Combination therapy involving antibodies against claudin 18.2 and immune checkpoint inhibitors for treatment of cancer

Abstract

To provide methods for effectively treating and / or preventing cancer diseases.SOLUTION: The present invention provides a combination therapy comprising an anti-Claudin (CLDN) 18.2 antibody and an immune checkpoint inhibitor 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-neck cancer, and cancer of the gallbladder and metastases thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Gastric and esophageal (gastroesophageal; GE) cancers are among the malignant tumors with the highest unmet medical needs. Gastric cancer is one of the leading causes of cancer death worldwide. The incidence of esophageal cancer has been increasing over the past few decades, in line with a shift in histological type and primary tumor location. Adenocarcinoma of the esophagus is now more prevalent than squamous cell carcinoma in the United States and Western Europe, and most tumors are located in the distal esophagus. The overall 5-year survival rate for GE cancer is 20-25%, despite aggressive established standard treatments with significant side effects.

[0002] The majority of patients present with locally advanced or metastatic disease. For these patients, the first-line treatment is chemotherapy. Treatment regimens are mainly based on the backbone of platinum and fluoropyrimidine derivatives in combination with a third compound (e.g., taxane or anthracycline). Nevertheless, a median progression-free survival of 5-7 months and a median overall survival of 9-11 months are the best that can be expected.

[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. In recent years, trastuzumab has been approved for Her2 / neu-positive gastroesophageal cancer. However, since only about 20% of patients are eligible for this treatment, 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 transmembrane domains with two small extracellular loops.

[0005] In normal tissues, except for the stomach, there is no detectable expression of CLDN18.2 by RT-PCR. Immunohistochemistry with CLDN18.2-specific antibodies reveals that the stomach is the only positive tissue.

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

[0007] The chimeric IgG1 antibody IMAB362 (zolbetuximab [previously called claudiximab]), directed against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. This antibody comprises a heavy chain having the sequence shown in SEQ ID NO: 51 and a light chain having the sequence shown in SEQ ID NO: 24. 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 two independent and highly potent mechanisms of action. Upon target binding, IMAB362 mediates cell death mainly by ADCC and CDC. Thus, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. The anti-tumor efficacy of IMAB362 has been demonstrated in mice bearing xenograft tumors inoculated with CLDN18.2-positive cancer cell lines.

[0008] IgG1 antibodies are typically involved in the cellular immune system through the interaction of the Fc domain with Fc gamma receptors (FcγR) expressed on various immune cells, including natural killer cells, the main effector of ADCC. However, IgG1 monoclonal antibodies (mAbs) that induce ADCC face several limitations, including the widespread distribution of low-affinity Fc receptor variants in the population (up to 80%) and in vivo IgG1 modifications that reduce mAb efficacy (Chames, P. et al., 2009, Br J Pharmacol, 157(2):220-233). Therapeutic antibodies also have to compete with patient IgG, leading to the need for high doses of mAbs in vivo. Furthermore, therapeutic antibodies can interact with FcγRIIb, an inhibitory FcγR expressed by B cells, macrophages, dendritic cells, and neutrophils, resulting in negative signaling that reduces their efficacy.

[0009] In the past few decades, immune checkpoint inhibitors have come to be regarded as powerful cancer treatment drugs. These therapeutic agents block inhibitory immune checkpoint signaling that restricts the function of the immune system. Thus, immune checkpoint inhibitors can bring about increased activation, proliferation, and / or signaling of T cells. However, it has been found that immune checkpoint inhibitors have low activity in some cancers and are beneficial only in a very small fraction of patients (Darvin et al., 2018, Exp Mol Med 50(12):165). Approaches to overcome these limitations include the co-administration of drugs that target co-inhibitory checkpoint receptors, anti-angiogenic therapeutic agents, small molecule inhibitors of tumor targets, and oncolytic viruses that promote tumor cell lysis (Longo et al., 2019, Cancers 11(4):539). Given the high complexity of the tumor microenvironment (TME) and the diverse mechanisms by which specific TME components synergistically induce immunosuppression in the context of significant tumor heterogeneity, it is difficult to accurately evaluate the TME and devise anti-cancer strategies applicable to the general population (Duan et al., 2019, Cancer Med 7(9):4517 - 4529). Therefore, there is an urgent need to increase the effectiveness of immune checkpoint inhibitor therapy, and the Society for Immunotherapy of Cancer (SITC) in the United States has convened a combination immunotherapy task force to address the prospects and challenges of combining immune checkpoint blockade with other therapies.

[0010] Here, the inventors present data demonstrating that the co-administration of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor results in improved effects. In a mouse tumor model, the administration of an anti-CLDN18.2 antibody and a checkpoint inhibitor showed superior efficacy compared to the administration of the anti-CLDN18.2 antibody or the immune checkpoint inhibitor as single agents. SUMMARY OF THE INVENTION

[0011] 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, colon 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.

[0012] In one aspect, the present invention provides a method of treating a patient, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

[0013] In one aspect, the present invention provides a method of treating or preventing cancer in a patient, the method comprising administering to the patient an anti-CLDN1 8.2 antibody and an immune checkpoint inhibitor.

[0014] In a further aspect, the present invention provides a method of inhibiting tumor growth in a patient having cancer, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

[0015] In a further aspect, the present invention provides a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient, e.g., a patient having cancer, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

[0016] In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is selected from PD-1 inhibitors and PD-L1 inhibitors. In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0017] In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is an anti-PD-1 antibody. In one embodiment of all aspects disclosed herein, the anti-PD-1 antibody is nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN2810), sparalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ63723283), toripalimab (JS001), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI754091, or SHR-1210.

[0018] In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In one embodiment of all aspects disclosed herein, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (bavencio), rodaplimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, or MDX-1105.

[0019] In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment of all aspects disclosed herein, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In one embodiment of all aspects disclosed herein, the anti-CTLA-4 antibody is ipilimumab (Yervoy; Bristol Myers Squibb), tremelimumab (Pfizer / MedImmune), trevilizumab, AGEN-1884 (Agenus) or ATOR-1015.

[0020] In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody binds to the native epitope of CLDN18.2 present on the surface of living cells. In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody is a monoclonal, chimeric or humanized antibody, or a fragment of an antibody. In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody is conjugated to a therapeutic agent such as a toxin, radioisotope, drug or cytotoxin.

[0021] In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody binds to the first extracellular loop of CL DN18.2.

[0022] In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody mediates cell death 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.

[0023] In one embodiment of all aspects disclosed herein, the anti-CLDN18.2 antibody is (i) an antibody produced by and / or obtained from a clone deposited under accession number 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 of (i), (iii) an antibody having the specificity of the antibody of (i), and (iv) an antigen-binding portion or antigen-binding site of the antibody of (i), particularly an antibody comprising a variable region and preferably having the specificity of the antibody of (i), and is an antibody selected from the group consisting of.

