects.

Background Art

[0002] Most patients present with locally advanced or metastatic disease and must be subjected to first-line chemotherapy. Treatment regimens are mostly based on a backbone of platinum derivatives and fluoropyrimidine derivatives combined with a third compound (e.g., taxane or anthracycline). Nevertheless, the best that can be expected is a median progression-free survival of 5 - 7 months and a median overall survival of 9 - 11 months.

[0003] The lack of significant benefit from various newer-generation combination chemotherapy regimens for these cancers has stimulated research into the use of targeted agents. Recently, trastuzumab was approved for Her2 / neu-positive gastroesophageal cancer. However, only about 20% of patients express this target and are eligible for this treatment, so the medical need remains high.

[0004] Tight junction molecule Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)) is a member of the Claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa transmembrane protein containing four membrane-spanning domains together with two small extracellular loops.

[0005] In normal tissues, with the exception of the stomach, there is no detectable expression of CLDN18.2 by RT-PCR. Immunohistochemical examination using a CLDN18.2-specific antibody reveals the stomach as the only positive tissue.

[0006] CLDN18.2 is a highly selective gastric lineage antigen that is exclusively expressed on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the process of malignant transformation and is thus often displayed on the surface of human gastric cancer cells. Furthermore, this pan-tumor antigen is ectopically activated at a significant level in adenocarcinomas of the esophagus, pancreas, and lung. The CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and particularly in distal metastases to the ovary (so-called Krukenberg tumors).

[0007] The chimeric IgG1 antibody IMAB362 directed against CLDN18.2 was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and combines four independent and highly potent mechanisms of action. Upon binding to the target, IMAB362 mediates cell killing by inducing apoptosis, which is induced by ADCC, CDC, and cross-linking of the target on the tumor cell surface, directly inhibiting proliferation. Thus, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. Mice bearing CLDN18.2-positive cancer cell lines have a survival benefit, and when treated with IMAB362, up to 40% of the mice show regression of their tumors.

[0008] The toxicity and PK / TK profiles of IMAB362 have been fully investigated in mice and cynomolgus monkeys, including a dose range finding study, a 28-day repeated dose toxicity study in cynomolgus monkeys, and a 3-month repeated dose toxicity study in mice. In both mice (dosed weekly for a maximum treatment duration of 3 months, maximum dose level 400 mg / kg) and cynomolgus monkeys (maximum of 100 mg / kg applied up to 5 times per week), repeated administration of IMAB362 i.v. was well tolerated. No signs of systemic or local toxicity were induced. Specifically, gastrointestinal toxicity was not observed in any of the toxicity studies. IMAB362 does not induce immune activation or cytokine release. No adverse effects on male or female genitalia were recorded. IMAB362 does not bind to tissues lacking the target. Biodistribution studies in mice indicate that the lack of gastrointestinal toxicity is most likely due to the compartmentalization of tight junctions at the luminal site in healthy gastric epithelium, thereby greatly impairing the accessibility of IMAB362 epitopes. This compartmentalization is lost during the malignant transformation that pharmacologically targets the epitope with IMAB362.

[0009] IMAB362 is in early clinical trials. A Phase I clinical trial is being conducted in humans. Five-dose cohorts of 3 patients each (33 mg / m 2 , 100 mg / m 2 , 300 mg / m 2 , 600 mg / m 2 , 1000 mg / m 2 ) received a single intravenous administration of IMAB362 and were observed for 28 days. IMAB362 was very well tolerated and was not associated with any safety findings in the patients. In one patient, all measured tumor markers decreased significantly within 4 weeks after treatment. In the ongoing Phase IIa clinical trial, IMAB362 is being administered repeatedly.

[0010] The data presented herein indicate that bisphosphonates, such as zoledronic acid (ZA), enhance the activity of anti-CLDN18.2 antibodies, such as IMAB362, particularly when administered in combination with recombinant interleukin-2 (IL-2). The underlying mechanism is the activation and expansion of a highly cytotoxic immune cell population (γ9δ2 T cells).

[0011] Furthermore, the inventors herein present data demonstrating that chemotherapeutic agents can stabilize or increase the expression of CLDN18.2 on the surface of cancer cells, enhancing the druggability of CLDN18.2 by anti-CLDN18.2 antibodies such as IMAB362. A synergistic effect of anti-CLDN18.2 antibodies, such as IMAB362, with specific chemotherapeutic agent regimens, particularly those used in the treatment of gastric cancer or the treatment of human solid tumors, was observed. Human cancer cells pre-treated with chemotherapy are sensitive to antibody-induced target-specific killing. In a mouse tumor model, tumor control using an anti-CLDN18.2 antibody and chemotherapy is superior to that using the anti-CLDN18.2 antibody as a single agent.

Prior Art Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0014] The present invention generally provides a combination therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer, as well as metastases thereof, particularly gastric cancer metastases such as Krukenberg tumors, peritoneal metastases, and lymph node metastases. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary.

Means for Solving the Problems

[0015] In one aspect, the present invention provides a method for treating or preventing a cancer disease, the method comprising administering to a patient an antibody having the ability to bind to CLDN18.2 in combination with an agent that stimulates γδT cells. The agent that stimulates γδT cells can be administered before, simultaneously with, or after the administration of the antibody having the ability to bind to CLDN18.2, or in combinations thereof.

[0016] In one embodiment, the γδ T cells are Vγ9Vδ2 T cells. In one embodiment, the agent that stimulates the γδ T cells is a bisphosphonate such as a nitrogen-containing bisphosphonate (aminobisphosphonate). In one embodiment, the agent that stimulates the γδ T cells is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid, and salts thereof. In one embodiment, the agent that stimulates the γδ T cells is administered in combination with interleukin-2.

[0017] In one embodiment, the method of the present invention further comprises a step of administering an agent that stabilizes or increases the expression of CLDN18.2. The expression of CLDN18.2 is preferably on the cell surface of cancer cells.

[0018] An agent that stabilizes or increases the expression of CLDN18.2 can be a cytotoxic agent and / or a cell growth inhibitor. In one embodiment, an agent that stabilizes or increases the expression of CLDN18.2 includes an agent that induces cell cycle arrest or cell accumulation in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the G1 phase. An agent that stabilizes or increases the expression of CLDN18.2 can include an agent selected from the group consisting of anthracyclines, platinum compounds, nucleoside analogs, taxanes, and camptothecin analogs, or prodrugs thereof, and combinations thereof. An agent that stabilizes or increases the expression of CLDN18.2 can include an agent selected from the group consisting of epirubicin, oxaliplatin, cisplatin, 5-fluorouracil or a prodrug thereof, such as capecitabine, docetaxel, irinotecan, and combinations thereof. An agent that stabilizes or increases the expression of CLDN18.2 can include a combination of oxaliplatin and 5-fluorouracil or a prodrug thereof, a combination of cisplatin and 5-fluorouracil or a prodrug thereof, a combination of at least one anthracycline and oxaliplatin, a combination of at least one anthracycline and cisplatin, a combination of at least one anthracycline and 5-fluorouracil or a prodrug thereof, a combination of at least one taxane and oxaliplatin, a combination of at least one taxane and cisplatin, a combination of at least one taxane and 5-fluorouracil or a prodrug thereof, or a combination of at least one camptothecin analog and 5-fluorouracil or a prodrug thereof. An agent that stabilizes or increases the expression of CLDN18.2 can be an agent that induces immunogenic cell death. An agent that induces immunogenic cell death can include an agent selected from the group consisting of anthracyclines, oxaliplatin, and combinations thereof. An agent that stabilizes or increases the expression of CLDN18.2 can include a combination of epirubicin and oxaliplatin.In one embodiment, the method of the present invention includes the step of administering at least one anthracycline, at least one platinum compound, and at least one of 5-fluorouracil and its prodrug. The anthracycline can be selected from the group consisting of epirubicin, doxorubicin, daunorubicin, idarubicin, and valrubicin. Preferably, the anthracycline is epirubicin. The platinum compound can be selected from the group consisting of oxaliplatin and cisplatin. The nucleoside analog can be selected from the group consisting of 5-fluorouracil and its prodrug. The taxane can be selected from the group consisting of docetaxel and paclitaxel. The camptothecin analog can be selected from the group consisting of irinotecan and topotecan. In one embodiment, the method of the present invention includes the step of administering (i) epirubicin, oxaliplatin, and 5-fluorouracil, (ii) epirubicin, oxaliplatin, and capecitabine, (iii) epirubicin, cisplatin, and 5-fluorouracil, (iv) epirubicin, cisplatin, and capecitabine, or (v) folinic acid, oxaliplatin, and 5-fluorouracil.

[0019] The method of the present invention may further include the step of administering at least one additional chemotherapeutic agent that can be a cytotoxic agent.

[0020] Antibodies having the ability to bind to CLDN18.2 can bind to the native epitopes of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, an antibody having the ability to bind to CLDN18.2 mediates cell killing by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cell-mediated cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment, an antibody having the ability to bind to CLDN18.2 is a monoclonal antibody, a chimeric antibody, or a humanized antibody, or a fragment of an antibody. In one embodiment, an antibody having the ability to bind to CLDN18.2 is selected from the group consisting of an antibody produced by and / or obtained from a clone deposited under deposit numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody that is a chimerized or humanized form of the antibody belonging to (i), (iii) an antibody having the specificity of the antibody belonging to (i), and (iv) an antigen-binding portion or antigen-binding site of an antibody belonging to (i) and preferably having the specificity of the antibody belonging to (i), particularly an antibody containing a variable region. In one embodiment, the antibody is conjugated to a therapeutic agent, such as a toxin, a radioisotope, a drug, or a cytotoxic agent.

[0021] In one embodiment, the method of the present invention comprises administering an antibody having the ability to bind to CLDN18.2 at a dose of up to 1000 mg / m 2 . In one embodiment, the method of the present invention comprises repeatedly administering an antibody having the ability to bind to CLDN18.2 at a dose of 300 - 600 mg / m 2 .

[0022] In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, the cancer disease is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof. The cancer disease can be Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer. The patient can be a HER2 / neu negative patient or a patient having a HER2 / neu positive status but not eligible for trastuzumab therapy.

[0023] According to the present invention, CLDN18.2 preferably has the amino acid sequence according to SEQ ID NO: 1.

[0024] In a further aspect, the present invention provides a pharmaceutical preparation comprising an antibody having the ability to bind to CLDN18.2 and an agent that stimulates γδT cells. The pharmaceutical preparation of the present invention may further comprise an agent that stabilizes or increases the expression of CLDN18.2. The antibody having the ability to bind to CLDN18.2, the agent that stimulates γδT cells, and optionally the agent that stabilizes or increases the expression of CLDN18.2 can be present in the pharmaceutical preparation as a mixture or separately from each other. The pharmaceutical preparation can be a kit comprising a first container containing an antibody having the ability to bind to CLDN18.2, a container containing an agent that stimulates γδT cells, and optionally a container containing an agent that stabilizes or increases the expression of CLDN18.2. The pharmaceutical preparation may further comprise printed instructions for using the preparation for treating cancer, particularly for using the preparation by the method of the present invention. Different embodiments of the pharmaceutical preparation, and particularly the agent that stimulates γδT cells and the agent that stabilizes or increases the expression of CLDN18.2, are as described above for the method of the present invention.

[0025] The present invention also provides an agent described herein, such as an antibody having the ability to bind to CLDN18.2, for use in the methods described herein, for example, for administration in combination with an agent that stimulates γδ T cells and optionally an agent that stabilizes or increases the expression of CLDN18.2.

[0026] Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0028] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these can be diverse. It should also be understood that the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] In the following, the elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only the explicitly described embodiments. It should be understood that this description supports and encompasses embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any rearrangement and combination of all the described elements in this application should be considered to be disclosed by the description of this application unless otherwise indicated by the context.

[0030] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0031] Unless otherwise indicated, the practice of the present invention uses conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature of this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0032] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted as implying the inclusion of the stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of any other member, integer, or step, or group of members, integers, or steps. That being said, in some embodiments, such other members, integers, or steps, or group of members, integers, or steps may be excluded, i.e., it is understood that the subject matter is intended to include the stated member, integer, or step, or group of members, integers, or steps. As used in connection with the description of the present invention (in particular, in connection with the claims), the terms "a", "an", and "the", and similar references are to be construed to cover both the singular and the plural forms unless otherwise indicated herein or clearly contradicted by the context. The description of a range of values herein is merely intended to serve as a convenient method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0033] Several documents are cited throughout the text of this specification. Each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety, whether supra or infra. None of the content in this specification should be construed as an admission that the present invention does not have the right to antedate such disclosure by virtue of a prior invention.

[0034] The term "CLDN18" refers to Claudin 18 and includes any variant, including Claudin 18 splice variant 1 (Claudin 18.1 (CLDN18.1)) and Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)).

[0035] The term "CLDN18.2" preferably refers to human CLDN18.2, particularly a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 in the Sequence Listing, or a variant of said amino acid sequence.

[0036] The term "CLDN18.1" preferably refers to human CLDN18.1, particularly a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 2 in the Sequence Listing, or a variant of said amino acid sequence.

[0037] The term "variant" according to the present invention particularly refers to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. Allelic variants relate to changes in the normal sequence of a gene, and their significance is often unclear. Complete gene sequencing often identifies multiple allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences for species of different origin from that of a given nucleic acid or amino acid sequence. The term "variant" shall include any post-translationally modified variant and conformational variant.

[0038] According to the present invention, the term "CLDN18.2-positive cancer" means a cancer involving cancer cells that express CLDN18.2, preferably on the surface of said cancer cells.

[0039] "Cell surface" is used in its ordinary meaning in the art and thus includes the outer side of the cell that is accessible to binding by proteins and other molecules.

[0040] When CLDN18.2 is located on the surface of a cell, it is expressed on the surface of said cell and is accessible to binding by a CLDN18.2-specific antibody added to the cell.

[0041] According to the present invention, CLDN18.2 is not substantially expressed intracellularly when the level of expression is lower compared to the expression in gastric cells or gastric tissue. Preferably, the level of expression is less than 10% of the expression in gastric cells or gastric tissue, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower. Preferably, CLDN18.2 is not substantially expressed intracellularly when the level of expression exceeds the level of expression in non-cancerous tissues other than the stomach by 2-fold or less, preferably 1.5-fold or less, and preferably does not exceed the level of expression in said non-cancerous tissues. Preferably, CLDN18.2 is not substantially expressed intracellularly when the level of expression is below the detection limit and / or the level of expression is too low to enable binding by a CLDN18.2-specific antibody added to the cell.

[0042] According to the present invention, CLDN18.2 is expressed intracellularly when the level of expression exceeds the level of expression in non-cancerous tissues other than the stomach by more than 2-fold, preferably more than 10-fold, more than 100-fold, more than 1000-fold, or more than 10000-fold. Preferably, CLDN18.2 is expressed intracellularly when the level of expression exceeds the detection limit and / or the level of expression is high enough to enable binding by a CLDN18.2-specific antibody added to the cell. Preferably, CLDN18.2 expressed intracellularly is expressed or exposed on the surface of said cell.

[0043] According to the present invention, the term "disease" refers to any pathological condition, including cancer, particularly the forms of cancer described herein. Any reference herein to cancer, or a particular form of cancer, also includes metastases of that cancer. In a preferred embodiment, the disease treated by the present application is associated with cells expressing CLDN18.2.

[0044] According to the present invention, the term "disease associated with cells expressing CLDN18.2" or similar expressions means that CLDN18.2 is expressed in the cells of the diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in the cells of the diseased tissue or organ is increased compared to the state in healthy tissue or organ. The increase refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more. In one embodiment, the expression is found only in the diseased tissue, while the expression in healthy tissue is suppressed. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Furthermore, according to the present invention, cancer diseases preferably have cancer cells that express CLDN18.2.

[0045] As used herein, "cancer disease" or "cancer" includes diseases characterized by abnormally controlled cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" mean abnormal cells that proliferate by rapid, uncontrolled cellular proliferation and continue to proliferate after the stimulus that initiated new growth has ended. Preferably, "cancer disease" is characterized by cells expressing CLDN18.2, and the cancer cells express CLDN18.2. The cells expressing CLDN18.2 are preferably the cancer cells of the cancer described herein.

[0046] "Adenocarcinoma" is cancer that originates from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes the skin, glands, and various other tissues that line the body's cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland as long as they have a secretory function. This form of cancer can occur in several higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly-differentiated adenocarcinomas may not. By staining cells from a biopsy, a pathologist can determine whether a tumor is an adenocarcinoma or one of several other types of cancer. Adenocarcinoma can occur in many tissues of the body due to the ubiquitous nature of glands within the body. Each gland may not secrete the same substance, but as long as it has an exocrine function to cells, it is considered a gland and thus its malignant form is called adenocarcinoma. Malignant adenocarcinomas often metastasize if given enough time to invade other tissues. Ovarian adenocarcinoma is the most common type of ovarian cancer. This includes serous adenocarcinoma and mucinous adenocarcinoma, clear cell adenocarcinoma, as well as endometrioid adenocarcinoma.

[0047] "Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then infiltration of the target organ after being transported by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after the primary tumor has been removed, as tumor cells or tumor components may remain and generate metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis", which relates to metastases distant from the primary tumor and the local lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis that can be treated using the therapies of the present invention is metastases originating from gastric cancer as the primary site. In a preferred embodiment, such gastric cancer metastases are Krukenberg tumors, peritoneal metastases, and / or lymph node metastases.

[0048] Krukenberg tumors are rare metastatic tumors of the ovary that account for 1% - 2% of all ovarian tumors. The prognosis of Krukenberg tumors remains very poor and there is no established treatment for Krukenberg tumors. Krukenberg tumors are metastatic signet ring cell adenocarcinomas of the ovary. The stomach is the primary site in most cases (70%) of Krukenberg tumors. Cancers of the large intestine, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumors originating from cancers of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urothelial tract have been reported. The interval between diagnosis of the primary cancer and subsequent discovery of ovarian involvement is usually less than 6 months, although longer periods have been reported. In many cases, the primary tumor is very small and can escape detection. A previous history of cancer in the stomach or another organ can be obtained in only 20% - 30% of cases.

[0049] Krukenberg tumors are, most commonly, an example of selective cancer spread along the stomach-ovary axis. This axis of tumor spread has historically drawn the interest of many pathologists, particularly when gastric neoplasms have been found to selectively metastasize to the ovaries without involvement of other tissues. The route of metastasis of gastric cancer to the ovaries has been a mystery for a long time, but it is now clear that retrograde lymph node spread is the most likely route of metastasis.

[0050] Women with Krukenberg tumors are generally within 50 years of their lives, with an average age of 45 years, so they tend to be unusually young for patients with metastatic cancer. This young age distribution can be partly related to the increasing frequency of gastric signet ring cell carcinoma in young women. The common main symptoms usually relate to ovarian involvement, the most common of which are abdominal pain and distension (mainly due to often bilateral and frequently large ovarian tumors). The remaining patients have non-specific gastrointestinal symptoms or are asymptomatic. Furthermore, Krukenberg tumors are reportedly associated with virilization resulting from hormone production by the ovarian stroma. Ascites is present in 50% of cases and usually shows malignant cells.

[0051] Krukenberg tumors are bilateral in over 80% of reported cases. The ovaries are usually enlarged asymmetrically with a bulging contour. The surface of the section is yellow or white and is usually solid, but may be cystic in some cases. Importantly, the capsular surface of the ovaries with Krukenberg tumors is generally smooth, without adhesions or peritoneal deposits. Notably, other metastatic tumors to the ovaries tend to be associated with surface implants. This can explain why the gross morphology of Krukenberg tumors can appear at first glance to be a primary ovarian tumor. However, the bilateral symmetry in Krukenberg tumors is consistent with their metastatic nature.

[0052] Patients with Krukenberg tumors have a significantly high overall mortality rate. Most patients die within two years (median survival time, 14 months). Some studies have shown that the prognosis is poor when the primary tumor is identified after ovarian metastasis is detected, and the prognosis worsens when the primary tumor remains hidden.

[0053] The optimal treatment strategy for Krukenberg tumors has not been clearly established in the literature at all. Whether surgical resection should be performed has not been adequately addressed. Chemotherapy or radiotherapy has no significant effect on the prognosis of patients with Krukenberg tumors.

[0054] "Treating" means administering to a subject a compound or composition, or a combination of compounds or compositions, to prevent or eliminate the disease, including reducing the size or number of tumors in the subject; stopping or slowing down the disease in the subject; inhibiting or slowing down the occurrence of a new disease in the subject; reducing the frequency or severity of symptoms and / or recurrence in a subject who currently has or has previously had the disease; and / or increasing the lifespan of the subject, i.e., increasing it.

[0055] In particular, the term "treatment of a disease" includes cure, shortening of duration, remission of progression or worsening, prevention, slowing down, or inhibition, or prevention or delay of the onset of the disease or its symptoms.

[0056] According to the present invention, the term "patient" means a subject to be treated, particularly a subject with a disease, including humans, non-human primates, or another animal, particularly a mammal, such as a cow, a horse, a pig, a sheep, a goat, a dog, a cat, or a rodent, such as a mouse and a rat. In a particularly preferred embodiment, the patient is a human.

[0057] γδ T cells (gamma-delta T cells) represent a small subset of T cells that have distinct T cell receptors (TCRs) on their surface. The majority of T cells have a TCR composed of two glycoprotein chains called the α- and β-TCR chains. In contrast, in γδ T cells, the TCR is composed of one γ-chain and one δ-chain. This group of T cells is generally far less common than αβ T cells. Human γδ T cells play important roles in stress-surveillance responses such as infectious diseases and autoimmunity. It has also been suggested that tumor transformation-induced changes trigger stress-surveillance responses mediated by γδ T cells, enhancing anti-tumor immunity. Importantly, after antigen engagement, activated γδ cells at the lesion site provide cytokines (e.g., INFγ, TNFα) and / or chemokines that mediate the recruitment of other effector cells and exhibit immediate effector functions such as cytotoxicity (via the cell death receptor and cytolytic granule pathways) and ADCC.

[0058] The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cell receptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates and are hypothesized to play an early and essential role in detecting "danger" by invading pathogens, as they expand dramatically in many acute infections and can outnumber all other lymphocytes within days, for example, in tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria.

[0059] γδ T cells respond to small non-peptidic phosphorylated antigens (phosphoantigens), such as pyrophosphate synthesized within bacteria, and isopentenyl pyrophosphate (IPP) produced within mammalian cells via the mevalonate pathway. IPP production within normal cells is not sufficient for γδ T cell activation, but when the mevalonate pathway is dysregulated within tumor cells, IPP accumulates and γδ T cells are activated. IPP may also be therapeutically increased by aminobisphosphonates, thereby inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). In particular, zoledronic acid (ZA, zoledronate, Zometa™, Novartis) represents such an aminobiphosphonate, which is already clinically administered to patients for treating osteoporosis and metastatic bone disease. After PBMCs are treated in vitro, ZA is taken up particularly by monocytes. IPP accumulates within monocytes, and these monocytes differentiate into antigen-presenting cells that stimulate the generation of γδ T cells. In this setting, the addition of interleukin-2 (IL-2) is suitable as a growth and survival factor for activated γδ T cells. Finally, certain alkylated amines have been described to activate Vγ9Vδ2 T cells in vitro, but only at millimolar concentrations.

[0060] According to the present invention, the term "agent that stimulates γδ T cells" relates to a compound that stimulates the generation of γδ T cells, particularly Vγ9Vδ2 T cells, in vitro and / or in vivo, particularly by inducing the activation and expansion of γδ T cells. Preferably, this term relates to a compound that increases isopentenyl pyrophosphate (IPP) produced within mammalian cells, preferably by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS), in vitro and / or in vivo.

[0061] A specific group of compounds that stimulate γδ T cells are bisphosphonates, in particular nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

[0062] For example, bisphosphonates suitable for use in the present invention include one or more of the following compounds, including analogs, derivatives, pharmaceutically acceptable salts, hydrates, esters, conjugates, and prodrugs of the following compounds: [1-Hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, for example, zoledronate; (Dichloro-phosphono-methyl)phosphonic acid, for example, clodronate {1-Hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronic acid, for example, ibandronate (3-Amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic acid, for example, pamidronate; (1-Hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid, risedronic acid, for example, risedronate; (1-Hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, minodronic acid; [3-(Dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid; [4-Amino-1-hydroxy-1-(hydroxy-oxide-phosphoryl)-butyl]phosphonic acid, alendronic acid, for example, alendronate; [(Cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid; (1-Hydroxyethane-1,1-diyl)bis(phosphonic acid), etidronic acid, for example, etidronate; and [(4-Chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic acid.

[0063] According to the present invention, zoledronic acid (INN) or zoledronate (sold by Novartis under the trade names Zometa, Zomera, Aclasta, and Reclast) is a particularly suitable bisphosphonate. Zometa is used to prevent skeletal damage in patients with cancers such as multiple myeloma and prostate cancer, and to treat osteoporosis. It can also be used to treat hypercalcemia of malignancy and may be useful in treating pain from bone metastases.

[0064] In a particularly preferred embodiment, the agent that stimulates γδ T cells according to the present invention is administered in combination with IL-2. Such a combination has been shown to be particularly useful in mediating the expansion and activation of γ9δ2 T cells.

[0065] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule within the immune system. It is a protein that attracts lymphocytes and is part of the body's natural response to microbial infection and in distinguishing foreign (non-self) from self. IL-2 mediates its effects by binding to the IL-2 receptor expressed by lymphocytes.

[0066] The IL-2 used according to the present invention can be any IL-2 that supports or enables the stimulation of γδ T cells and can be derived from any species, preferably human. Il-2 may be isolated, recombinantly produced, or synthetic IL-2, and may be naturally occurring or modified IL-2.

[0067] The term "substance that stabilizes or increases the expression of CLDN18.2" refers to a substance or combination of substances that, when supplied to a cell, increases the RNA and / or protein level of CLDN18.2 compared to a situation where the cell is not supplied with the substance or combination of substances, and preferably increases the level of CLDN18.2 protein on the cell surface. Preferably, the cell is a cancer cell, particularly a cancer cell that expresses CLDN18.2, such as a cell of the cancer types described herein. The term "substance that stabilizes or increases the expression of CLDN18.2" particularly refers to a substance or combination of substances that, when supplied to a cell, results in a higher density of CLDN18.2 on the surface of the cell compared to a situation where the cell is not supplied with the substance or combination of substances. "Stabilizing the expression of CLDN18.2" particularly means that a substance or combination of substances prevents or reduces the decrease in the expression of CLDN18.2. For example, the expression of CLDN18.2 decreases when the substance or combination of substances is not supplied, and the decrease in the expression of CLDN18.2 is prevented or reduced when the substance or combination of substances is supplied. "Increasing the expression of CLDN18.2" particularly means that a substance or combination of substances increases the expression of CLDN18.2. For example, the expression of CLDN18.2 decreases when the substance or combination of substances is not supplied, remains essentially constant or increases, and when the substance or combination of substances is supplied, the expression of CLDN18.2 increases compared to a situation where the substance or combination of substances is not supplied, and as a result, the resulting expression is higher compared to a situation where the expression of CLDN18.2 decreases, remains essentially constant or increases when the substance or combination of substances is not supplied.

[0068] According to the present invention, the term "substance that stabilizes or increases the expression of CLDN18.2" includes chemotherapeutic agents such as cell growth inhibitors or combinations of chemotherapeutic agents. The chemotherapeutic agent can affect cells in one of the following ways: (1) damage the DNA of the cells so that the cells can no longer reproduce, (2) inhibit the synthesis of new DNA strands so that cell replication is not possible at all, (3) stop the mitotic process of the cells so that the cells cannot divide into two cells.

[0069] According to the present invention, the term "substance that stabilizes or increases the expression of CLDN18.2" preferably refers to a substance or combination of substances such as a cell growth inhibitory compound or a combination of cell growth inhibitory compounds, which, when supplied to cells, particularly cancer cells, causes the cells to stop or accumulate in one or more phases of the cell cycle, preferably in one or more phases other than the G1 and G0 phases, preferably other than the G1 phase, preferably in the G2 or S phase of the cell cycle, for example, in one or more of the G1 / G2, S / G2, G2, or S phases of the cell cycle. The term "the cells stop or accumulate in one or more phases of the cell cycle" means that the percentage of cells within the one or more phases of the cell cycle increases. Each cell passes through a cycle that includes four phases to replicate itself. The first phase, called G1, is when the cell prepares to replicate its chromosomes. The second stage is called S, and during this phase, DNA synthesis occurs and the DNA is replicated. The next phase is the G2 phase, when RNA and proteins are replicated. The final stage is the M phase, which is the stage of actual cell division. In this final stage, the replicated DNA and RNA divide and move to opposite ends of the cell, and the cell actually divides into two identical functional cells. Chemotherapeutic agents that are DNA-damaging agents usually accumulate cells in the G1 phase and / or G2 phase. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, usually accumulate cells in the S phase. Examples of these drugs are 6-mercaptopurine and 5-fluorouracil.

[0070] According to the present invention, the term "substance that stabilizes or increases the expression of CLDN18.2" includes anthracyclines such as epirubicin, platinum compounds such as oxaliplatin and cisplatin, nucleoside analogs such as 5-fluorouracil or its prodrugs, taxanes such as docetaxel, camptothecin analogs such as irinotecan and topotecan, and combinations of drugs, for example, combinations of drugs containing oxaliplatin and 5-fluorouracil, combinations of drugs containing one or more of anthracyclines such as epirubicin, oxaliplatin and 5-fluorouracil, or other combinations of drugs described herein.

[0071] In a preferred embodiment, the "substance that stabilizes or increases the expression of CLDN18.2" is a "substance that induces immunogenic cell death".

[0072] In certain situations, cancer cells can enter a lethal stress pathway linked to the release of a spatiotemporally defined combination of signals that are decoded by the immune system to activate a tumor-specific immune response (Zitvogel L. et al., (2010) Cell 140: 798-804). In such a scenario, cancer cells are induced by innate immune effectors such as dendritic cells to emit signals that trigger an adaptive immune response involving CD8+ T cells and IFN-γ signaling. As a result, tumor cell death can elicit a productive anti-cancer immune response. These signals include the pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1. Collectively, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and γ-irradiation can induce all the signals that define ICD, while cisplatin, for example, which lacks the ability to induce processes that require ER stress, such as the translocation of CRT from the ER to the surface of dying cells, requires complementation with tunicamycin, an ER stress inducer.