[0024] In one embodiment of all aspects disclosed in this specification, the anti-CLDN18.2 antibody comprises a heavy-chain variable region CDR1 comprising the sequence at positions 45-52 of the sequence shown in SEQ ID NO: 17, a heavy-chain variable region CDR2 comprising the sequence at positions 70-77 of the sequence shown in SEQ ID NO: 17, a heavy-chain variable region CDR3 comprising the sequence at positions 116-126 of the sequence shown in SEQ ID NO: 17, a light-chain variable region CDR1 comprising the sequence at positions 47-58 of the sequence shown in SEQ ID NO: 24, a light-chain variable region CDR2 comprising the sequence at positions 76-78 of the sequence shown in SEQ ID NO: 24, and a light-chain variable region CDR3 comprising the sequence at positions 115-123 of the sequence shown in SEQ ID NO: 24.

[0025] In one embodiment of all aspects disclosed in this specification, the anti-CLDN18.2 antibody comprises a heavy-chain variable region comprising the sequence shown in SEQ ID NO: 32 or a functional variant thereof, or a fragment of the amino acid sequence or a functional variant, and / or a light-chain variable region comprising the sequence shown in SEQ ID NO: 39 or a functional variant thereof, or a fragment of the amino acid sequence or a functional variant.

[0026] In one embodiment of all aspects disclosed in this specification, the anti-CLDN18.2 antibody comprises a heavy-chain constant region comprising the sequence shown in SEQ ID NO: 13 or 52, or a functional variant thereof, or a fragment of the amino acid sequence or a functional variant.

[0027] In one embodiment of all aspects disclosed in this specification, the anti-CLDN18.2 antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO: 17 or 51, or a functional variant thereof, or a fragment of the amino acid sequence or a functional variant, and / or a light chain comprising the sequence shown in SEQ ID NO: 24 or a functional variant thereof, or a fragment of the amino acid sequence or a functional variant.

[0028] In one embodiment of all aspects disclosed in this specification, the method is up to 1000 mg / m 2administering an anti-CLDN18.2 antibody at a dose of. In one embodiment of all aspects disclosed herein, the method is repeated at a dose of 300-600 mg / m 2 comprises administering an anti-CLDN18.2 antibody at a dose of.

[0029] In one embodiment of all aspects disclosed herein, the cancer is CLDN18.2 positive. In one embodiment of all aspects disclosed herein, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof. In one embodiment of all aspects disclosed herein, the cancer is Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis. In one embodiment of all aspects disclosed herein, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment of all aspects disclosed herein, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly lower esophageal cancer, gastro-esophageal junction cancer, and gastro-esophageal cancer.

[0030] In one embodiment of all aspects disclosed herein, CLDN18.2 has the amino acid sequence according to SEQ ID NO: 1.

[0031] In a further aspect, the invention provides a pharmaceutical preparation comprising an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

[0032] In one embodiment of all aspects disclosed herein, the pharmaceutical preparation is a kit comprising a first container containing an anti-CLDN18.2 antibody and a second container containing an immune checkpoint inhibitor.

[0033] In one embodiment of all aspects disclosed herein, the pharmaceutical preparation further comprises printed instructions for using the preparation to treat cancer.

[0034] In one embodiment of all aspects disclosed herein, the pharmaceutical preparation is a composition comprising an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

[0035] In one embodiment of all aspects disclosed herein, the method of the present invention further comprises administering a cytotoxic agent and / or a cytostatic agent. In one embodiment of all aspects disclosed herein, the pharmaceutical preparation of the present invention further comprises a cytotoxic agent and / or a cytostatic agent.

[0036] The cytotoxic agent and / or cell growth inhibitor can be 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. In one embodiment, the cytotoxic agent and / or cell growth inhibitor comprises an agent that induces cell cycle arrest or the accumulation of cells in one or more phases of the cell cycle, preferably one or more phases of the cell cycle other than the G1 phase. The cytotoxic agent and / or cell growth inhibitor can comprise an agent selected from the group consisting of anthracyclines, platinum compounds, nucleoside analogs, taxanes and camptothecin analogs, or prodrugs thereof, and combinations thereof. The cytotoxic agent and / or cell growth inhibitor can comprise 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. The cytotoxic agent and / or cell growth inhibitor can comprise 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. The cytotoxic agent and / or cell growth inhibitor can be an agent that induces immunogenic cell death. An agent that induces immunogenic cell death is anthracycli It may contain an agent selected from the group consisting of, oxaliplatin, and combinations thereof. The cytotoxic agent and / or cytostatic agent may 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 5-fluorouracil and its prodrug. In one embodiment, the medical preparation of the present invention includes at least one anthracycline, at least one platinum compound, and at least one 5-fluorouracil and its prodrug. The anthracycline may be selected from the group consisting of epirubicin, doxorubicin, daunorubicin, idarubicin, and valrubicin. Preferably, the anthracycline is epirubicin. The platinum compound may be selected from the group consisting of oxaliplatin and cisplatin. The nucleoside analog may be selected from the group consisting of 5-fluorouracil and its prodrug. The taxane may be selected from the group consisting of docetaxel and paclitaxel. The camptothecin analog may 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, (v) folic acid, oxaliplatin, and 5-fluorouracil, (vi) folic acid, oxaliplatin, and capecitabine, or (vii) oxaliplatin and capecitabine. In one embodiment, the medical preparation of the present invention includes (i) epirubicin, oxaliplatin, and 5-fluorouracil, (ii) epirubicin, oxaliplatin, and capecitabine, (iii) epirubicin, cisplatin, and 5-fluorouracil, (iv) epirubicin, cisplatin, and capecitabine, (v) folic acid, oxaliplatin, and 5-fluorouracil, (vi) folic acid, oxaliplatin, and capecitabine, or (vii) oxaliplatin and capecitabine.

[0037] An anti-CLDN18.2 antibody, an immune checkpoint inhibitor, and optionally a cytotoxic agent and / or a cytostatic agent can be present in a mixture or separately from each other in a pharmaceutical preparation. The pharmaceutical preparation can be a kit comprising a first container containing the CLDN18.2 antibody, a container containing the immune checkpoint inhibitor, and optionally a container containing the cytotoxic agent and / or the cytostatic agent. The pharmaceutical preparation can further comprise printed instructions for using the preparation, in particular for using the preparation in the methods of the present invention, for treating cancer. The pharmaceutical preparations, and in particular the various embodiments of the immune checkpoint inhibitor and the cytotoxic agent and / or the cytostatic agent, are as described above for the methods of the present invention.

[0038] The present invention also provides agents as described herein, such as anti-CLDN18.2 antibodies and immune checkpoint inhibitors, for use in therapy. In one embodiment, such therapy comprises treating and / or preventing a cancer disease, such as a disease associated with cells expressing CLDN18.2, including those described herein.