[0073] According to the present invention, the term "agent that induces immunogenic cell death" refers to an agent or combination of agents that can induce cells, particularly cancer cells, to enter a lethal stress pathway that ultimately leads to a tumor-specific immune response when supplied to the cells. In particular, an agent that induces immunogenic cell death when supplied to cells induces the cells to emit a spatiotemporally defined combination of signals, including the pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1.

[0074] According to the present invention, the term "agent that induces immunogenic cell death" includes anthracyclines and oxaliplatin.

[0075] Anthracyclines are a class of drugs that are also antibiotics and are commonly used in cancer chemotherapy. Structurally, all anthracyclines share a common 4-ringed 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[0076] Anthracyclines bring about one or more of the following mechanisms of action: 1. Inhibiting DNA synthesis and RNA synthesis by intercalating between the base pairs of DNA / RNA strands, thus preventing the replication of rapidly proliferating cancer cells. 2. Inhibiting the topoisomerase II enzyme and preventing the relaxation of supercoiled DNA, thus blocking DNA transcription and replication. 3. Creating iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0077] According to the present invention, the term "anthracycline" preferably refers to an agent, preferably an anticancer agent, for inducing apoptosis by inhibiting the recombination of DNA in topoisomerase II.

[0078] Preferably, according to the present invention, the term "anthracycline" generally refers to a class of compounds having the following cyclic structure,

[0079]

Chemical formula

[0080] including these analogs, and derivatives, pharmaceutical salts, hydrates, esters, conjugates, and prodrugs.

[0081] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pirarubicin, valrubicin, N-trifluoro-acetyl doxorubicin-14-valerate, aclacinomycin, morpholino doxorubicin (morpholino-DOX), cyano morpholino-doxorubicin (cyano morpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and aclacinomycin A (aclacinomycin). Mitoxantrone is a member of the anthracenedione class of compounds that lacks the sugar moiety of anthracyclines but retains a planar polycyclic aromatic ring structure that permits intercalation into DNA.

[0082] Particularly preferred as the anthracyline according to the present invention is a compound of the following formula:

[0083]

Chemical formula

[0084] In the formula, R1 is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and OMe, R3 is selected from the group consisting of H and OH, and R4 is selected from the group consisting of H and OH.

[0085] In one embodiment, R1 is H, R2 is OMe, R3 is H, and R4 is OH. In another embodiment, R1 is OH, R2 is OMe, R3 is H, and R4 is OH. In another embodiment, R1 is OH, R2 is OMe, R3 is OH, and R4 is H. In another embodiment, R1 is H, R2 is H, R3 is H, and R4 is OH.

[0086] Particularly contemplated as an anthracycline in connection with the present invention is epirubicin. Epirubicin is an anthracycline drug having the following formula:

[0087] [Chemical formula]

[0088] It is sold in the United States under the trade name Ellence, and elsewhere under Pharmorubicin or Epirubicin Ebewe. In particular, the term "epirubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. Epirubicin is preferred over doxorubicin, the most prevalent anthracycline in some chemotherapy regimens, as it appears to cause fewer side effects.

[0089] According to the present invention, the term "platinum compound" refers to compounds containing platinum in their structure, such as platinum complexes, and includes compounds such as cisplatin, carboplatin, and oxaliplatin.

[0090] The term "cisplatin" or "cisplatinum" refers to the compound cis-diamminedichloroplatinum(II) (CDDP) of the following formula:

[0091] [Chemical formula]

[0092] The term "carboplatin" refers to the compound cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) of the following formula:

[0093] [Chemical formula]

[0094] The term "oxaliplatin" refers to a compound that is a platinum compound complexed to a diaminocyclohexane carrier ligand of the following formula:

[0095]

Chem.

[0096] In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II). Oxaliplatin for injection is also sold under the trade name Eloxatine.

[0097] The term "nucleoside analog" refers to a category that includes structural analogs of nucleosides, i.e., both purine analogs and pyrimidine analogs. In particular, the term "nucleoside analog" refers to fluoropyrimidine derivatives that include fluorouracil and its prodrugs.

[0098] The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the brand names Adrucil, Carac, Efudix, Efudex, and Fluoroplex) is a compound that is a pyrimidine analog of the following formula:

[0099]

Chem.

[0100] In particular, this term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0101] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted to 5-FU in tissue. Capecitabine, which can be administered orally, has the following formula:

[0102] [Chem.]

[0103] In particular, this term refers to the compound pentyl [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

[0104] Taxanes are a class of diterpene compounds first derived from natural sources such as plants of the genus Taxus, although some are synthetically produced. The main mechanism of action of the taxane class of drugs is disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0105] According to the present invention, the term "docetaxel" refers to a compound having the following formula:

[0106] [Chem.]

[0107] According to the present invention, the term "paclitaxel" refers to a compound having the following formula:

[0108] [Chem.]

[0109] According to the present invention, the term "camptothecin analog" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog" refers to a compound having the following structure:

[0110] [Chem.]

[0111] According to the present invention, suitable camptothecin analogs are inhibitors of DNA enzyme topoisomerase I (topo I). Suitable camptothecin analogs according to the present invention are irinotecan and topotecan.

[0112] Irinotecan is a drug that prevents DNA from unwinding by inhibiting topoisomerase I. Chemically, it is a semi-synthetic analog of the natural alkaloid camptothecin having the following formula:

[0113]

Chemical formula

[0114] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0115] Topotecan is a topoisomerase inhibitor of the formula:

[0116]

Chemical formula

[0117] ...

[0118] In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0119] According to the present invention, the substance that stabilizes or increases the expression of CLDN18.2 can be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, and can be a part of a combination of drugs such as a combination of drugs established for use in cancer treatment. Such a combination of drugs can be a combination of drugs used in chemotherapy, and can be a combination of drugs used in a chemotherapeutic agent regimen selected from the group consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCF chemotherapy, and FLOT chemotherapy.

[0120] The combination of drugs used in EOX chemotherapy is composed of epirubicin, oxaliplatin, and capecitabine. The combination of drugs used in ECF chemotherapy is composed of epirubicin, cisplatin, and 5-fluorouracil. The combination of drugs used in ECX chemotherapy is composed of epirubicin, cisplatin, and capecitabine. The combination of drugs used in EOF chemotherapy is composed of epirubicin, oxaliplatin, and 5-fluorouracil.

[0121] Over a total of 8 three-week cycles, epirubicin is usually at a dose of 50 mg / m 2 cisplatin is at 60 mg / m 2 oxaliplatin is at 130 mg / m 2 the continuous intravenous infusion of 5-fluorouracil is at 200 mg / m 2 / day, and oral capecitabine is at 625 mg / m 2 administered twice daily.

[0122] The combination of drugs used in FLO chemotherapy is composed of 5-fluorouracil, folinic acid, and oxaliplatin (usually 5-fluorouracil 2,600 mg / m 2 24-hour infusion, folinic acid 200 mg / m 2 and oxaliplatin 85 mg / m 2 , every two weeks).

[0123] FOLFOX is a chemotherapy regimen composed of folic acid (leucovorin), 5-fluorouracil, and oxaliplatin. The recommended dosing schedule administered every two weeks is as follows: Day 1: Oxaliplatin 85 mg / m 2 IV infusion and leucovorin 200 mg / m 2 IV infusion, followed by 5-FU 400 mg / m 2 IV bolus injection, followed by 5-FU 600 mg / m as a 22-hour continuous infusion 2 IV infusion; Day 2: Leucovorin 200 mg / m 2 IV infusion over 120 minutes, followed by 5-FU 400 mg / m administered over 2 - 4 minutes 2 IV bolus injection, followed by 5-FU 600 mg / m as a 22-hour continuous infusion 2 IV infusion.

[0124] The drug combination used in FOLFIRI chemotherapy consists of 5-fluorouracil, leucovorin, and irinotecan.

[0125] The drug combination used in DCF chemotherapy consists of docetaxel, cisplatin, and 5-fluorouracil.

[0126] The drug combination used in FLOT chemotherapy consists of docetaxel, oxaliplatin, 5-fluorouracil, and folic acid.

[0127] The term "folic acid" or "leucovorin" refers to a compound useful in a synergistic combination with the chemotherapeutic agent 5-fluorouracil. Folic acid has the following formula:

[0128]

Chemical formula

[0129] In particular, this term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}pentanedioic acid.

[0130] In one embodiment, for administering an agent that stabilizes or increases the expression of CLDN18.2, a standard chemotherapy with an EOX regimen, in particular in combination with IMAB362, an antibody having the ability to bind to CLDN18.2, is administered over a maximum of 8 cycles. The dosage and schedule can be as follows: · On day 1 of each cycle during the EOX phase, epirubicin at 50 mg / m 2 is administered i.v. as a 15-minute infusion. · On day 1 of each cycle during the EOX phase, oxaliplatin at 130 mg / m 2 is administered i.v. as a 2-hour infusion. · Starting in the evening of day 1 of each cycle during the EOX phase, capecitabine at 625 mg / m 2 is taken p.o. twice daily for 21 days, in the morning and evening. · On day 1 of cycle 1, the antibody at 1000 mg / m 2 is administered i.v. as a 2-hour infusion. Thereafter, after the infusion of oxaliplatin is completed, on day 1 of each cycle, the antibody at 600 mg / m 2 is administered i.v. as a 2-hour infusion. · After chemotherapy, the patient continues with the antibody at 600 mg / m 2 as a 2-hour infusion every 3 or 4 weeks.

[0131] In one embodiment of the present invention, a standard chemotherapy with an EOX regimen, in combination with ZA / IL-2 and an antibody having the ability to bind to CLDN18.2, in particular IMAB362, is administered over a maximum of 8 cycles (24 weeks).

[0132] The term "antigen" relates to an agent such as a protein or peptide that contains an epitope to which an immune response is directed and / or can be directed. In a preferred embodiment, the antigen is a tumor-associated antigen such as CLDN18.2, i.e., a component of cancer cells that can be derived from the cytoplasm, cell surface, and cell nucleus, particularly an antigen that is preferably produced in large quantities intracellularly or as a surface antigen of cancer cells.

[0133] In the context of the present invention, the term "tumor-associated antigen" preferably relates to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or at a specific stage of development and is expressed or abnormally expressed in one or more tumor or cancer tissues. In the context of the present invention, the tumor-associated antigen preferably relates to the cell surface of cancer cells and is preferably not expressed or is only rarely expressed in normal tissues.

[0134] The term "epitope" refers to an antigenic determinant within a molecule, i.e., a part of a molecule that is recognized by the immune system, e.g., recognized by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features and specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former rather than the latter is lost in the presence of a denaturing solvent. The epitopes of a protein such as CLDN18.2 preferably include a continuous or discontinuous portion of the protein and preferably have an amino acid length between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, and most preferably between 10 and 25. For example, the epitope can preferably have a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0135] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule containing its antigen-binding portion. The term "antibody" includes, without limitation, monoclonal antibodies, as well as antibody fragments or derivatives, including human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies such as scFv, and antigen-binding antibody fragments such as Fab fragments and Fab' fragments, and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain is composed of a heavy-chain variable region (abbreviated as VH herein) and a heavy-chain constant region. Each light chain is composed of a light-chain variable region (abbreviated as VL herein) and a light-chain constant region. The VH region and the VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells), and the first component of the classical complement system (Clq).

[0136] The antibodies described herein can be human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0137] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can include a complete variable domain fused onto a constant domain, or can include only the complementarity-determining regions (CDRs) grafted onto an appropriate framework region in the variable domain. The antigen-binding site can be wild-type or modified by one or more amino acid substitutions, for example, modified to be more closely similar to a human immunoglobulin. Some forms of humanized antibodies conserve all of the CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more CDRs that are changed relative to the original antibody.

[0138] The term "chimeric antibody" refers to an antibody in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to the corresponding sequences in another. Generally, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one species of mammal, while the constant portions are homologous to the sequences of an antibody derived from another species. One obvious advantage of such chimeric forms is that the variable regions can be derived from readily available sources, conveniently using, for example, B cells or hybridomas from a non-human host organism, in combination with constant regions, advantageously, from a human cell specimen. The variable regions have the advantage of ease of preparation and the specificity is not affected by the source, while the constant regions, being human, are less likely to induce an immune response in a human subject when the antibody is injected than are constant regions derived from non-human sources. However, the definition is not limited to this particular example.

[0139] The terms "antigen-binding portion" (or simply "binding portion") of an antibody, or "antigen-binding fragment" (or simply "binding fragment") of an antibody, or similar terms, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody are: (i) a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) an F(ab')2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature, 341: 544-546); (vi) an isolated complementarity-determining region (CDR), and (vii) a combination of two or more isolated CDRs that can optionally be joined by a synthetic linker. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but these can be joined using recombinant methods by a synthetic linker that enables the VL and VH regions to pair to form a monovalent molecule as a single polypeptide chain (known as a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science, 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. Further examples are binding domain immunoglobulin fusion proteins comprising: (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to a hinge region; and (iii) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region.The conjugative domain immunoglobulin fusion proteins are further disclosed in US2003 / 0118592 and US2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0140] The term "bispecific molecule" is intended to include any agent having two different binding specificities, such as a protein, peptide, or protein complex or peptide complex. For example, this molecule can bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent having more than two different binding specificities, such as a protein, peptide, or protein complex or peptide complex. For example, this molecule can bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Thus, the present invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules directed to, inter alia, CLDN18.2 and other targets such as Fc receptors on effector cells. The term "bispecific antibody" includes diabodies. Diabodies are expressed using a linker where the VH and VL domains are on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thereby forcing these domains to pair with complementary domains on another chain to create two antigen-binding sites, a bivalent bispecific antibody (see, for example, Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448; Poljak, R. J. et al., (1994) Structure, 2: 1121-1123).

[0141] Antibodies can be conjugated to a therapeutic moiety or agent such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. Cytotoxins or cytotoxic agents include any agent that is harmful to cells, particularly those that kill cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Suitable therapeutic agents for forming antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, 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 mitotic inhibitors (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0142] Antibodies can also be conjugated to radioisotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals.

[0143] The antibody conjugates of the present invention can be used to modify a given biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins include, for example, enzymatically active toxins, or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors.

[0144] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

[0145] As used herein, an antibody is obtained from one source by immunizing an animal or by screening an immunoglobulin gene library, and an antibody is "derived" from a particular germline sequence if the selected antibody has an amino acid sequence that is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, an antibody derived from a particular germline sequence will exhibit 10 or fewer amino acid differences, more preferably 5 or fewer, or even more preferably 4, 3, 2, or 1 or fewer amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0146] As used herein, the term "heteroantibody" refers to a preparation of two or more antibodies, their derivatives, or antigen-binding regions linked together, wherein at least two of these have different specificities. These different specificities include binding specificities for Fc receptors on effector cells and for antigens or epitopes on target cells, such as tumor cells.

[0147] The antibodies described herein can be monoclonal antibodies. The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising B cells obtained from a non-human animal, such as a mouse, fused to immortalized cells.

[0148] The antibodies described herein can be recombinant antibodies. As used herein, the term "recombinant antibody" refers to any antibody prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to an immunoglobulin gene or hybridoma prepared from itself, (b) antibodies isolated from a host cell transformed to express an antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies prepared, expressed, created, or isolated by any other means that splices an immunoglobulin gene sequence to another DNA sequence, and the like.