[0039] The present invention also provides agents as described herein, such as anti-CLDN18.2 antibodies, for use in the methods described herein, for administration in combination with, for example, an immune checkpoint inhibitor and optionally a cytotoxic agent and / or a cytostatic agent. The present invention also provides the use of agents as described herein, such as anti-CLDN18.2 antibodies, for preparing a pharmaceutical composition for administration in combination with, for example, an immune checkpoint inhibitor and optionally a cytotoxic agent and / or a cytostatic agent, for use in the methods described herein.

[0040] In one aspect, the present invention is a method of treating or preventing cancer in a patient, comprising an anti-CLDN1 Provided is an anti-CLDN18.2 antibody for use in a method comprising the step of administering an 8.2 antibody and an immune checkpoint inhibitor to a patient. In a further aspect, the invention provides an anti-CLDN18.2 antibody for use in a method of inhibiting tumor growth in a patient having cancer, the method comprising the step of administering an anti-CLDN18.2 antibody and an immune checkpoint inhibitor to the patient. In a further aspect, the invention provides an anti-CLDN18.2 antibody for use in a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer, the method comprising the step of administering an anti-CLDN18.2 antibody and an immune checkpoint inhibitor to the patient. Preferred embodiments of these aspects are as described above for the methods of the invention.

[0041] In one aspect, the invention provides an immune checkpoint inhibitor for use in a method of treating or preventing cancer in a patient, the method comprising the step of administering an anti-CLDN18.2 antibody and an immune checkpoint inhibitor to the patient. In a further aspect, the invention provides an immune checkpoint inhibitor for use in a method of inhibiting tumor growth in a patient having cancer, the method comprising the step of administering an anti-CLDN18.2 antibody and an immune checkpoint inhibitor to the patient. In a further aspect, the invention provides an immune checkpoint inhibitor for use in a method of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer, the method comprising the step of administering an anti-CLDN18.2 antibody and an immune checkpoint inhibitor to the patient. Preferred embodiments of these aspects are as described above for the methods of the invention.

[0042] In one aspect, the present invention provides an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for use in a method of treating or preventing cancer in a patient. In a further aspect, the present invention provides an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for use in a method of inhibiting tumor growth in a patient having cancer. In a further aspect, the present invention provides an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for use in a method of inducing antibody-dependent cell cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer. Preferred embodiments of these aspects are as described above for the methods of the present invention.

[0043] In one aspect, the present invention provides the use of an anti-CLDN18.2 antibody for preparing a pharmaceutical composition for treating or preventing cancer in a patient, wherein the anti-CLDN18.2 antibody is administered together with an immune checkpoint inhibitor. In a further aspect, the present invention provides the use of an anti-CLDN18.2 antibody for preparing a pharmaceutical composition for inhibiting tumor growth in a patient having cancer, wherein the anti-CLDN18.2 antibody is administered together with an immune checkpoint inhibitor. In a further aspect, the present invention provides the use of an anti-CLDN18.2 antibody for preparing a pharmaceutical composition for inducing antibody-dependent cell cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer, wherein the anti-CLDN18.2 antibody is administered together with an immune checkpoint inhibitor. Preferred embodiments of these aspects are as described above for the methods of the present invention.

[0044] In one aspect, the present invention provides the use of an immune checkpoint inhibitor for preparing a pharmaceutical composition for treating or preventing a patient's cancer, wherein the immune checkpoint inhibitor is administered together with an anti-CLDN18.2 antibody. In a further aspect, the present invention provides the use of an immune checkpoint inhibitor for preparing a pharmaceutical composition for inhibiting tumor growth in a patient having cancer, wherein the immune checkpoint inhibitor is administered together with an anti-CLDN18.2 antibody. In a further aspect, the present invention provides the use of an immune checkpoint inhibitor for preparing a pharmaceutical composition for inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer, wherein the immune checkpoint inhibitor is administered together with an anti-CLDN18.2 antibody. The preferred embodiments of these aspects are as described above for the methods of the present invention.

[0045] In one aspect, the present invention provides the use of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for preparing a pharmaceutical composition for treating or preventing a patient's cancer. In a further aspect, the present invention provides the use of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for preparing a pharmaceutical composition for inhibiting tumor growth in a patient having cancer. In a further aspect, the present invention provides the use of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor for preparing a pharmaceutical composition for inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient, such as a patient having cancer. The preferred embodiments of these aspects are as described above for the methods of the present invention.

[0046] In one embodiment of the aspects described herein, the treatment described herein involves an immunotherapeutic treatment of a patient. In one embodiment of the aspects described herein, the treatment described herein involves inducing immune-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, the treatment described herein involves inducing immune cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, the treatment described herein involves inducing T cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, the treatment described herein involves inducing NK cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, the treatment described herein involves inducing antibody-dependent cell cytotoxicity (ADCC) against cancer cells in a patient. In one embodiment, ADCC is mediated, at least in part, by NK cells. In one embodiment of the aspects described herein, the treatment described herein involves inducing complement-dependent cell cytotoxicity (CDC) against cancer cells in a patient.

[0047] In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the antitumor efficacy of an anti-CLDN18.2 antibody. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces immune-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces immune cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces T cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces NK cell-mediated inhibition or destruction of cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces antibody-dependent cell cytotoxicity (ADCC) against cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor increases the efficacy of an anti-CLDN18.2 antibody and induces complement-dependent cell cytotoxicity (CDC) against cancer cells in a patient. In one embodiment of the aspects described herein, administration of an immune checkpoint inhibitor synergistically increases the efficacy of an anti-CLDN18.2 antibody.

[0048] Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief Description of the Drawings

[0049]

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DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention will be described in detail below. However, it should be understood that the specific methodologies, protocols, and reagents described herein may vary, and the present invention is not limited thereto. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is 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.

[0051] Elements of the present invention are described below. Although these elements are recited in specific embodiments, it should be understood that these elements 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. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Further, any permutation and combination of all the recited elements in this application should be considered to be disclosed by the description of this application, unless the context specifically indicates otherwise.

[0052] 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).

[0053] The practice of the present invention, unless otherwise indicated, uses conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0054] Throughout the following specification and claims, unless the context requires otherwise,[[]] The word "comprise", and variations such as "comprises" and "comprising", are understood to mean the inclusion of the stated members, integers or steps, or groups of members, integers or steps, but not the exclusion of other members, integers or steps, or groups of members, integers or steps, although in some embodiments such other members, integers or steps, or groups of members, integers or steps may be excluded, i.e., the subject matter consists of the inclusion of the stated members, integers or steps, or groups of members, integers or steps. The terms "a", "an" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural unless specifically indicated otherwise herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand way of referring individually to each separate value falling within the range. Unless specifically indicated otherwise herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless specifically indicated otherwise herein or clearly contradicted by the context. Any and all examples, or exemplary language (e.g., the use of "such as") provided herein are intended merely to better illustrate the invention and are not to be construed as limiting the scope of the claimed invention. None of the language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0055] Throughout the text of this specification several documents are cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0056] The term "CLDN18" relates 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)).