[0149] The antibodies described herein can be derived from a variety of species including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0150] The antibodies described herein include polyclonal antibodies and monoclonal antibodies, and antibodies of IgA, e.g., IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD. In various embodiments, the antibody is an IgG1 antibody, more specifically, an IgG1, kappa, or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

[0151] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express an antibody, such as fungi including CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or yeast cells.

[0152] As used herein, "heterologous antibody" is defined with respect to a transgenic organism that produces such an antibody. This term refers to an antibody having an amino acid sequence or a coding nucleic acid sequence corresponding to that found in an organism that is not composed of the transgenic organism, and generally an antibody derived from a species other than the transgenic organism.

[0153] As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain associated with a mouse light chain is a heterohybrid antibody.

[0154] For the purposes of the present invention, the term "antibody" encompasses all antibodies and antibody derivatives described herein. The term "antibody derivative" refers to any modified form of an antibody, such as an antibody conjugated with another agent or conjugated with an antibody, or an antibody fragment.

[0155] The antibodies described herein are preferably isolated. "Isolated antibody" is intended to refer, as used herein, to an antibody that substantially does not contain other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 substantially does not contain an antibody that specifically binds to an antigen other than CLDN18.2). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may cross-react with, for example, other related antigens from other species (e.g., CLDN18.2 homologs). Further, an isolated antibody may substantially not contain other cellular substances and / or chemical substances. In one embodiment of the present invention, a combination of "isolated" monoclonal antibodies relates to antibodies having different specificities and combined in a particular composition or mixture.

[0156] The term "binding" according to the present invention preferably relates to specific binding.

[0157] According to the present invention, an antibody can bind to a predetermined target if it has a significant affinity for the predetermined target and binds to the predetermined target in a standard assay. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant (K D ). Preferably, the term "significant affinity" refers to binding to a predetermined target with a dissociation constant (K D ) of 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less.

[0158] An antibody cannot (substantially) bind to a target if it does not have a significant affinity for the target and does not bind significantly to the target in a standard assay, particularly not to a detectable extent. Preferably, the antibody does not bind to the target to a detectable extent when present at a concentration of up to 2, preferably 10, more preferably 20, particularly 50, or 100 μg / ml or more. Preferably, an antibody has no significant affinity for a target if it binds to a single target with a K D that is at least 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, or 10 6 -fold higher K D for binding to a single target. For example, if the K D for an antibody's binding to a target that the antibody can bind is 10 -7 M, the K D for the antibody's binding to a single target with no significant affinity is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 M.

[0159] An antibody is specific for a given target if it can bind to the given target while being unable to bind to other targets, i.e., having no significant affinity for other targets and not significantly binding to other targets in a standard assay. According to the present invention, an antibody is specific for CLDN18.2 if it can bind to CLDN18.2 but is unable to (substantially) bind to other targets. Preferably, if the affinity and binding to such other targets do not significantly exceed the affinity or binding to a CLDN18.2-unrelated protein, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or a non-claudin transmembrane protein such as an MHC molecule or a transferrin receptor, or any other designated polypeptide, the antibody is specific for CLDN18.2. Preferably, the antibody has a K D that is at least one-tenth, one-hundredth, one 3 -thousandth, one 4 -ten-thousandth, one 5 -hundred-thousandth, or one 6 -millionth lower than the K D for binding to the given target, and is specific for the given target. For example, if the K D for the binding of the antibody to the target for which it is specific is 10 -7 M, the K D for its binding to a non-specific target should be at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 M.

[0160] The binding of an antibody to its target can be determined experimentally using any suitable method, for example, see Berzofsky et al., "Antibody-Antigen Interactions", Fundamental Immunology, Paul, W.E. ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W.H. Freeman and Company New York, N.Y. (1992), and the methods described herein. Affinity can be determined by equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to those of skill in the art, etc., using conventional techniques. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary when measured under different conditions, such as salt concentration, pH. Thus, affinity as well as other antigen-binding parameters, such as K D i 50 is preferably measured using a standardized solution of the antibody and antigen, as well as a standardized buffer.

[0161] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0162] As used herein, "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0163] As used herein, the term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by a person in the laboratory is naturally occurring.

[0164] As used herein, the term "rearranged" when referring to an immunoglobulin locus of a heavy or light chain refers to a configuration in which a V segment is positioned directly adjacent to a D-J segment or a J segment within a conformation that essentially encodes a complete VH domain or VL domain, respectively. A rearranged immunoglobulin (antibody) locus can be identified by comparison to germline DNA, and a rearranged locus will have at least one recombined heptamer / nonamer homology element.

[0165] As used herein, the term "unrearranged" or "germline configuration" when referring to a V segment refers to a configuration in which the V segment has not been rearranged such that it is directly adjacent to a D segment or a J segment.

[0166] According to the present invention, an antibody having the ability to bind to CLDN18.2 is an antibody that can bind to an epitope present in CLDN18.2, preferably the extracellular domain of CLDN18.2, particularly the first extracellular domain, preferably an epitope located within amino acid positions 29 to 78 of CLDN18.2. In certain embodiments, an antibody having the ability to bind to CLDN18.2 is an antibody that can bind to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NOs: 3, 4, and 5, (ii) an epitope localized on CLDN18.2-loop1, preferably SEQ ID NO: 8, (iii) an epitope localized on CLDN18.2-loop2, preferably SEQ ID NO: 10, (iv) an epitope localized on CLDN18.2-loop D3, preferably SEQ ID NO: 11, (v) an epitope encompassing CLDN18.2-loop1 and CLDN18.2-loop D3, or (vi) a non-glycosylated epitope localized on CLDN18.2-loop D3, preferably SEQ ID NO: 9.

[0167] According to the present invention, an antibody having the ability to bind to CLDN18.2 is preferably an antibody having the ability to bind to CLDN18.2 but not having the ability to bind to CLDN18.1. Preferably, an antibody having the ability to bind to CLDN18.2 is specific for CLDN18.2. Preferably, an antibody having the ability to bind to CLDN18.2 is preferably an antibody having the ability to bind to CLDN18.2 expressed on the cell surface. In certain preferred embodiments, an antibody having the ability to bind to CLDN18.2 binds to the native epitope of CLDN18.2 present on the surface of live cells. Preferably, an antibody having the ability to bind to CLDN18.2 binds to one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, an antibody having the ability to bind to CLDN18.2 is specific for the above-mentioned protein, peptide, or immunogenic fragment or derivative thereof. An antibody having the ability to bind to CLDN18.2 can be obtained by a method comprising the step of immunizing an animal with a protein or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50, or a nucleic acid or host cell expressing said protein or peptide. Preferably, the antibody binds to cancer cells, particularly cells of the cancer types described above, and preferably does not bind to non-cancerous cells.

[0168] Preferably, when an antibody having the ability to bind to CLDN18.2 binds to cells expressing CLDN18.2, killing of the cells expressing CLDN18.2 is induced or mediated. The cells expressing CLDN18.2 are preferably cancer cells, and in particular are selected from the group consisting of tumor-forming gastric, esophageal, pancreatic, lung, ovarian, colorectal, liver, head and neck, and gallbladder cancer cells. Preferably, the antibody induces or mediates cell killing by inducing one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cell-mediated cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation of cells expressing CLDN18.2. Preferably, ADCC-mediated lysis of cells occurs in the presence of effector cells, which in certain embodiments are selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs. Inhibition of cell proliferation can be measured in vitro by determining cell proliferation in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is an analog of thymidine and is a synthetic nucleoside that can be incorporated into newly synthesized DNA of replicating cells (during the S phase of the cell cycle) and replaces thymidine during DNA replication. For example, detection of the incorporated chemical using an antibody specific for BrdU indicates cells that were actively replicating their own DNA.

[0169] In preferred embodiments, the antibodies described herein can be characterized by one or more of the following properties: a) Specificity for CLDN18.2; b) Binding affinity for CLDN18.2 of about 100 nM or less, preferably about 5 - 10 nM or less, more preferably about 1 - 3 nM or less; c) Ability to induce or mediate CDC on CLDN18.2-positive cells; d) Ability to induce or mediate ADCC on CLDN18.2-positive cells; e) Ability to inhibit the proliferation of CLDN18.2-positive cells; f) Ability to induce apoptosis of CLDN18.2-positive cells.

[0170] In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 is deposited with DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and is generated by hybridomas having the following names and accession numbers: a.182-D1106-055, accession number DSM ACC2737, deposited on October 19, 2005 b.182-D1106-056, accession number DSM ACC2738, deposited on October 19, 2005 c.182-D1106-057, accession number DSM ACC2739, deposited on October 19, 2005 d.182-D1106-058, accession number DSM ACC2740, deposited on October 19, 2005 e.182-D1106-059, accession number DSM ACC2741, deposited on October 19, 2005 f.182-D1106-062, accession number DSM ACC2742, deposited on October 19, 2005, g.182-D1106-067, accession number DSM ACC2743, deposited on October 19, 2005 h.182-D758-035, accession number DSM ACC2745, deposited on November 17, 2005 i.182-D758-036, accession number DSM ACC2746, deposited on November 17, 2005 j.182-D758-040, accession number DSM ACC2747, deposited on November 17, 2005 k.182-D1106-061, accession number DSM ACC2748, deposited on November 17, 2005 l.182-D1106-279, accession number DSM ACC2808, deposited on October 26, 2006 m.182-D1106-294, accession number DSM ACC2809, deposited on October 26, 2006, n. 182-D1106-362, Deposit No. DSM ACC2810, deposited on October 26, 2006.

[0171] Preferred antibodies according to the present invention are those produced by the above-described hybridomas, i.e., 37G11 in the case of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in the case of 182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in the case of 182-D1106-067, 26B5 in the case of 182-D758-035, 26D12 in the case of 182-D758-036, 28D10 in the case of 182-D758-040, 42E12 in the case of 182-D1106-061, 125E1 in the case of 182-D1106-279, 163E12 in the case of 182-D1106-294, and 175D10 in the case of 182-D1106-362; and those obtained therefrom, as well as their chimeric and humanized forms.

[0172] Preferred chimeric antibodies and their sequences are shown in the following table.

[0173] [Table 1]

[0174] In a preferred embodiment, the antibody, particularly the chimerized form of the antibody according to the present invention, includes a heavy chain constant region (CH) that includes an amino acid sequence derived from a human heavy chain constant region such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof. In a further preferred embodiment, the antibody, particularly the chimerized form of the antibody according to the present invention, includes a light chain constant region (CL) that includes an amino acid sequence derived from a human light chain constant region such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In a specific preferred embodiment, the antibody, particularly the chimerized form of the antibody according to the present invention, includes a CH that includes an amino acid sequence derived from a human CH such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof, and a CL that includes an amino acid sequence derived from a human CL such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.

[0175] In one embodiment, the antibody having the ability to bind to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody that includes a kappa mouse variable light chain, a human kappa light chain constant region allotype Km(3), a mouse heavy chain variable region, and a human IgG1 constant region allotype G1m(3).

[0176] In certain preferred embodiments, the chimerized form of the antibody includes a heavy chain that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, and fragments thereof, and / or a light chain that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, and fragments thereof.

[0177] In certain preferred embodiments, the chimerized form of the antibody includes an antibody that includes a combination of a heavy chain and a light chain selected from the following possibilities (i) to (ix): (i) a heavy chain that includes the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, and a light chain that includes the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof, (ii) a heavy chain that includes the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, and a light chain that includes the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof, (iii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof, (iv) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof, (v) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof, (vi) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof, (vii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof, (viii) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof, and (ix) a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 28 or a fragment thereof.

[0178] The "fragment" or "fragment of an amino acid sequence" used above represents a part of the antibody sequence, i.e., an antibody sequence shortened at the N-terminus and / or C-terminus, and when it replaces the antibody sequence in the antibody, it relates to the sequence that retains the binding ability of the antibody to CLDN18.2, and preferably, the function of the antibody described herein, such as CDC-mediated lysis or ADCC-mediated lysis. Preferably, the fragment of the amino acid sequence contains at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues derived from the amino acid sequence. The fragment of the amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 preferably relates to the sequence in which the N-terminal 17, 18, 19, 20, 21, 22, or 23 amino acids are removed.

[0179] In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a heavy chain variable region (VH) selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 29, 30, 31, 32, 33, 34, and fragments thereof.

[0180] In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a light chain variable region (VL) selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, and fragments thereof.

[0181] In certain preferred embodiments, an antibody having the ability to bind to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i)-(ix): (i) VH comprising the amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof, and VL comprising the amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof, (ii) VH comprising the amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, and VL comprising the amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, (iii) A VH comprising the amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof, (iv) A VH comprising the amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof, (v) A VH comprising the amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, (vi) A VH comprising the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof, (vii) A VH comprising the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof, (viii) A VH comprising the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof, (ix) A VH comprising the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and a VL comprising the amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

[0182] In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a VH comprising a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (vi): (i) CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, (ii) CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, (iii) CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, (iv) CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, (v) CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, and (vi) CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19.

[0183] In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a VL comprising a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (ix): (i) CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (ii) CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (iii) CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (v) CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0184] In a preferred embodiment, an antibody having the ability to bind to CLDN18.2 comprises a combination of VH and VL each containing a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) to (ix): (i) VH: CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, VL: CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (ii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, VL: CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (iii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, VL: CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, VL: CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, VL: CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (vii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0185] In a further preferred embodiment, an antibody having the ability to bind to CLDN18.2 preferably comprises one or more of the complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy-chain variable region (VH) and / or the light-chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, and preferably comprises one or more of the complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy-chain variable region (VH) and / or the light-chain variable region (VL) described herein. In one embodiment, said one or more of the complementarity-determining regions (CDRs) are selected from the set of complementarity-determining regions CDR1, CDR2, and CDR3 described herein. In a particularly preferred embodiment, an antibody having the ability to bind to CLDN18.2 preferably comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy-chain variable region (VH) and / or the light-chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, and preferably comprises the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy-chain variable region (VH) and / or the light-chain variable region (VL) described herein.

[0186] In one embodiment, an antibody comprising one or more of the CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs together with these intervening framework regions. Preferably, this portion comprises at least about 50% of either or both of the first and fourth framework regions, and this 50% is the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. When constructing an antibody produced by recombinant DNA techniques, cloning or other manipulation steps are facilitated, including the introduction of linkers that join the variable regions of the invention to immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or additional protein sequences including protein labels, and residues N-terminal or C-terminal can be introduced into the variable regions encoded by the linkers introduced.

[0187] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs described herein comprises said CDRs within a human antibody framework.

[0188] References herein to an antibody that includes a particular chain, or particular region or sequence, with respect to its own heavy chain preferably relate to circumstances in which all of the heavy chains of said antibody include said particular chain, region, or sequence. This applies correspondingly to the light chains of the antibody.

[0189] The term “nucleic acid” as used herein is intended to include DNA and RNA. The nucleic acid may be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0190] According to the present invention, the term “expression” is used in its most general sense and includes the production of RNA, or RNA and protein / peptide. This includes partial expression of the nucleic acid. Further, expression may be carried out transiently or stably.