[0057] The term "CLDN18.2" preferably relates to human CLDN18.2, and in particular relates to a protein preferably consisting of or including an amino acid sequence according to SEQ ID NO: 1 in the sequence listing or a variant of said amino acid sequence.

[0058] The term "CLDN18.1" preferably relates to human CLDN18.1, and in particular relates to a protein preferably consisting of or including an amino acid sequence according to SEQ ID NO: 2 in the sequence listing or a variant of said amino acid sequence.

[0059] 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 occurring naturally. Allelic variants relate to changes in the normal sequence of a gene, the significance of which is usually unclear. Complete gene sequencing often identifies a number of allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences of a species of different origin from that of a given nucleic acid or amino acid sequence. The term "variant" shall be taken to include any post-translational modification variant and conformational variant.

[0060] According to the present invention, the term "CLDN18.2 positive cancer" preferably means a cancer accompanied by cancer cells expressing CLDN18.2 on the surface of the cancer cells.

[0061] "Cell surface" is used according to its ordinary meaning in the art, and thus includes the outer side of the cell available for binding by proteins and other molecules.

[0062] CLDN18.2 is located on the surface of the cell and is expressed on the surface of the cell when available for binding by a CLDN18.2 specific antibody added to the cell.

[0063] According to the present invention, CLDN18.2 is not substantially expressed in the cell when the expression level is lower compared to the expression in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% or lower than the expression in gastric cells or gastric tissue, or even lower. Preferably, CLDN18.2 is not substantially expressed in the cell when the expression level exceeds the expression level in a non-cancerous tissue other than the stomach by no more than 2-fold, preferably no more than 1.5-fold, preferably not exceeding the expression level in the non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in the cell when the expression level is below the detection limit and / or the expression level is too low to allow binding by a CLDN18.2 specific antibody added to the cell.

[0064] According to the present invention, CLDN18.2 is expressed in the cell when the expression level exceeds the expression level in a non-cancerous tissue other than the stomach by more than 2-fold, preferably by 10-fold, 100-fold, 1000-fold or 10000-fold. Preferably, CLDN18.2 is expressed in the cell when the expression level is above the detection limit and / or the expression level is high enough to allow binding by a CLDN18.2 specific antibody added to the cell. Preferably, CLDN18.2 expressed in the cell is expressed on the surface of the cell or exposed on the surface of the cell.

[0065] 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 includes metastasis of that cancer. In a preferred embodiment, the disease treated by the present application is associated with cells expressing CLDN18.2.

[0066] The term "disease associated with cells expressing CLDN18.2" or similar expressions means, according to the present invention, that CLDN18.2 is expressed in the cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in the cells of a 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 even more. In one embodiment, the expression is only seen in the diseased tissue and the expression in healthy tissue is suppressed. According to the present invention, the diseases associated with cells expressing CLDN18.2 include cancer diseases. Further, according to the present invention, the cancer disease is preferably a cancer disease in which cancer cells express CLDN18.2.

[0067] As used herein, "cancer disease" or "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion and / or migration. "Cancer cell" means an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimulus that initiated the new growth has ceased. Preferably, "cancer disease" is characterized by cells expressing CLDN18.2, and cancer cells express CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells of the cancers described herein.

[0068] "Adenocarcinoma" is a cancer that originates in 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 an adenocarcinoma, cells do not necessarily have to be part of a gland as long as they have secretory properties. This form of carcinoma can occur in some 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 the cells from a biopsy, a pathologist can determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can occur in many tissues of the body due to the widespread nature of glands within the body. Each gland may not secrete the same substance, but as long as it has an exocrine function for cells, it is considered a gland, and thus its malignant form is called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize if given enough time. Ovarian adenocarcinoma is the most common type of ovarian cancer. This includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0069] "Metastasis" means the spread of cancer cells from the 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, after being transported by the blood vessels, invasion of the target organ. Finally, the growth of new tumors at the target site depends on angiogenesis. Since tumor cells or components remain and can develop metastatic ability, tumor metastasis often occurs even after removal of the primary tumor. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis" with respect to metastases distant from the primary tumor and the associated lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One specific form of metastasis that can be treated using the treatment of the present invention is a metastasis derived 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.

[0070] Krukenberg tumors are rare metastatic tumors of the ovary, accounting 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. Carcinomas of the colon, appendix, and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumors derived from carcinomas of the gallbladder, biliary tract, pancreas, small intestine, cecum, cervix, and bladder / urethra have been reported. The interval between the diagnosis of the primary carcinoma and the subsequent discovery of ovarian involvement is usually less than 6 months, although longer periods have also been reported. In many cases, the primary tumor is very small and can escape detection. A previous history of carcinoma of the stomach or another organ can be obtained in only 20% - 30% of cases. Krukenberg tumors are most commonly an example of the selective spread of cancer along the stomach-ovary axis. This axis of tumor spread has historically attracted the interest of many pathologists, particularly as it has been found that gastric neoplasms selectively metastasize to the ovary without the involvement of other tissues. The route of metastasis of gastric cancer to the ovary has long been a mystery, but it is now clear that retrograde lymphatic spread is the most likely route of metastasis. Women with Krukenberg tumors are typically of an average age of 45 years and are in their 40s, so they tend to be unusually young for patients with metastatic cancer. This young age distribution may in part be related to the increasing frequency of gastric signet-ring cell carcinoma in young women. The symptoms commonly presented are usually related to ovarian involvement and its most common ones are abdominal pain and distension (mainly due to usually bilateral, often large ovarian tumors). The remaining patients have non-specific gastrointestinal symptoms or are asymptomatic. Additionally, Krukenberg tumors are reportedly associated with virilization due to hormone production by ovarian stroma. Ascites is present in 50% of cases and usually reveals malignant cells. Krukenberg tumors are bilateral in over 80% of reported cases. The ovaries are usually asymmetrically enlarged and have a nodular contour. The transected surface is yellow or white; these are usually solid but sometimes cystic. Importantly, the capsular surface of the ovaries with Krukenberg tumors is typically 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, like primary ovarian tumors. However, the bilateralism of Krukenberg tumors is consistent with their metastatic nature. Patients with Krukenberg tumors have a significantly high overall mortality rate. Most patients die within 2 years (median survival, 14 months). Some studies have shown that when the primary tumor is identified after the metastasis to the ovaries is discovered, the prognosis is poor, and when the primary tumor remains hidden, the prognosis is even worse. The optimal treatment strategy for Krukenberg tumors has not been clearly established in the literature. Whether surgical resection should be performed has not been adequately studied. Chemotherapy or radiotherapy has no significant effect on the prognosis of patients with Krukenberg tumors.

[0071] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The terms are intended to include the full range of treatments for a given condition from which a patient suffers, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, alleviate or relieve symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual for the purpose of combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0072] The term "therapeutic treatment" refers to any treatment that improves the health status and / or extends (increases) the life span of an individual. Said treatment can eliminate the disease in an individual, halt or slow the development of the disease in an individual, inhibit or slow the development of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce the recurrence of an individual who currently or previously had the disease.