[0191] Instructions provided herein with respect to a specific amino acid sequence, such as those shown in the Sequence Listing, should be interpreted to also relate to variants of said specific sequence that are functionally equivalent to said specific sequence, such as amino acid sequences that exhibit the same or similar properties as the properties of the specific amino acid sequence. One important property is retaining the ability of the antibody to bind to its target, or maintaining the effector function of the antibody. Preferably, a sequence that is a variant with respect to the specific sequence, when it replaces the specific sequence in the antibody, retains the ability of the antibody to bind to CLDN18.2, and preferably, the functions of said antibody described herein, such as CDC-mediated lysis or ADCC-mediated lysis.

[0192] In particular, it is understood by those skilled in the art that the sequences of the CDRs, hypervariable regions, and variable regions can be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions are identical or highly homologous to the regions of the antibodies specified herein. By "highly homologous," it is contemplated that substitutions such as 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2, etc., can be made within the CDRs. Further, the hypervariable regions and variable regions can be modified to exhibit substantial homology to the regions of the antibodies specifically disclosed herein.

[0193] For the purposes of the present invention, a "variant" of an amino acid sequence includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of a protein are also referred to as N-terminal and / or C-terminal truncation variants.

[0194] Amino acid insertion variants include the insertion of single or two or more amino acids into a particular amino acid sequence. In the case of an amino acid sequence variant having an insertion, one or more amino acid residues are inserted within a particular site in the amino acid sequence, although it is also possible to insert them randomly using appropriate screening of the resulting products.

[0195] Amino acid addition variants include the addition of one or more amino acids, such as amino and / or carboxy terminal fusions of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0196] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, by the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be within any position of the protein.

[0197] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications within positions in the amino acid sequence that are not conserved between homologous proteins or peptides, and / or the replacement of an amino acid with another amino acid having similar properties are preferred. Preferably, the amino acid changes in the protein variant are conservative amino acid changes, i.e., substitutions of amino acids that are similarly charged or uncharged. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in terms of their side chains. Naturally occurring amino acids are generally classified into four families, namely acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids.

[0198] The degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably contiguous amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, for example, using Align with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0199] "Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0200] The term "percentage identity" is intended to represent the percentage of amino acid residues that are identical between two sequences being compared, after the best alignment has been obtained, and this percentage is purely statistical and the differences between the two sequences are distributed randomly and over their entire lengths. Sequence comparison between two amino acid sequences is customarily carried out by comparing these sequences after they have been optimally aligned, and said comparison is carried out by segments or by a "window of comparison" in order to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be generated by, in addition to by hand, the local homology algorithms of Smith and Waterman, 1981, Ads App. Math., 2, 482, the local homology algorithms of Neddleman and Wunsch, 1970, J. Mol. Biol., 48, 443, the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0201] Percentage identity is calculated by determining the number of positions that are identical between two sequences being compared, dividing this number by the number of positions compared, and multiplying the resulting quotient by 100, thereby obtaining the percentage identity between these two sequences.

[0202] The term "transgenic animal" refers to an animal having a genome that contains one or more introduced genes, preferably a heavy chain introduced gene and / or a light chain introduced gene, or a transchromosome (either integrated or not integrated into the animal's native genomic DNA), and preferably capable of expressing the introduced gene. For example, a transgenic mouse can have a human light chain introduced gene, and a human heavy chain introduced gene or a human heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with the CLDN18.2 antigen and / or cells expressing CLDN18.2. The human heavy chain introduced gene can be integrated into the chromosomal DNA of a transgenic mouse, such as in the case of a HuMAb mouse, such as an HCo7 mouse or an HCol2 mouse, or the human heavy chain introduced gene can be maintained extrachromosomally as in the case of a transchromosomal (e.g., KM) mouse described in WO02 / 43478. Such transgenic mice and transchromosomal mice can produce multiple isotypes (e.g., IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN18.2 by undergoing V-D-J recombination and isotype switching.

[0203] "Reduce", "decrease", or "inhibit", as used herein, means an overall decrease of at least, for example, at least 5%, at least 10%, at least 20%, more preferably at least 50%, most preferably at least 75% in the level, e.g., the expression level, or the level of cell proliferation, or the ability to cause such an overall decrease.

[0204] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more.

[0205] Mechanism of mAb action The following provides considerations regarding the mechanisms underlying the therapeutic efficacy of the antibodies of the invention, but should in no way be construed as limiting the invention.

[0206] The antibodies described herein preferably interact with components of the immune system, preferably by ADCC or CDC. The antibodies described herein can also be used to target a payload (e.g., a radioisotope, a drug, or a toxin) to kill tumor cells directly, or used synergistically with conventional chemotherapeutic agents to attack tumors by complementary mechanisms of action that can include an anti-tumor immune response that may have been impaired due to the cytotoxic side effects of chemotherapeutic agents on T lymphocytes. However, the antibodies described herein can also simply bind to CLDN18.2 on the cell surface and thus exert an effect, for example, by blocking cell proliferation.

[0207] Antibody-dependent cell-mediated cytotoxicity ADCC describes the cell killing ability of effector cells, particularly lymphocytes, described herein that preferably require target cells marked by an antibody.

[0208] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain engages with an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism that directly induces varying degrees of immediate tumor destruction that present antigen and induce a tumor-directed T-cell response. Preferably, induction of ADCC in vivo will lead to a tumor-directed T-cell response and a host-derived antibody response.

[0209] Complement-dependent cytotoxicity CDC is another method of cell killing that can be directed by an antibody. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are highly effective at directing CDC via the classical complement activation pathway. Preferably, in this cascade, formation of the antigen-antibody complex results in the uncloaking of multiple Clq-binding sites that are in close proximity on the C H 2 domain of the participating antibody molecule such as an IgG molecule (C1q is one of three small components of complement C1). Preferably, these uncloaked C1q-binding sites convert the previously low-affinity C1q-IgG interaction into one of high binding activity, which induces a cascade of events involving a series of other complement proteins and results in the proteolytic release of the effector chemotactic / activating agents C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, thereby creating pores in the cell membrane that facilitate the free passage of water and solutes in and out of the cell.

[0210] The antibodies described herein can be generated by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature, 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for generating monoclonal antibodies, such as viral transformation or carcinogenesis of B-lymphocytes, or phage display techniques using antibody gene libraries, can also be used.

[0211] A preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma generation in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. The fusion partner (e.g., mouse myeloma cells), and the fusion procedure are also known.

[0212] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat system and the rabbit system (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A., 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol., 124: 295 (2005)).

[0213] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic mice or transchromosomal mice that carry a part of the human immune system instead of the mouse system. These transgenic mice and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice". The production of human antibodies in such transgenic mice can be carried out as described in detail for CD20 in WO2004 035607.

[0214] Yet another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from lymphocytes that produce antibodies of defined specificity, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details on recombinant antibody manipulation, see also Welschof and Kraus, Recombinant antibodes for cancer therapy, ISBN-0-89603-918-8, and Benny K.C. Lo, Antibody Engineering, ISBN 1-58829-092-1.

[0215] To generate antibodies, as described, mice can be immunized with a carrier-conjugated peptide derived from the antigen sequence, i.e., the sequence to which the antibody is directed, a recombinant expressed antigen or a fragment thereof in a concentrated preparation, and / or cells expressing the antigen. Alternatively, mice can be immunized with DNA encoding the antigen or a fragment thereof. If immunization with a purified or concentrated preparation of the antigen does not result in antibodies, mice can also be immunized with cells expressing the antigen, e.g., cell lines, to enhance the immune response.

[0216] The immune response can be monitored throughout the immunization protocol using plasma and serum samples obtained by tail vein bleeding or retro-orbital bleeding. Mice with sufficient titers of immunoglobulins can be used for fusion. To increase the proportion of specific antibody-secreting hybridomas, mice can be boostered intraperitoneally or intravenously with antigen-expressing cells 3 days prior to sacrifice and spleen removal.

[0217] To generate hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line such as a mouse myeloma cell line. The resulting hybridomas can then be screened to produce antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies specific for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. The antibody-secreting hybridomas can be replated and screened again, and if still positive for the monoclonal antibody, can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to produce and characterize the antibody in tissue culture medium.

[0218] Antibodies can also be generated in host cell transfectomas, as is well known in the art, for example, using a combination of recombinant DNA techniques and gene transfection methods (Morrison, S. (1985) Science, 229: 1202).

[0219] For example, in one embodiment, the target gene(s), e.g., an antibody gene, can be ligated into an expression vector such as a eukaryotic expression plasmid used by the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338841, or other expression systems well-known in the art. A purified plasmid containing the cloned antibody gene can be introduced into a eukaryotic host cell, e.g., CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, etc., or alternatively other eukaryotic cell-like plant-derived cells, fungal cells or yeast cells. The methods used to introduce these genes can be those described in the art, e.g., electroporation, lipofectine, lipofectamine, etc. After introducing these antibody genes into the host cell, cells expressing the antibody can be identified and selected. These cells represent transfectomas and can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0220] Alternatively, the cloned antibody gene can be expressed in other expression systems including prokaryotic cells such as Escherichia coli (E. coli). Furthermore, antibodies can be produced in transgenic non-human animals, e.g., in milk from sheep and rabbits, in eggs from hens, or in transgenic plants, etc. See, for example, Verma, R. et al., (1998) J. Immunol. Meth., 216: 165-181; Pollock et al., (1999) J. Immunol. Meth., 231: 147-157; and Fischer, R. et al., (1999) Biol. Chem., 380: 825-839.

[0221] Chimerization Mouse monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with toxins or radioisotopes. Unlabeled mouse antibodies are highly immunogenic in humans when repeatedly applied and reduce the therapeutic effect. The major immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided when each antibody is chimerized or humanized. Chimeric antibodies are antibodies in which different parts are derived from different animal species, for example, those having a variable region derived from a mouse antibody and a human immunoglobulin constant region. Chimerization of an antibody is achieved by joining the variable regions of the mouse heavy and light chains to the constant regions of the human heavy and light chains (as described, for example, by Kraus et al., Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, the chimeric antibody is produced by joining the human kappa-light chain constant region to the mouse light chain variable region. Also in a preferred embodiment, the chimeric antibody can be produced by joining the human lambda-light chain constant region to the mouse light chain variable region. Suitable heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other suitable heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0222] Humanized Antibodies interact with target antigens mainly through amino acid residues located within six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Since CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors containing CDR sequences derived from specific naturally occurring antibodies grafted onto framework sequences from different antibodies with different properties (see, for example, Riechmann, L. et al., (1998) Nature, 332: 323-327; Jones, P. et al., (1986) Nature, 321: 522-525; and Queen, C. et al., (1989) Proc. Natl. Acad. Sci. U. S. A., 86: 10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B cell maturation. The germline gene sequences will also differ from the sequences of high-affinity secondary repertoire antibodies at individual locations evenly distributed across the variable region.

[0223] The ability of an antibody to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry analysis).

[0224] To purify the antibody, the selected hybridoma can be grown in a 2-liter spinner flask for monoclonal antibody purification. Alternatively, the antibody can be produced in a dialysis-based bioreactor. Before affinity chromatography using protein G-Sepharose or protein A-Sepharose, the supernatant can be filtered and concentrated if necessary. The eluted IgG can be confirmed by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be exchanged to PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80 °C.

[0225] Site-directed or multi-site-directed mutagenesis can be used to determine whether the selected monoclonal antibody binds to a unique epitope.

[0226] To determine the isotype of the antibody, isotype ELISA can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics). The wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate can be reacted with the monoclonal antibody or purified isotype control for 2 hours at ambient temperature. The wells can then be reacted with a mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or mouse IgM-specific peroxidase-conjugated probe. After washing, the plate can be developed with an ABTS substrate (1 mg / ml) and analyzed at an OD of 405 - 650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, catalog number 1493027) can be used as described by the manufacturer.

[0227] Flow cytometry can be used to demonstrate the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to live cells expressing an antigen. Cell lines that express the antigen either naturally or after transfection, and negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with monoclonal antibodies at various concentrations in hybridoma supernatants or in PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, an APC or Alexa 647-labeled anti-IgG antibody can be bound to the antigen-binding monoclonal antibody under the same conditions as the primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument, using light and side scatter properties to gate single live cells. A co-transfection method can be used to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement. Cells transiently transfected with a plasmid encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescent channel from the antibody-stained cells. Since most of the transfected cells express both transgenes, the antigen-specific monoclonal antibody preferentially binds to the fluorescent marker-expressing cells, while non-specific antibodies bind to non-transfected cells at an equivalent ratio. As an alternative or in addition to the flow cytometry assay, a fluorescence microscopy-based alternative assay may be used. Cells can be stained precisely as described above and examined by fluorescence microscopy.

[0228] Immunofluorescence microscopy analysis can be used to demonstrate the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to live cells expressing an antigen. For example, cell lines that express an antigen spontaneously or after transfection, and negative controls lacking antigen expression, can be grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with a monoclonal antibody to the antigen for 30 minutes at 25°C. After washing, the cells can be reacted with an Alexa 555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0229] Cell extracts can be prepared from cells expressing the antigen and appropriate negative controls and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens can be transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody being tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with an ECL substrate.

[0230] Antibodies can be further tested for reactivity with the antigen by immunohistochemistry using paraformaldehyde or acetone-fixed frozen sections, or paraffin-embedded tissue sections fixed with paraformaldehyde, derived from non-cancerous or cancerous tissue samples obtained from mice bearing xenograft tumors inoculated with cell lines that express an antigen spontaneously or after transfection, or from patients during conventional surgery, or spontaneously, in a manner well known to those skilled in the art. For immunostaining, an antibody reactive with the antigen can be incubated according to the supplier's instructions, and then incubated with a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (DAKO).

[0231] The antibody can be tested for its ability to mediate phagocytosis and killing of cells expressing CLDN18.2. Testing monoclonal antibody activity in vitro provides an initial screening before testing in an in vivo model.

[0232] Antibody-dependent cell-mediated cytotoxicity (ADCC): Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells, or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation and then lysed of contaminating red blood cells. The washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or alternatively 5% heat-inactivated human serum and mixed with 51 Cr-labeled target cells expressing CLDN18.2 at various ratios of effector cells to target cells. Alternatively, the target cells may be labeled with a fluorescence-enhancing ligand (BATDA). A highly fluorescent europium chelate containing the enhancing ligand released from dead cells can be measured by a fluorometer. In another alternative technique, transfection of the target cells with luciferase may be utilized. The added luciferin can then be oxidized only by viable cells. Then, purified anti-CLDN18.2 IgG can be added at various concentrations. Irrelevant human IgG can be used as a negative control. The assay can be performed at 37 °C for 4 - 20 hours depending on the effector cell type used. 51 Samples can be assayed for cell lysis by measuring the presence of

[0233] To determine whether cell lysis is enhanced by multiple monoclonal antibodies, anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations.

[0234] Complement-dependent cytotoxicity (CDC): The monoclonal anti-CLDN18.2 antibody can be tested for these abilities to mediate CDC using various known techniques. For example, complement-containing serum can be obtained from blood in a manner known to those skilled in the art. Various methods can be used to determine the CDC activity of the mAb. 51 Cr release can be measured, for example, and the increase in membrane permeability can be evaluated using a propidium iodide (PI) exclusion assay. Briefly, the target cells are washed and can be incubated at 5×10 5 cells / ml with various concentrations of the mAb at room temperature or 37°C for 10 - 30 minutes. Serum or plasma can then be added to a final concentration of 20% (v / v), and the cells can be incubated at 37°C for 20 - 30 minutes. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be immediately analyzed by flow cytometry using a FACSArray.