[0073] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0074] The terms "individual" and "subject" are used interchangeably herein. These refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse or primate) that can be affected by, or is susceptible to, a disease or disorder (e.g., cancer), and may or may not have the disease or disorder. In many embodiments, the individual is human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus include adults, elderly, pediatric and neonatal individuals. In embodiments of the present disclosure, an "individual" or a "subject" is a "patient".

[0075] The term "patient" means an individual or subject for treatment, particularly an individual or subject with a disease.

[0076] As used herein, "immune checkpoint" refers to regulators of the immune system, particularly co-stimulatory and inhibitory signals that regulate the magnitude and amount of antigen recognition by the T cell receptor. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to replace CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG-3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands, such as galectin 9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the inhibitory signal is the interaction between one or several KIRs and their ligands. In certain embodiments, the inhibitory signal is the interaction between TIGIT and one or more of its ligands, PVR, PVRL2, and PVRL3. In certain embodiments, the inhibitory signal is the interaction between CD94 / NKG2A and HLA-E. In certain embodiments, the inhibitory signal is the interaction between VISTA and its binding partner(s). In certain embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In certain embodiments, the inhibitory signal is the interaction between GARP and one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction between CD47 and SIRPα. In certain embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In certain embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In certain embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In certain embodiments, the inhibitory signal is the interaction between a part of the adenosine signaling pathway, such as A2AR and / or A2BR, and adenosine produced by CD39 and CD73.In certain embodiments, the inhibitory signal is an interaction between B7-H3 and its receptor and / or between B7-H4 and its receptor. In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX or TDO.

[0077] The "Programmed Death-1 (PD-1)" receptor is an immunosuppressive receptor belonging to the CD28 family. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one epitope common to at least one hPD-1. "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 that downregulates T cell activation and cytokine secretion when bound to PD-1 (the other being PD-L2). As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one epitope common to at least one hPD-L1. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isoforms and species homologs of hPD-L2, and analogs having at least one epitope common to at least one hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are antigen-presenting cells such as dendritic cells or macrophages, and other immune cells It is expressed on the surface. The binding of PD-1 to PD-L1 or PD-L2 results in the downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can switch off T cells expressing PD-1, which can lead to the suppression of the anti-cancer immune response. The interaction between PD-1 and its ligand results in a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is also blocked.

[0078] "Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)" (also known as CD152) is a T cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms and species homologs of hCTLA-4, and analogs having at least one epitope in common with at least one hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28, which has a much higher binding affinity for CD80 and CD86. CTLA4 is expressed on the surface of activated T cells, and its ligand is expressed on the surface of specialized antigen-presenting cells. The binding of CTLA-4 to its ligand interferes with the co-stimulatory signal of CD28 and generates an inhibitory signal. Thus, CTLA-4 downregulates T cell activation.

[0079] The "T cell immunoreceptor with Ig and ITIM domain" (also known as TIGIT, WUCAM or Vstm3) is an immunoreceptor on T cells and natural killer (NK) cells, binds to PVR (CD155), such as DC and macrophages, and PVRL2 (CD112; nectin-2) and PVRL3 (CD113; nectin-3), and regulates T cell-mediated immunity. As used herein, the term "TIGIT" includes human TIGIT (hTIGIT), variants, isoforms and species homologs of hTIGIT, and analogs having an epitope common to at least one hTIGIT. As used herein, the term "PVR" includes human PVR (hPVR), variants, isoforms and species homologs of hPVR, and analogs having an epitope common to at least one hPVR. As used herein, the term "PVRL2" includes human PVRL2 (hPVRL2), variants, isoforms and species homologs of hPVRL2, and analogs having an epitope common to at least one hPVRL2. As used herein, the term "PVRL3" includes human PVRL3 (hPVRL3), variants, isoforms and species homologs of hPVRL3, and analogs having an epitope common to at least one hPVRL3.

[0080] The "B7 family" refers to inhibitory ligands of undefined receptors. The B7 family includes B7-H3 and B7-H4, which are both upregulated on tumor cells and tumor-infiltrating cells. As used herein, the terms "B7-H3" and "B7-H4" include human B7-H3 (hB7-H3) and human B7-H4 (hB7-H4), their variants, isoforms and species homologs, and analogs having an epitope common to at least one B7-H3 and B7-H4, respectively.

[0081] "B and T Lymphocyte Attenuator" (also known as BTLA, CD272) is a TNFR family member that is expressed in Th1 cells but not in Th2 cells. BTLA expression is induced during T cell activation and is expressed, in particular, on the surface of CD8+ T cells. As used herein, the term "BTLA" includes human BTLA (hBTLA), variants, isoforms and species homologs of hBTLA, and analogs having an epitope common to at least one hBTLA. BTLA expression is gradually downregulated during the differentiation of human CD8+ T cells into an effector cell phenotype. Tumor-specific human CD8+ T cells express high levels of BTLA. BTLA binds to "Herpesvirus entry mediator" (also known as HVEM, TNFRSF14 or CD270) and is involved in T cell suppression. As used herein, the term "HVEM" includes human HVEM (hHVEM), variants, isoforms and species homologs of hHVEM, and analogs having an epitope common to at least one hHVEM. The BTLA-HVEM complex negatively regulates the T cell immune response.

[0082] "Killer-cell Immunoglobulin-like Receptor" (KIR) is a receptor for MHC class I molecules on NK T cells and NK cells that is involved in distinguishing between healthy and diseased cells. KIR binds to human leukocyte antigens (HLA) A, B, and C, which inhibits normal immune cell activation. As used herein, the term "KIR" includes human KIR (hKIR), variants, isoforms and species homologs of hKIR, and analogs having an epitope common to at least one hKIR. As used herein, the term "HLA" includes variants, isoforms and species homologs of HLA, and analogs having an epitope common to at least one HLA. KIR as used herein specifically refers to KIR2DL1, KIR2DL2 and / or KIR2DL3.

[0083] "Lymphocyte Activation Gene-3" (LAG-3), also known as CD223, is an inhibitory receptor associated with the inhibition of lymphocyte activation by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits CD8+ effector T cell function, resulting in immune response suppression. LAG-3 is expressed on activated T cells, NK cells, B cells and DCs. As used herein, the term "LAG-3" includes human LAG-3 (hLAG-3), variants, isoforms and species homologs of hLAG-3, and analogs having at least one common epitope.

[0084] "T Cell Membrane Protein-3 (TIM-3)", also known as HAVcr-2, is an inhibitory receptor that is involved in the inhibition of lymphocyte activity by inhibiting Th1 cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancer. Other TIM-3 ligands include phosphatidylserine (PtdSer), high-mobility group protein 1 (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). As used herein, the term "TIM-3" includes human TIM3 (hTIM-3), variants, isoforms and species homologs of hTIM-3, and analogs having at least one common epitope. As used herein, the term "GAL9" includes human GAL9 (hGAL9), variants, isoforms and species homologs of hGAL9, and analogs having at least one common epitope. As used herein, the term "PdtSer" includes variants and analogs having at least one common epitope. As used herein, the term "HMGB1" includes human HMGB1 (hHMGB1), variants, isoforms and species homologs of hHMGB1, and analogs having at least one common epitope. As used herein, the term "CEACAM1" includes human CEACAM1 (hCEACAM1), variants , isoforms and species homologs of hCEACAM1, and analogs having at least one common epitope.

[0085] "CD94 / NKG2A" is an inhibitory receptor that is predominantly expressed on the surface of natural killer cells and CD8+ T cells. As used herein, the term "CD94 / NKG2A" includes human CD94 / NKG2A (hCD94 / NKG2A), variants, isoforms and species homologs of hCD94 / NKG2A, and analogs having at least one common epitope. The CD94 / NKG2A receptor is a heterodimer comprising CD94 and NKG2A. It suppresses NK cell activation and CD8+ T cell function, presumably by binding to ligands such as HLA-E. CD94 / NKG2A restricts cytokine release and cytotoxic responses of natural killer cells (NK cells), natural killer T cells (NK-T cells) and T cells (α / β and γ / δ). NKG2A is frequently expressed in tumor-infiltrating cells and HLA-E is overexpressed in some cancers.

[0086] "Indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. As used herein, the term "IDO" includes human IDO (hIDO), variants, isoforms and species homologs of hIDO, and analogs having at least one common epitope. IDO is the rate-limiting enzyme in tryptophan degradation that catalyzes the conversion to kynurenine. Thus, IDO is involved in the depletion of essential amino acids. This is known to be involved in the suppression of T and NK cells, the production and activation of Tregs and myeloid-derived immunosuppressive cells, and the promotion of tumor angiogenesis. IDO is overexpressed in many cancers, promotes immune evasion of tumor cells, and has been shown to promote chronic tumor progression when induced by local inflammation.

[0087] As used herein, the "adenosinergic pathway" or "adenosine signaling pathway" refers to the conversion of ATP to adenosine by ectonucleotidases CD39 and CD73, which results in inhibitory signaling through adenosine binding to one or more of the inhibitory adenosine receptors, the "Adenosine A2A Receptor" (also known as A2AR, ADORA2A) and the "Adenosine A2B Receptor" (also known as A2BR, ADORA2B). Adenosine is a nucleoside with immunosuppressive properties and is present at high concentrations in the tumor microenvironment, limiting immune cell infiltration, cytotoxicity, and cytokine production. Thus, adenosine signaling is a strategy by which cancer cells evade host immune system clearance. Adenosine signaling through A2AR and A2BR is an important checkpoint in cancer therapy that is activated by the high adenosine concentrations typically present in the tumor microenvironment. CD39, CD73, A2AR, and A2BR are expressed by most immune cells, including T cells, invariant natural killer cells, B cells, platelets, mast cells, and eosinophils. Adenosine signaling through A2AR and A2BR interferes with T cell receptor-mediated activation of immune cells, resulting in an increase in the number of Tregs and a decrease in the activation of DCs and effector T cells. As used herein, the term "CD39" includes human CD39 (hCD39), variants, isoforms, and species homologs of hCD39, and analogs having at least one common epitope. As used herein, the term "CD73" includes human CD73 (hCD73), variants, isoforms, and species homologs of hCD73, and analogs having at least one common epitope. As used herein, the term "A2AR" includes human A2AR (hA2AR), variants, isoforms, and species homologs of hA2AR, and analogs having at least one common epi tope. As used herein, the term "A2BR" includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs having at least one common epitope. As used herein, the term "adenosine" includes adenosine and pharmaceutically acceptable salts, esters, prodects, and metabolites thereof. As used herein, the term "inhibitor" refers to a compound that inhibits the activity of CD39, CD73, A2AR, or A2BR, or a combination thereof. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It includes analogs having a tope. As used herein, the term "A2BR" includes human A2BR (hA2BR), variants, isoforms and species homologs of hA2BR, and analogs having at least one common epitope.

[0088] "V-domain Ig suppressor of T cell activation" (VISTA, also known as C10orf54) has homology with PD-L1 but shows a unique expression pattern restricted to the hematopoietic compartment. As used herein, the term "VISTA" includes human VISTA (hVISTA), variants, isoforms and species homologs of hVISTA, and analogs having at least one common epitope. VISTA induces T cell suppression and is expressed by leukocytes within tumors.

[0089] "Sialic acid binding immunoglobulin type lectin" (Siglec) family members recognize sialic acid and are involved in the discrimination between "self" and "non-self". As used herein, the term "Siglec" includes human Siglec (hSiglec), variants, isoforms and species homologs of hSiglec, and analogs having at least one epitope common to one or more hSiglecs. The human genome contains 14 Siglecs, some of which, including but not limited to Siglec-2, Siglec-3, Siglec-7 and Siglec-9, are involved in immunosuppression. Siglec receptors bind to glycans containing sialic acid but differ in the recognition of the binding radiochemistry and spatial distribution of sialic acid residues. Family members also have distinct expression patterns. A wide range of malignancies overexpress one or more Siglecs.

[0090] "CD20" is an antigen expressed on the surface of B and T cells. High expression of CD20 can be seen in cancers such as B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. As used herein, the term "CD20" includes human CD20 (hCD20), variants, isoforms and species homologs of hCD20, and analogs having at least one common epitope.

[0091] "Glycoprotein A repetitions predominant" (GARP) plays a role in immune tolerance and the ability of tumors to evade the patient's immune system. As used herein, the term "GARP" includes human GARP (hGARP), variants, isoforms and species homologs of hGARP, and analogs having at least one common epitope. GARP is expressed on lymphocytes including Treg cells in peripheral blood and tumor-infiltrating T cells at the tumor site. This likely binds to the latent "transforming growth factor β" (TGF-β). Disruption of GARP signaling in Tregs results in decreased tolerance and inhibits the migration of Tregs to the intestine and increased proliferation of cytotoxic T cells.

[0092] "CD47" is a transmembrane protein that binds to the ligand "signal-regulatory protein alpha" (SIRPα). As used herein, the term "CD47" includes human CD47 (hCD47), variants, isoforms and species homologs of hCd47, and analogs having at least one common epitope with hCD47. As used herein, the term "SIRPα" includes human SIRPα (hSIRPα), variants, isoforms and species homologs of hSIRPα, and analogs having at least one common epitope with hSIRPα. CD47 signaling is involved in apoptosis, proliferation, adhesion It is involved in a range of cellular processes including movement. CD47 is overexpressed in many cancers and functions as a "don't eat me" signal to macrophages. Blocking CD47 signaling through inhibitory anti-CD47 or anti-SIRPα antibodies enables macrophage phagocytosis of cancer cells and promotes the activation of cancer-specific T lymphocytes.