[0235] In an alternative assay, the induction of CDC can be determined in adherent cells. In one embodiment of this assay, in a tissue culture flat-bottom microtiter plate, 3×10 4Seed cells at a density of cells per well 24 hours prior to the assay. The next day, remove the growth medium and incubate the cells in triplicate with the antibody. Incubate control cells with growth medium or growth medium containing 0.2% saponin to determine background lysis and maximum lysis, respectively. After incubating for 20 minutes at room temperature, remove the supernatant and add 20% (v / v) human plasma or serum in DMEM (pre-warmed to 37°C) to the cells and incubate for an additional 20 minutes at 37°C. Add all cells from each sample to propidium iodide solution (10 μg / ml). Then replace the supernatant with PBS containing 2.5 μg / ml ethidium bromide and measure the fluorescence emission at 600 nm using a Tecan Safire when excited at 520 nm. Calculate the percentage specific lysis as follows: % specific lysis = (fluorescence of sample - fluorescence of background) / (fluorescence of maximum lysis - fluorescence of background) × 100.

[0236] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies: To test the ability to initiate apoptosis, monoclonal anti-CLDN18.2 antibodies can be incubated with, for example, CLDN18.2-positive tumor cells such as SNU-16, DAN-G, KATO-III, or tumor cells transfected with CLDN18.2 at 37°C for about 20 hours. Cells can be harvested, washed in annexin-V binding buffer (BD biosciences), and incubated for 15 minutes in the dark with annexin V conjugated to FITC or APC (BD biosciences). All cells from each sample can be added to PI solution (10 μg / ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, catalog number AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Incorporated BrdU is detected using a europium-labeled monoclonal antibody. To enable antibody detection, cells are fixed and DNA is denatured using Fix solution. Unbound antibody is washed away and DELFIA enhancer is added to dissociate europium ions from the labeled antibody into solution, where they form highly fluorescent chelates with components of the DELFIA enhancer. Fluorescence measured using time-resolved fluorometry in detection is proportional to DNA synthesis within the cells of each well.

[0237] Preclinical trials Monoclonal antibodies that bind to CLDN18.2 can also be tested in in vivo models (e.g., in immunodeficient mice bearing xenograft tumors inoculated with cell lines that express CLDN18.2 such as DAN-G, SNU-16, or KATO-III, or cell lines that express CLDN18.2 after transfection such as HEK293) to determine these potencies in the control of the growth of CLDN18.2-expressing tumor cells.

[0238] In vivo tests after xenotransplanting CLDN18.2-expressing tumor cells into immunodeficient mice or other animals can be carried out using the antibodies described herein. Antibodies can be administered to tumor-free mice, and then tumor cells can be injected to measure the effect of the antibodies in preventing tumor formation or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice to determine the therapeutic efficacy of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibody application can be combined with the application of other substances, such as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies, to determine the synergistic efficacy and potential toxicity of the combination. To analyze the toxic side effects mediated by the antibodies, animals can be inoculated with the antibodies or control reagents to fully investigate the symptoms possibly associated with CLDN18.2-antibody therapy. Possible side effects of the in vivo application of CLDN18.2 antibodies include, in particular, toxicity in CLDN18.2-expressing tissues including the stomach. Antibodies that recognize CLDN18.2 in humans and in other species, such as mice, are particularly useful for predicting the potential side effects mediated by the application of monoclonal CLDN18.2-antibodies in humans.

[0239] Mapping of the epitopes recognized by the antibodies can be carried out as described in detail in "Epitope Mapping Protocols" (Methods in Molecular Biology), Glenn E. Morris, ISBN-089603-375-9, and "Epitope Mapping: A Practical Approach" Practical Approach Series, 248, Olwyn M. R. Westwood, Frank C. Hay.

[0240] The compounds and agents described herein can be administered in the form of any suitable pharmaceutical composition.

[0241] Pharmaceutical compositions are usually provided in a uniform dosage form and can be prepared in a manner known per se. The pharmaceutical composition can be, for example, in the form of a solution or a suspension.

[0242] The pharmaceutical composition can contain salts, buffering substances, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the action of the active ingredient of the pharmaceutical composition.

[0243] Pharmaceutically unacceptable salts may be used to prepare pharmaceutically acceptable salts and are included in the present invention. This type of pharmaceutically acceptable salt includes, in a non-limiting manner, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts, etc.

[0244] Buffering substances suitable for use in pharmaceutical compositions include acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt.

[0245] Preservatives suitable for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0246] Injectable preparations can contain pharmaceutically acceptable excipients such as Ringer's lactate.

[0247] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature with which the active ingredient is combined to facilitate, enhance, or enable its application. According to the present invention, the term "carrier" also includes one or more compatible solid fillers or liquid fillers, diluents, or encapsulating substances suitable for administration to a patient.

[0248] Carrier substances suitable for parenteral administration are, for example, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalene, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0249] The term "excipient", as used herein, is intended to denote all substances that can be present in a pharmaceutical composition and are not active ingredients, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents, etc.

[0250] The agents and compositions described herein can be administered via any conventional route, for example, by parenteral administration including, for example, by injection or infusion. Administration is preferably parenteral, for example, intravenous, intraarterial, subcutaneous, intradermal, or intramuscular.

[0251] Compositions suitable for parenteral administration usually include sterile aqueous or non-aqueous formulations of the active compound that are preferably isotonic with respect to the recipient's blood. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. Furthermore, usually, sterile fixed oils are used as solutions or suspension media.

[0252] The agents and compositions described herein are administered in an effective amount. "Effective amount" refers to the amount that, alone or together with further doses, achieves the desired reaction or desired effect. In the case of treating a particular disease or a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This includes slowing down the progression of the disease, and in particular, interruption or reversal of the progression of the disease. The desired reaction in the treatment of a disease or condition can also be delay in the onset or prevention of the onset of the said disease or the said condition.

[0253] The effective amount of the agent or composition described herein will depend on the condition being treated, the severity of the disease, individual parameters of the patient including age, physiological condition, size, and weight, the duration of treatment, the type of adjunctive therapy (if any), the particular route of administration, and like factors. Accordingly, the dosage administered of the agent described herein can depend on a variety of such parameters. If the patient's response is inadequate at the initial dosage, higher dosages (or effectively higher dosages achieved by a different more localized route of administration) can be used.

[0254] The agents and compositions described herein can be administered, for example, in vivo to a patient to treat or prevent various disorders such as those described herein. Suitable patients include human patients having a disorder that can be corrected or ameliorated by administration of the agents and compositions described herein. This includes disorders involving cells characterized by an altered pattern of CLDN18.2 expression.

[0255] For example, in one embodiment, the antibodies described herein can be used to treat patients having a cancer disease such as those described herein characterized by the presence of cancer cells that express CLDN18.2.

[0256] The pharmaceutical compositions and methods of treatment described by the present invention can also be used for immunization or vaccination to prevent the diseases described herein.

[0257] The present invention is further illustrated by the following examples which should not be construed as limiting the scope of the invention.

Example

[0258] (Example 1) CLDN18.2 expression in human gastric cancer cell lines is stabilized by in vitro treatment with chemotherapeutic agents The human gastric tumor cell line, KatoIII cells, were cultured at 37°C and 5% CO2 in RPMI 1640 medium (Invitrogen) containing 20% FCS (Perbio) and 2 mM Glutamax (Invitrogen), either with or without cell division - arresting compounds. Epirubicin (Pfizer) was tested at concentrations of 10 or 100 ng / ml, 5 - FU (NeoFluor from NeoCorp AG) was tested at concentrations of 10 or 100 ng / ml, and oxaliplatin (Hospira) was tested at concentrations of 50 or 500 ng / ml. Combinations of all three compounds (EOF; epirubicin 10 ng / ml, oxaliplatin 500 ng / ml, 5 - FU 10 ng / ml) were also used. 8×10 5 individual KatoIII cells were cultured for 96 hours or 72 hours without medium change and then for 24 hours in standard medium to release the cells from cell cycle arrest at 37°C and 5% CO2 in a 6 - well tissue culture plate. Cells were harvested using EDTA / trypsin, washed, and analyzed.

[0259] For extracellular detection of CLDN18.2, cells were stained with the monoclonal anti - CLDN18.2 antibody IMAB362 (Ganymed) or an isotype - matched control antibody (Ganymed). The secondary reagent, goat anti - huIgG - APC from Dianova, was used.

[0260] The cell cycle stage was determined based on the measurement of cellular DNA content. This allows the identification of cells in the G1, S, or G2 phases of the cell cycle. During the S phase, DNA replication occurs, and during the G2 phase, the cell grows and prepares for mitosis. Cell cycle analysis was performed using the CycleTEST PLUS DNA reagent kit from BD Biosciences according to the manufacturer's protocol. Flow cytometry acquisition and analysis were carried out using BD FACS CantoII (BD Biosciences) and FlowJo (Tree Star) software.

[0261] The columns in FIGS. 1a and 1b show the respective percentages of cells in the G1, S, or G2 phase of the cell cycle. KatoIII cells cultured in medium mainly show cell cycle arrest in the G1 phase. Cells treated with 5-FU are mainly blocked in the S phase. KatoIII cells treated with epirubicin or EOF mainly show cell cycle arrest in the G2 phase. KatoIII cells treated with oxaliplatin mainly show enrichment of cells in the G1 and G2 phases. As can be seen in FIG. 1c, CLDN18.2 is stabilized or upregulated by cell cycle arrest in the S or G2 phase. Immediately after cells are released from any stage of the cell cycle (FIG. 1b), the expression of CLDN18.2 on the cell surface of KatoIII cells is upregulated (FIG. 1d).

[0262] NUGC-4 and KATO III cells were treated with 5-FU+OX (10 ng / ml of 5-FU and 500 ng / ml of oxaliplatin), EOF (10 ng / ml of epirubicin, 500 ng / ml of oxaliplatin and 10 ng / ml of 5-FU) or FLO (10 ng / ml of 5-FU, 50 ng / ml of folic acid and 500 ng / ml of oxaliplatin) for 96 hours. RNA of pretreatment NUGC-4 and KATO III cells before chemotherapy was isolated and converted to cDNA. CLDN18.2 transcript levels were analyzed by quantitative real-time PCR. The results are shown in FIG. 2a as relative expression compared to the transcript level of the housekeeping gene HPRT. FIG. 2b shows Western blots of CLDN18.2 and actin loading controls in untreated and treated NUGC-4 cells. The intensity of the luminescence signal is shown as a percentage relative to actin.

[0263] Pretreatment of NUGC-4 and KATO III cells with EOF, FLO and 5-FU+OX combination chemotherapy increases the RNA and protein levels of CLDN18.2 as shown by quantitative real-time PCR (FIG. 2a) and Western blot (FIG. 2b).

[0264] Binding of IMAB362 to NUGC-4 and KATO III gastric cancer cells treated with EOF (10 ng / ml epirubicin, 500 ng / ml oxaliplatin and 10 ng / ml 5-FU) or FLO (10 ng / ml 5-FU, 50 ng / ml folic acid and 500 ng / ml oxaliplatin) for 96 h by flow cytometry was analyzed. The amount of CLDN18.2 protein targetable by IMAB362 on the surface of gastric cancer cell lines increased as shown in Fig. 2c. This effect was most prominent in cells pretreated with EOF or FLO.

[0265] KatoIII cells were pretreated with irinotecan or docetaxel for 4 days and analyzed for CLDN18.2 expression and cell cycle arrest. Treatment of cells with irinotecan resulted in dose-dependent inhibition of cell proliferation and cell cycle arrest in the S / G2 phase (Fig. 3). Treatment of cells with docetaxel resulted in dose-dependent inhibition of cell proliferation and cell cycle arrest in the G2 phase (Fig. 3).

[0266] (Example 2) Pretreatment of human gastric cancer cells with chemotherapeutic drugs increases the efficiency of IMAB362-mediated ADCC IMAB361-mediated ADCC was examined using NUGC-4 gastric cancer cells as targets, and the gastric cancer cells were either untreated or pretreated with 10 ng / ml 5-FU and 500 ng / ml oxaliplatin (5-FU+OX), 10 ng / ml epirubicin, 500 ng / ml oxaliplatin and 10 ng / ml 5-FU (EOF) or 10 ng / ml 5-FU, 50 ng / ml folic acid and 500 ng / ml oxaliplatin (FLO) for 96 h (effector to target ratio 40 to 1). EC 50 Values were obtained from 7 healthy donors untreated and from NUGC-4 cells pretreated with EOF, FLO or 5-FU+OX.

[0267] As shown in Fig. 4a, the dose / response curves of the pretreated cells shifted upward and to the left compared to the untreated target cells. This resulted in a higher maximum lysis and EC 50The value decreased to one-third of that of the untreated cells (Figure 4b).

[0268] Peripheral blood mononuclear cells (PBMCs) containing NK cells, monocytes, mononuclear cells, or other effector cells derived from healthy human donors were purified by Ficoll-Hypaque density centrifugation. The washed effector cells were seeded in X-Vivo medium. KatoIII cells, which endogenously express CLDN18.2 and are of gastric origin, were used as target cells in this setting. The target cells stably expressed luciferase and luciferase yellow, which is oxidized only by living cells. Purified anti-CLDN18.2 antibody IMAB362 was added at various concentrations, and an irrelevant chim huIgG1 antibody was used as an isotype control antibody. Samples were assayed for cell lysis by measuring the luminescence resulting from the oxidation of luciferase yellow, a value representing the amount of living cells remaining after IMAB362-induced cytotoxicity. KatoIII pretreated with irinotecan (1000 ng / ml), docetaxel (5 ng / ml), or cisplatin (2000 ng / ml) for 3 days was compared with untreated medium-cultured target cells to quantify IMAB362-induced ADCC.

[0269] KatoIII pretreated with irinotecan, docetaxel, or cisplatin for 3 days showed lower levels of living cells compared to medium-cultured target cells (Figure 5a), and claudin 18.2 expression in cells pretreated with irinotecan, docetaxel, or cisplatin was increased compared to medium-cultured target cells (Figure 5b).

[0270] Furthermore, pretreatment of KatoIII cells with irinotecan, docetaxel, or cisplatin increased the potency of IMAB362 to induce ADCC (Figures 5c, 5d).

[0271] (Example 3) The efficiency of IMAB362-induced CDC is increased by chemotherapy The effect of chemotherapeutic agents on IMAB362-induced CDC was analyzed by pre-treating KATO III gastric cancer cells with 10 ng / ml of 5-FU and 500 ng / ml of oxaliplatin (5-FU+OX) for 48 hours. The representative dose-response curve of IMAB362-induced CDC using KATO III cells pre-treated with chemotherapeutic agents is shown in Figure 6. Pre-treatment of tumor cells for 48 hours increased the potency of IMAB362 to induce CDC, and the maximum cell lysis of pre-treated tumor cells was higher compared to non-treated cells.

[0272] (Example 4) The ability of immune effector cells to perform IMAB362-induced ADCC is not impaired by treatment with chemotherapeutic agents The chemotherapeutic agents used in the EOF or FLO regimen are highly potent in inhibiting target cell proliferation. To examine the detrimental effects of chemotherapy on effector cells, PBMCs from healthy donors were treated with 10 ng / ml of epirubicin, 500 ng / ml of oxaliplatin and 10 ng / ml of 5-FU (EOF) or 10 ng / ml of 5-FU, 50 ng / ml of folic acid and 500 ng / ml of oxaliplatin (FLO) for 72 hours prior to application in the ADCC assay. Figure 7a shows the EC 50 values of four healthy donors, and Figure 7b shows the representative dose / response curve of IMAB362-induced ADCC using EOF or FLO pre-treated effector cells. IMAB362-induced ADCC of NUGC-4 gastric cancer cells is not impaired by EOF or FLO chemotherapy.