[0093] "Poliovirus receptor related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "Poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in some cancers. PVRIG expression also induces TIGIT and PD-1 expression, and PVPL2 and PVR (the TIGIT ligand) are co-overexpressed in some cancers. Blocking the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses, and thus decreased immunosuppression and increased interferon responses. As used herein, the term "PVRIG" includes human PVRIG (hPVRIG), variants, isoforms and species homologs of hPVRIG, and analogs having an epitope common to at least one hPVRIG. As used herein, "PVRL2" includes hPVRL2 as defined above.

[0094] The "colony-stimulating factor 1" pathway is another checkpoint that can be targeted according to the present disclosure. CSF1R is a myeloproliferative factor that binds to CSF1. Blocking CSF1R signaling can functionally reprogram the macrophage response, thereby enhancing antigen presentation and anti-tumor T cell responses. As used herein, the term "CSF1R" includes human CSF1R (hCSF1R), variants, isoforms and species homologs of hCSF1R, and analogs having an epitope common to at least one hCSF1R. As used herein, the term "CSF1" includes human CSF1 (hCSF1), variants, isoforms and species homologs of hCSF1, and analogs having an epitope common to at least one hCSF1.

[0095] "Nicotinamide adenine dinucleotide phosphate NADPH oxidase" refers to enzymes of the NOX family of enzymes in myeloid cells that generate immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1-NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels are detected in almost all cancers and promote many aspects of tumor development and progression. ROS generated by NOX weakens NK and T cell function, and inhibition of NOX in myeloid cells improves the anti-tumor function of adjacent NK and T cells. As used herein, the term "NOX" includes human NOX (hNOX), variants, isoforms and species homologs of hNOX, and analogs having an epitope common to at least one hNOX.

[0096] Another immune checkpoint that can be targeted by the present disclosure is the signal mediated by "tryptophan-2,3-dioxygenase" (TDO). TDO is an alternative pathway to IDO in tryptophan degradation and is involved in immunosuppression. Since tumor cells can catabolize tryptophan via TDO instead of IDO, TDO can be an additional target for checkpoint blockade. In fact, some cancer cell lines have been found to upregulate TDO, and TDO can complement IDO inhibition. As used herein, the term "TDO" refers to human T DO (hTDO), variants, isoforms and species homologs of hTDO, and analogs having an epitope common to at least one hTDO.

[0097] Many of the immune checkpoints are regulated by the interaction between specific receptor and ligand pairs, such as those described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation and / or T cell function. Cancer cells often utilize these checkpoint pathways to prevent themselves from being attacked by the immune system. Therefore, the functions of checkpoint proteins regulated by the present disclosure typically involve the regulation of T cell activation, T cell proliferation and / or T cell function. Thus, immune checkpoint proteins regulate and maintain self-tolerance as well as the duration and magnitude of the physiological immune response. Many of the immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily and activate signaling molecules mobilized to the cytoplasmic domain by binding to specific ligands (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

[0098] As used herein, the term "immune checkpoint modulator" or "checkpoint modulator" refers to a molecule or compound that modulates the function of one or more checkpoint proteins. An immune checkpoint modulator can typically regulate self-tolerance and / or the magnitude and / or duration of an immune response. Preferably, the immune checkpoint modulator used in accordance with the present disclosure modulates the function of one or more human checkpoint proteins and is thus a "human checkpoint modulator". In a preferred embodiment, the human checkpoint modulator used herein is an immune checkpoint inhibitor.

[0099] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more checkpoint proteins, or completely or partially reduces, inhibits, interferes with, or negatively regulates the expression of one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to a precursor of one or more checkpoint proteins, for example, at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used.

[0100] As used herein, the term "partially" means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98% or 99% at a level, e.g., the level of inhibition of a checkpoint protein.

[0101] In certain embodiments, an immune checkpoint inhibitor suitable for use in the methods disclosed herein is an antibody that targets an antagonist of an inhibitory signal, e.g., PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4 or TIM-3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12:252~264, 2012. Additional immune checkpoint proteins that can be targeted according to the present disclosure are described herein.

[0102] In certain embodiments, the immune checkpoint inhibitor interferes with an inhibitory signal associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts inhibitory signal transduction associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts inhibitory signal transduction. In certain embodiments, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts inhibitory signal transduction. In certain embodiments, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts inhibitory signal transduction.

[0103] In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between checkpoint blocker proteins, such as an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1 or PD-L2. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between LAG-3 and its ligand, or between TIM-3 and its ligand. In certain embodiments, the immune checkpoint inhibitor interferes with inhibitory signaling through CD39 and / or CD73, and / or the interaction between A2AR and / or A2BR and adenosine. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between B7-H3 and its receptor, and / or between B7-H4 and its receptor. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between BTLA and its ligand HVEM. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between one or more KIRs and their respective ligands. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between LAG-3 and one or more of its ligands. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between TIM-3 and one or more of its ligands galectin-9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between TIGIT and one or more of its ligands PVR, PVRL2, and PVRL3. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between CD94 / NKG2A and HLA-E. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between VISTA and one or more of its binding partners. In certain embodiments, the immune checkpoint inhibitor interferes with the interaction between one or more Siglecs and their respective ligands. In certain embodiments, the immune checkpoint inhibitor interferes with CD20 signaling.In certain embodiments, an immune checkpoint inhibitor interferes with the interaction of GARP and one or more of its ligands. In certain embodiments, an immune checkpoint inhibitor interferes with the interaction of CD47 and SIRPα. In certain embodiments, an immune checkpoint inhibitor interferes with the interaction of PVRIG and PVRL2. In certain embodiments, an immune checkpoint inhibitor interferes with the interaction of CSF1R and CSF1. In certain embodiments, an immune checkpoint inhibitor interferes with NOX signaling. In certain embodiments, an immune checkpoint inhibitor interferes with IDO and / or TDO signaling.

[0104] Inhibition or blockade of inhibitory immune checkpoint signaling as described herein results in the prevention or reversal of immunosuppression and the establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of immune checkpoint signaling as described herein reduces or inhibits immune system dysfunction. In one embodiment, inhibition of immune checkpoint signaling as described herein makes dysfunctional immune cells less dysfunctional. In one embodiment, inhibition of immune checkpoint signaling as described herein makes dysfunctional T cells less dysfunctional.

[0105] As used herein, the term "dysfunction" refers to a state of reduced immune responsiveness to antigen stimulation. This term includes common elements of both exhaustion and / or anergy where antigen recognition can occur but the subsequent immune response is ineffective at controlling either infection or tumor growth. Dysfunction also includes states where antigen recognition is delayed by dysfunctional immune cells.

[0106] As used herein, the term "dysfunctional" refers to immune cells that are in a state of reduced immune responsiveness to antigenic stimulation. Dysfunctionality includes being unresponsive to antigen recognition as well as impairment of the ability to convert antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2) and / or target cell killing.