[0273] (Example 5) The combination of ZA / IL-2 treatment results in optimized expansion of peripheral blood mononuclear cell (PBMC) cultures The effect of ZA / IL-2 on the proliferation of PBMC cultures was evaluated in vitro. PBMC were harvested from healthy human donors and the cultures were treated with a single dose of ZA. IL-2 was added every 3 - 4 days. Specifically, PBMC from three different healthy human donors (#1, #2, #3) were cultured in RPMI medium for 14 days with 1 μM ZA plus high (300 U / ml) or low (25 U / ml) doses of IL-2 (cells 1×10 6 cells / ml) (see Figure 8a). PBMC from the same donors were further cultured in RPMI medium for 14 days with 300 U / ml of IL-2 plus ZA or without ZA (see Figure 8b). The increase in cell number was determined by counting viable cells on days 6, 8, 11, and 14.

[0274] In the medium supplied with high-dose IL-2, approximately 2 - 5 times more cells expanded compared to the cultures supplied with low IL-2 dose (Figure 8a). The expansion of cells in the medium without ZA was approximately half of that of the cells grown in the medium with ZA (Figure 8b). From these data, the necessity of applying both ZA and IL-2 compounds in combination to ensure proper expansion of cells has been revealed.

[0275] (Example 6) ZA / IL-2 treatment expands a large amount of Vγ9Vδ2 T cells in PBMC cultures PBMC were cultured for 14 days in RPMI medium supplemented with 300 U / ml IL-2 and 1 μM of ZA or without it. The percentage of Vγ9+Vδ2+ T cells within the CD3+ lymphocyte population (Figure 9a) and the percentage of CD16+ cells within the CD3+Vγ9+Vδ2+ T cell population (Figure 9b) were determined by multicolor FACS on day 0 and day 14. The results were scored as scatter plots for each donor. Figure 9c shows a scatter plot depicting the time-dependent increase (enrichment) in the number of CD3+Vγ9+Vδ2+ and CD3+CD16+Vγ9+Vδ2+ T cells within the lymphocyte population. The amount of cells seeded on day 0 and the amount of cells harvested on day 14 were taken into account.

[0276] Addition of IL-2 in PBMC cultures is necessary for lymphocyte survival and growth. Lymphocytes expanded efficiently in cultures supplied with 300 U / ml of IL-2. By FACS analysis using Vγ9- and Vδ2-specific antibodies, it has been revealed that addition of ZA / IL-2 specifically induces accumulation of Vγ9Vδ2 T cells (Figure 9a). After 14 days, the CD3+ lymphocyte population can contain up to 80% Vγ9Vδ2 T cells. Some of the Vγ9Vδ2 T cells express CD16, and the enrichment of these cells within the CD3+ lymphocyte population is 10 - to 700-fold depending on the donor (Figures 9b and 9c). The enrichment of CD16+Vγ9+Vδ2+ T cells in the cultures is 10 - to 600-fold compared to cultures grown without ZA (Figure 9c). We conclude that in vitro treatment of PBMC with ZA / IL-2 upregulates the ADCC-mediating FcγIII receptor CD16 in a significant proportion of γδ T cells.

[0277] (Example 7) IL-2 affects the expansion of Vγ9Vδ2 T cells in a dose-dependent manner Addition of ZA in the cultures is the most important factor inducing the development of Vγ9Vδ2 T cells. It is well known that IL-2 is required for T cell growth and survival.

[0278] PBMC were cultured for 14 days in RPMI medium supplemented with 1 μM ZA and increasing concentrations of IL-2. IL-2 was added on days 0 and 4. The enrichment of CD16+Vγ9+Vδ2+ T cells within the CD3+ lymphocyte population was determined by multicolor FACS staining on days 0 and 14. To compare different donors, the amount of CD16+Vγ9+Vδ2+ T cells harvested after culturing with 600 U / ml of IL-2 was set to 100% (see left in Figure 10). Furthermore, the ADCC activity of isolated cultures grown for 14 days with increasing concentrations of IL-2 was tested (see right in Figure 10).

[0279] By dose-response analysis, it was confirmed that IL-2 also stimulates the growth and survival of the Vγ9Vδ2 T cell subset. By adding a low IL-2 concentration to the medium, a correlation was found between the IL-2 dose and the percentage of CD16+Vγ9Vδ2 T cells within the CD3+ lymphocyte population (Figure 10, left). Furthermore, the ADCC activity of cells grown at higher IL-2 concentrations (150 - 600 U / ml) was improved compared to cells grown at low IL-2 concentrations (Figure 10, right).

[0280] (Example 8) ZA induces IPP production in monocytes and cancer cells and stimulates the expansion of both Vγ9Vδ2 T cells Fresh PBMC (Experiment #1) or 14-day ZA / IL-2-stimulated Vγ9Vδ2 T cell cultures (Experiments #2 - 5) were incubated without monocytes (effector to monocyte ratio 1 to 0) with either 0.2-fold (4 to 1) or 5-fold (1 to 4) amounts of monocytes ± 1 μM ZA. The enrichment of Vγ9Vδ2 T cells in the co-cultures after 14 days was determined by multicolor FACS, and the expansion of the cultures was considered in the calculations. The enrichment factor of Vγ9Vδ2 T cells cultured at a 1 to 4 ratio with monocytes was set to 100% for each experiment. With the increase in monocytes in the culture, Vγ9Vδ2 T cells were enriched by more than 10-fold. This effect was clearly ZA-dependent (see Figure 11a).

[0281] Furthermore, human gastric cancer cells (NUGC-4-luciferase) and mouse gastric cancer cells (CLS103-calcein staining) were pretreated with 5 μM ZA or without ZA for 2 days. Human Vγ9Vδ2 T cells were MACS-purified (day 14) and co-cultured with the cancer cells for 24 hours. The cytotoxicity of Vγ9Vδ2 T cells against untreated and ZA-treated target cells was determined by measuring the remaining luciferase activity or calcein fluorescence (see Figure 11b). Target cells (NUGC-4 and CLS103) were pretreated with 5 μM ZA or without ZA for 2 days, followed by incubation with mitomycin c (50 MI) for 4 hours to arrest proliferation. MACS-purified human day 14 resting Vγ9Vδ2 T cells and 3H thymidine was added to the target cells and the co-cultures were incubated at 37 °C for 48 h. Proliferation was determined by measuring the 3 H thymidine incorporation into DNA using a MicroBeta scintillation counter. Proliferation of target cells not treated with ZA and without Vγ9Vδ2 T cells was set to 100% (see Fig. 11c).

[0282] As shown in Figs. 11b and 11c, ZA-pulsed human cancer cells activated Vγ9Vδ2 T cells in terms of cytotoxicity (5 - 10-fold) and proliferation (1.4 - 1.8-fold), while the mouse cancer cell line CLS103 did not induce these effects on Vγ9Vδ2 T cells.

[0283] (Example 9) ZA / IL-2 treatment affects the composition of PBMC cultures The growth and differentiation of specific cell types in PBMC cultures are cytokine-dependent. These components are added to the medium (e.g., growth factors present in serum, IL-2) or secreted by the immune cells themselves. Which cell types evolve also depends on the initial composition and genetic endowment of the PBMC. To analyze the overall increase in effector cells (NK cells and Vγ9Vδ2 T cells), PBMC from 10 different donors were grown for 14 days in the presence of 300 U / ml IL-2, with or without 1 μM ZA. The amount of effector cells within the lymphocyte population was confirmed by multi-color FACS staining using CD3, CD16, CD56, Vγ9 and Vδ2 antibodies. CD3-CD56+CD16+ cells represent NK cells and CD3+Vγ9+Vδ2+ represent Vγ9Vδ2 T cells.

[0284] By multi-color FACS analysis, IL-2 treatment mainly led to the development of NK cells, while in ZA / IL-2-treated cultures, Vγ9Vδ2 T cells mainly expanded (Fig. 12).

[0285] (Example 10) ZA / IL-2 treatment produces Vγ9Vδ2+ effector memory T cells Subpopulations of T lymphocytes can be described with the help of two surface markers, the high molecular weight isoform of the common lymphocyte antigen CD45RA and the chemokine receptor CCR7. CCR7+ naive and central memory (CM) T cells are characterized by their ability to repeatedly circulate in lymph nodes and encounter antigens. In contrast, effector memory (EM) and effector T lymphocyte RA+ (TEMRA) downregulate CCR7 and appear to be specialized for migration to peripheral non-lymphoid tissues, such as sites of infection or tumors. EM cells can be further subdivided based on differential CD27 and CD28 expression. The progressive loss of CD28 and CD27 surface expression occurs concomitantly with upregulation of the cell's cytolytic capacity. Furthermore, the level of CD57 correlates with the expression of granzyme and perforin and thus represents a third marker indicative of cytotoxicity / cell maturation.

[0286] PBMCs were cultured for 14 days with 1 μM ZA or without ZA and with 300 U / ml IL-2. Expression of different surface markers was determined by polychromatic FACS analysis on day 0 (PBMC) and day 14. Untreated cells were CD45RA+CCR7+, central memory cells (CM) were CD45RA-CCR7+, TEMRA were CD45RA+CCR7-, and effector memory cells (EM) were negative for both markers (see Fig. 13a). Furthermore, the cytolytic activity of Vγ9Vδ2 T cells was determined by staining for the CD27 and CD57 markers (see Fig. 13b, Fig. 13c). Furthermore, the development of NK cell-like features important for ADCC activity was analyzed by staining CD3+ cells with CD16 (antibody binding) and CD56 (adhesion) (see Fig. 13d).

[0287] Multicolor FACS analysis of Vγ9Vδ2 T cells revealed that ZA / IL-2 treatment clearly stimulates the development of EM-type Vγ9Vδ2 T cells that are CD27- and CD57+ (Figures 13b - 13c). In addition to enhanced cytolytic activity, an increase in the levels of CD16 and CD56, which are known to be involved in ADCC from NK cells (CD3-CD16+CD56+), was observed in the CD3+ population (Figure 13d).

[0288] Collectively, these data imply that treatment of PBMC with ZA results in the development of CD16+Vγ9+Vδ2+ effector memory T cells that can migrate to peripheral non-lymphoid tissues and display markers of high cytolytic activity. In combination with the IMAB362 tumor-targeting antibody, these cells are very well utilized to migrate to and target tumor cells for killing.

[0289] (Example 11) ZA / IL-2-expanded Vγ9Vδ2 T cells are potent effectors of IMAB362-mediated CLDN18.2-dependent ADCC Similar to NK cells, ZA / IL-2-expanded Vγ9Vδ2 T cells are positive for CD16, an FcγRIII receptor through which cell-bound antibodies initiate ADCC (see Figures 9 and 13). To evaluate whether Vγ9Vδ2 T cells can induce potent ADCC in combination with IMAB362, a series of experiments were conducted.

[0290] PBMCs from two different donors (#1 and #2) were cultured in medium with 300 U / ml IL-2 and with or without 1 μM ZA. After 14 days, the cells were harvested and added together with increasing concentrations (0.26 ng / ml to 200 μg / ml) of IMAB362 to NUGC-4 cells expressing CLDN18.2. Specific killing was determined in a luciferase assay (see Figure 14a). Figures 14b and 14c give a schematic of the ADCC assay performed using 27 donors grown either with 300 U / ml IL-2 and with or without ZA. NUGC-4 served as the target cell. For each donor, the EC 50 values (b) and the maximum specific killing rate at a dose of 200 μg / ml IMAB362 (c) were scored on a scatter plot.

[0291] Strong IMAB362-dependent ADCC activity was observed against CLDN18.2-positive NUGC-4 cells using PBMCs cultured with ZA / IL-2 for 14 days (Figure 14a). Using ZA / IL-2-treated PBMC cultures, ADCC is dependent on the presence of Vγ9Vδ2 T cells (Figures 12 and 15). When the cells are cultured without ZA, ADCC activity decreases in most donors. In these cultures, the remaining ADCC activity is NK cell-dependent (Figures 11 and 14). By testing more than 20 donors, the ADCC assay has revealed that treating PBMCs with ZA / IL-2 improves the EC 50 values and the maximum specific killing rate compared to PBMCs cultured with IL-2 alone.

[0292] Furthermore, PBMCs from two different donors (#1 and #2) were cultured with 1 μM ZA and 300 U / ml IL-2. These effector cell cultures were used in an ADCC assay together with CLDN18.2-positive (NUGC-4, KATO III) and negative (SK-BR-3) human target cell lines (E:T ratio 40:1). The amount of the IMAB362 antibody was added while increasing (0.26 ng / ml to 200 μg / ml). ADCC was measured in a luciferase assay (see Figure 15a). The same experiment described in (a) was performed using NUGC-4 target cells and effector cells harvested from cultures treated with ZA / IL-2 at various time points (see Figure 15b). The same experiment described in (a) was performed using NUGC-4 as the target cell (see Figure 15c). ZA / IL-2-expanded cells were used directly or Vγ9Vδ2 T cells were purified from the cultures using TCRγδ MACS sorting (Miltenyi Biotech). Vγ9Vδ2 T cells with a purity exceeding 97.0% in lymphocytes were obtained.

[0293] Strong ADCC activity was observed against CLDN18.2-positive human tumor cell lines, but not against CLDN18.2-negative human tumor cell lines (Figure 15a). Furthermore, no ADCC activity was obtained with an isotype control antibody (not shown). During the course of ZA / IL-2 treatment, the ADCC lysis activity increased over time in some donors (Figure 15b). The dose / effect curve of IMAB362 shifted upward and to the left, indicating that the EC 50 value and the maximum lysis rate were improved over time. Compared with unconditioned PBMCs, Vγ9Vδ2 effector T cells enriched by ZA / IL-2 treatment were able to reach a higher maximum killing rate of CLDN18.2-positive target cells, and the concentration of IMAB362 required for the same killing rate could be lower.

[0294] To confirm that Vγ9Vδ2 T cells are the storage site of lytic activity, these cells were isolated from the ZA / IL-2 cultured PBMC population by magnetic cell sorting on day 14 with a purity exceeding 97%. The ADCC activity in combination with IMAB362 was retained and was somewhat improved due to the higher purity. From these data, it is confirmed that Vγ9Vδ2 T cells are the main cause of the ADCC activity observed in the PBMC cultures on day 14 (Figure 15c).

[0295] (Example 12) Treatment of target cell lines with ZA / IL-2 doses does not affect the surface expression of CLDN18.2 The IMAB362 initiating mechanism of action is strictly dependent on the presence and amount of extracellularly detectable CLDN18.2. Therefore, the effect of ZA / IL-2 treatment on CLDN18.2 surface density has been analyzed by flow cytometry using the endogenous CLDN18.2-expressing NUGC-4 and KATO III cell lines. Specifically, flow cytometry analysis of IMAB362 binding on permeabilized untreated NUGC-4 gastric cancer cells pre-treated 72 hours with ZA / IL-2 or ZA / IL-2 + EOF or ZA / IL-2 + 5-FU / OX was performed.

[0296] It has been revealed that ZA / IL-2 in vitro treatment does not result in a change in the amount of CLDN18.2 surface localization (see Figure 16).

[0297] (Example 13) The increase in IMAB362-mediated ADCC by ZA / IL-2 treatment of PBMCs is not impaired by pre-treatment with EOF Chemotherapeutic agents impair cell proliferation. In contrast, ZA / IL-2 treatment initiates the expansion of Vγ9Vδ2 T cells. To analyze the effect of these opposing interactions on effector cells, PBMCs from six healthy donors were cultured with ZA / IL-2 or ZA / IL-2 + EOF for 8 days prior to application in the ADCC assay (E to T ratio 15 to 1). 50% ADCC-mediated lysis of untreated NUGC-4 target cells (EC 50Determined the IMAB362 concentration that brings about

[0298] The increase in IMAB362-induced ADCC of NUGC-4 cells due to PBMC treatment with ZA / IL-2 is not significantly altered by the combined treatment of PBMC with EOF (Figure 17).

[0299] (Example 14) In vivo targeting of IMAB362 to CLDN18.2-positive tumors in nude mice and antitumor effect of IMAB362 on human tumor cell xenografts To examine the in vivo tumor cell targeting of IMAB362, 80 μg of Dyelight® 680-labeled antibody was intravenously administered to nude mice xenografted subcutaneously with the human gastric cancer cell line NUGC-4. NUGC-4 cells show surface expression of CLDN18.2 as well as HER2 / Neu (target of trastuzumab), but are negative for CD20. Control studies were performed by injecting either Dyelight680-labeled trastuzumab (positive control group) or Dyelight® 680-labeled rituximab (negative control) into a group of NUGC-4 xenografted mice. Twenty-four hours after intravenous injection of the antibody, as shown by live imaging of the mice using a Xenogen® fluorescence imaging system, IMAB362 accumulates strongly and exclusively in the tumor xenografts (Figure 18). IMAB362 is efficiently retained in the target positive tumors and can be detected at comparable intensities even after 120 hours (Figure 18). Trastuzumab is also detected exclusively in the tumor xenografts 24 hours after injection. The trastuzumab signal is rapidly washed out within 120 hours after injection. No signal is detected with rituximab.

[0300] Furthermore, nude mice bearing CLDN18.2-positive xenograft tumors were treated with IMAB362. An early treatment model study (administering IMAB362 just 3 days after tumor cell inoculation) was conducted. Furthermore, the advanced tumor treatment experiment was initiated by 9 days after tumor cell inoculation when the tumors reached a volume of approximately 60 - 120 mm 3 in size.

[0301] Nude mice were subcutaneously inoculated with 1×10 7 individual HEK293~CLDN18.2 transfectants. Treatment of 10 mice per group was started 3 days after tumor inoculation. The mice were treated twice weekly for 6 weeks with 200 μg of IMAB362, infliximab as an isotype control, and PBS, applied alternately via the intravenous and intraperitoneal routes. All mice in the groups treated with either PBS or the isotype control died within 70 - 80 days, while there was a life-prolonging effect in the animals treated with IMAB362 (Figure 19). Not only was the time to death prolonged, but 4 out of 10 mice survived the entire 210-day observation period.

[0302] Treatment of 9 to 10 mice per group was started when the mean tumor volume reached 88 mm 3 (62 - 126 mm 3 ). Prior to treatment, the mice were classified into test groups to ensure comparable tumor sizes in all groups. The mice were treated twice weekly for 6 weeks with 200 μg of IMAB362, an isotype control, or PBS, applied alternately via the intravenous and intraperitoneal routes. All mice in the groups treated with either PBS or the isotype control died within 50 - 100 days. There was a life-prolonging effect in the animals treated with IMAB362, and the mean survival time was almost doubled (47 vs. 25 days). Three of these mice survived the entire observation period (Figure 20). Importantly, the in vivo anti-tumor efficacy depends on the presence of the target on the tumor cells. The anti-tumor effect of IMAB362 treatment was not seen in mice transplanted with CLDN18.2-negative HEK293 tumor cells.

[0303] The NUGC-4 gastric tumor model was used to examine the efficacy of IMAB362 against cancer cells that endogenously express CLDN18.2. NUGC-4 cells grow aggressively in nude mice.

[0304] 1×10 7Individual NUGC-4 gastric cancer cells were injected subcutaneously into the left flank of thymus-deficient nude mice (n = 9 in the IMAB362 group and n = 8 in the control group). IMAB362 (200 μg per injection) and the control were started 6 days after tumor inoculation by intravenous injection and applied alternately intravenously and intraperitoneally twice a week. Tumor size was monitored twice a week. The data presented in Figure 21a are the means with SEM. Tumor growth treated with IMAB362 was significantly inhibited compared to mice treated with the control (*p < 0.05). Figure 21b shows the tumor volume on day 21 after tumor inoculation. The tumor volume of IMAB362-treated mice was significantly smaller than that of the tumors of control mice (*p < 0.05).

[0305] 1×10 7 When 1×10 tumor cells are inoculated into mice, the average survival period of untreated mice will not be longer than 25 days. When the tumor volume reaches an average size of about 109 mm 3 (63 - 135 mm 3 ), treatment with IMAB362, cetuximab, trastuzumab, or isotype and buffer control was started. Mice were classified into treatment groups size-dependently (Figure 21). IMAB362 was shown to significantly reduce the tumor growth rate. In this aggressively growing tumor model, no significant decrease in tumor growth was observed compared to saline or antibody control. The delay in tumor growth was associated with a non-significant increase in the median survival period of IMAB362-treated mice (31 days vs. 25 days).

[0306] The antitumor activity of IMAB362 was investigated in two human gastric cancer xenograft models using NCI-N87 or NUGC-4 cells transduced with lentivirus expressing the IMAB362 target CLDN18.2 (NCI-N87-CLDN18.2 and NUGC-4-CLDN18.2).

[0307] NCI-N87-CLDN18.2 xenograft tumors were 1×10 7Individual NCI-N87~CLDN18.2 cells were subcutaneously inoculated by injecting them into the flanks of 8 nude mice (female, 6 weeks old) per treatment group. Treatments were initiated 5 days after tumor inoculation by intravenous injection of 800 μg of IMAB362 or using 200 μl of 0.9% NaCl in the saline control group. Intravenous administration was continued weekly throughout the entire observation period. Tumor size and animal health were monitored twice a week. Figure 22a shows the effect of IMAB362 treatment on tumor growth. The size of the subcutaneous injection tumors was measured twice a week (mean + SEM, ***p<0.001). Figure 22b shows a Kaplan-Meier survival plot. Mice were sacrificed when the tumor reached a volume of 1400 mm 3 3.

[0308] Thus, continuous IMAB362 treatment significantly (p<0.001) inhibited the tumor growth of NCI-N87~CLDN18.2 gastric cancer xenografts (Figure 22a). The delay in tumor growth was associated with a significantly (p<0.05) longer survival period in IMAB362-treated mice (Figure 22b).

[0309] IMAB362 immunotherapy of rapidly growing NUGC-4~CLDN18.2 xenografts significantly (p<0.05) reduced the tumor size on day 14 of treatment. After the first 2 weeks of IMAB362 treatment, the tumor progression of NUGC-4~CLDN18.2 was very aggressive. However, the inhibition of NUGC-4~CLDN18.2 tumor growth until day 14 of treatment significantly (p<0.05) prolonged the survival period of IMAB362-treated mice.

[0310] In summary, IMAB362 was highly effective in treating gastric cancer xenografts, showing a significant delay in tumor progression and long-term survival in an endogenous CLDN18.2-positive tumor model. In a very aggressive tumor model system, although these antitumor effects of IMAB362 are not so prominent, they are still significant, highlighting the strong antitumor ability of IMAB362.

[0311] (Example 15) Antitumor Effect of IMAB362 in Combination with Chemotherapy in a Mouse Tumor Model In vitro, IMAB362-mediated ADCC was more efficient on human gastric cancer cells pretreated in combination with chemotherapeutic agents including EOF and 5-FU+OX. Therefore, the antitumor effects of combining these compounds with IMAB362 were investigated in vivo in a mouse tumor model.

[0312] NCI-N87~CLDN18.2 xenograft tumors were inoculated by subcutaneous injection of 1×10 7 cells of NCI-N87~CLDN18.2 into the flanks of 9 mice per treatment group. Tumor-bearing mice were treated intraperitoneally with 1.25 mg / kg epirubicin, 3.25 mg / kg oxaliplatin, and 56.25 mg / kg 5-fluorouracil on days 4, 11, 18, and 25 after tumor inoculation according to the EOF regimen, followed by intravenous injection of 800 μg of IMAB362 24 hours after chemotherapy administration. IMAB362 treatment was continued weekly. Tumor size and animal health were monitored twice a week. Figure 23a shows the effect of the combined treatment on tumor growth. The size of subcutaneous injection tumors was measured twice a week (mean + SEM, *p<0.05). Figure 23b shows a Kaplan-Meier survival plot. Mice were sacrificed when the tumor reached a volume of 1400 mm 3 .

[0313] NCI-N87~CLDN18.2 tumor nude mice treated with IMAB362 or the EOF regimen showed that tumor growth was extremely significantly inhibited compared to control mice. Additional IMAB362 treatment in combination with EOF chemotherapy significantly (p<0.05) inhibited tumor growth higher than treatment with the EOF regimen alone (Figure 23a). The median survival time of mice in the physiological saline control group was 59 days. Similar to the survival of mice in the EOG group with a median survival time of 76 days, weekly IMAB362 treatment of mice significantly extended the median survival time to 76 days. However, the combined treatment of IMAB362 and EOF increased the median survival time to 81 days (Figure 23b).

[0314] Xenograft tumors were inoculated by injecting 1×10 7 NUGC-4~CLDN18.2 cells per mouse into the subcutaneous tissue of the flanks of 10 nude mice (female, 6 weeks old) per treatment group. Mice were treated with chemotherapeutic agents on days 3, 10, 17, and 24. IMAB362 treatment was continued weekly. Figure 24a shows the tumor growth curve of subcutaneous NUGC-4~CLDN18.2 xenografts (mean + SEM). Figure 24b shows the Kaplan-Meier survival plot (log-rank (Mantel-Cox) test, **p < 0.01).

[0315] Subcutaneous NUGC-4~CLDN18.2 xenograft tumors grew extremely aggressively. Nevertheless, treatment of tumor-bearing nude mice with IMAB362 significantly inhibited tumor growth compared to the saline-treated control group. In combination therapy with EOF, the effect of IMAB362 on NUGC-4~CLDN18.2 tumor growth was masked by the growth inhibition caused by EOF treatment, and no increase in tumor growth inhibition was shown compared to treatment with EOF alone (Figure 24a). However, the median survival time of mice treated with IMAB362 and EOF was extremely significantly (p < 0.01) prolonged compared to the survival of mice treated with EOF alone (Figure 24b).

[0316] (Example 16) ZA / IL-2-expanded Vγ9Vδ2 T cells improve IMAB362-mediated control of progressive tumors in vivo To examine the combined activity of IMAB362 and ZA / IL-2-producing γδ T cells in a mouse model, NSG mice were used. NSG mice lack mature T cells, B cells, natural killer (NK) cells, multiple cytokine signaling pathways, and have many deficiencies in innate immunity, and the niches in primary and secondary immunological tissues are permissive for colonization by human immune cells.

[0317] NSG mice were subcutaneously inoculated with 1×10 7 CLDN18.2-transfected HEK293 cells per mouse. On the same day, Vγ9Vδ2 T cells cultured in ZA-supplemented medium for 14 days were concentrated to 8×106 Individual human PBMCs were administered to mice. Furthermore, the mice were injected with 50 μg / kg ZA and 5000 U IL-2 (Proleukin). To maintain human T cells in a functional state, IL-2 was administered twice a week and ZA was administered weekly. When the HEK293~CLDN18.2 tumors became macroscopically visible, twice-weekly treatment with 200 μg of IMAB362 was initiated. In addition to the nine mice treated as described, two control groups of mice were established. One group was not administered human γδ T cells, and the other group was treated with an isotype control antibody instead of IMAB362. Tumor growth of CLDN18.2-positive tumors in mice treated with IMAB362 in the presence of human γδ T cells and ZA was significantly inhibited and almost suppressed. In either mice treated with the isotype control antibody or lacking human T cell effectors, the tumors grew aggressively and the mice had to be sacrificed prematurely (Figure 25).

[0318] (Example 17) Antitumor effect of IMAB362 in combination with chemotherapy in a mouse tumor model The antitumor activity of IMAB362 in combination with chemotherapy was investigated in subcutaneous gastric cancer xenografts of immunocompetent non-inbred NMRI mice using CLS-103 cells transduced with lentivirus expressing mouse cldn18.2 (CLS-103~cldn18.2).

[0319] CLS-103~cldn18.2 xenograft tumors were 1×10 6Individual CLS-103~cldn18.2 cells were inoculated by subcutaneous injection into the flanks of 10 NMRI mice for each treatment group. Tumor-bearing mice were treated intraperitoneally with 1.25 mg / kg epirubicin, 3.25 mg / kg oxaliplatin and 56.25 mg / kg 5-fluorouracil (EOF) on days 3, 10, 17 and 24 after tumor inoculation, and subsequently 800 μg of IMAB362 was injected intravenously 24 hours after each chemotherapy administration. IL-2 was administered twice a week by subcutaneous injection at 3000 IE. After the end of chemotherapy, IMAB362 and IL-2 treatments were continued throughout the observation period. Tumor size and animal health were monitored twice a week. Mice were sacrificed when the tumor reached 1400 mm 3 in volume or when the tumor became ulcerated.

[0320] As can be seen in Figure 26, CLS-103~cldn18.2 tumor NMRI mice treated with IMAB362 or EOF alone showed no significant inhibition of tumor growth compared to the saline control group. In contrast, when EOF chemotherapy was combined with IMAB362 treatment, tumor growth was significantly more inhibited and the survival of tumor-bearing mice was prolonged. These findings indicate that the combination of EOF chemotherapy and IMAB362 immunotherapy has an additive or even synergistic therapeutic effect. IL-2 treatment showed no effect on tumor growth.

[0321]

Table 2

[0322] New international patent application Ganymed Pharmaceuticals AG et al. 「Combined therapy with an antibody against Claudin 18.2 for treating cancer」 Our reference: 342-75 PCT Additional sheet for biological substances Identification of further deposits: 1) Name and address of the depository institution for the deposited materials (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC-2745, DSM ACC2746, DSM ACC2747, DSM ACC2748): DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg 1b 38124 Braunschweig DE 2) Name and address of the recipient institution for the deposited materials (DSM ACC2808, DSM ACC2809, DSM ACC2810): DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7 B 38124 Braunschweig DE

[0323]

Table 3

[0324] Additional indications for all of the above-mentioned deposited materials: - Mouse (Mus musculus) myeloma P3X63Ag8U.1 fused with mouse (Mus musculus) spleen cells - Hybridoma-secreted antibody against human Claudin-18A2 3) Depositor: All of the above-mentioned deposits were made as follows. Ganymed Pharmaceuticals AG Freiligrathstrasse 12 55131 Mainz DE

Claims

[Claim 1] A method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2 in combination with an agent that stimulates γδ T cells.

Images