[0107] As used herein, the term "anergy" refers to a state of non-responsiveness to antigenic stimulation that results from incomplete or insufficient signals delivered through the T cell receptor (TCR). T cell anergy also occurs upon stimulation by antigen in the absence of co-stimulation and can result in cells that become refractory to subsequent activation by antigen even in co-stimulatory settings. The non-responsive state can often be abrogated by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or do not acquire effector functions.

[0108] As used herein, the term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that results from persistent TCR signaling that occurs during many chronic infections and cancers. This is distinguished from anergy in that it results from persistent signaling rather than through incomplete or insufficient signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors and a transcriptional state distinct from functional effector or memory T cells. Exhaustion impedes the optimal control of disease (e.g., infections and tumors). Exhaustion can be caused by both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and cell-intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways as described herein).

[0109] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have a sustained or amplified biological function, or regenerating or reactivating depleted or inactive T cells. Examples of enhancing T cell function include increased secretion of gamma-interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance), compared to pre-intervention levels. In one embodiment, the level of enhancement is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or more. Methods for measuring this enhancement are known to those of skill in the art.

[0110] Immune checkpoint inhibitors can be inhibitory nucleic acid molecules. As used herein, the terms "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" refer to nucleic acid molecules, such as DNA or RNA, that completely or partially reduce, inhibit, interfere with, or negatively regulate one or more checkpoint proteins. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0111] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule that can decrease protein expression, particularly the expression of checkpoint proteins, such as the checkpoint proteins described herein. Oligonucleotides are typically short DNA or RNA molecules containing 2 to 50 nucleotides. Oligonucleotides can be single-stranded or double-stranded. Checkpoint inhibitor oligonucleotides can be antisense oligonucleotides.

[0112] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly the nucleic acid sequence (or a fragment thereof) of a checkpoint protein. Antisense RNA is typically used to prevent the protein translation of mRNA, such as the mRNA encoding a checkpoint protein, by binding to the mRNA. Antisense DNA is typically used to target specific, complementary (coding or non-coding) RNA. When binding occurs, such DNA / RNA hybrids can be degraded by the enzyme RNase H. Furthermore, morpholino antisense oligonucleotides can be used for gene knockdown in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed a B7-H4-specific morpholino that specifically blocks B7-H4 expression in macrophages, resulting in increased T cell proliferation and decreased tumor volume in mice by tumor-associated antigen (TAA)-specific T cells.

[0113] The terms "siRNA" or "small interfering RNA" or "small inhibitory RNA" are used interchangeably herein and refer to double-stranded RNA molecules having a typical length of 20 to 25 base pairs that interfere with the expression of a specific gene, for example, a gene encoding a checkpoint protein having a complementary nucleotide sequence. In one embodiment, the siRNA interferes with the mRNA and thus blocks translation, for example, the translation of an immune checkpoint protein. Transfection of exogenous siRNA can be used for gene knockdown, but its effect may be only transient, especially in cells that are dividing rapidly. Stable transfection can be achieved, for example, by the use of RNA modifications or expression vectors. Modifications and vectors useful for stable transfection of cells with siRNA are known in the art. The siRNA sequence can also be modified to introduce a short loop between the two strands to obtain "small hairpin RNA" or "shRNA". shRNA can be processed by Dicer into functional siRNA. shRNA has a relatively low degradation rate and metabolic turnover. Thus, an immune checkpoint inhibitor can be an shRNA.

[0114] As used herein, the term "aptamer" refers to a single-stranded nucleic acid molecule, typically 25 to 70 nucleotides in length, such as DNA or RNA, that can bind to a target molecule, such as a polypeptide. In one embodiment, the aptamer binds to an immune checkpoint protein, such as an immune checkpoint protein described herein. For example, an aptamer according to the present disclosure can specifically bind to an immune checkpoint protein or polypeptide, or a molecule in a signaling pathway that regulates the expression of an immune checkpoint protein or polypeptide. The production and therapeutic use of aptamers are well known in the art (see, for example, U.S. Patent No. 5,475,096).

[0115] The terms "small molecule inhibitor" or "small molecule" are used interchangeably herein, and refer to a low molecular weight organic compound, usually up to 1000 Daltons, that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more of the above checkpoint proteins. Such small molecule inhibitors are usually synthesized by organic chemistry but can also be isolated from natural sources such as plants, fungi, and microorganisms. The low molecular weight allows the small molecule inhibitor to rapidly diffuse across cell membranes. For example, various A2AR antagonists known in the art are organic compounds with a molecular weight of less than 500 Daltons.

[0116] An immune checkpoint inhibitor can be an antibody, an antigen-binding fragment thereof, an antibody mimetic, or a fusion protein comprising an antibody portion having the required specificity antigen-binding fragment. The antibody or its antigen-binding fragment is as described herein. An antibody or its antigen-binding fragment that is an immune checkpoint inhibitor particularly includes an antibody or its antigen-binding fragment that binds to an immune checkpoint protein such as an immune checkpoint receptor or an immune checkpoint receptor ligand. The antibody or antigen-binding fragment can also be conjugated to additional moieties described herein. In particular, the antibody or its antigen-binding fragment is a chimeric, humanized, or human antibody. Preferably, the immune checkpoint inhibitor antibody or its antigen-binding fragment is an antagonist of an immune checkpoint receptor or an immune checkpoint receptor ligand.

[0117] In a preferred embodiment, the antibody that is an immune checkpoint inhibitor is an isolated antibody. An antibody or antigen-binding fragment thereof that is an immune checkpoint inhibitor according to the present disclosure can also be an antibody that cross-competes for antigen binding with any known immune checkpoint inhibitor antibody. In certain embodiments, the immune checkpoint inhibitor antibody cross-competes with one or more of the immune checkpoint inhibitor antibodies described herein. The ability of an antibody to cross-compete for binding to an antigen indicates that these antibodies can bind to the same epitope region of the antigen, or, if binding to a different epitope, can sterically hinder the known immune checkpoint inhibitor antibody from binding to that particular epitope region. These cross-competing antibodies are expected to block the binding of the immune checkpoint to its ligand by binding to the same epitope or by sterically hindering ligand binding, and thus can have functional properties very similar to those of the antibodies with which they cross-compete. Cross-competing antibodies can be readily identified based on their ability to cross-compete with one or more known antibodies in standard binding assays such as surface plasmon resonance analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0118] In certain embodiments, the antibody or antigen-binding fragment thereof that cross-competes for binding to a given antigen with one or more known antibodies, or that binds to the epitope region of the same given antigen as one or more known antibodies, is a monoclonal antibody. For administration to a human patient, these cross-competing antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0119] The checkpoint inhibitor can also be in a soluble form of the molecule (or variant thereof) itself, such as in the form of soluble PD-L1 or a PD-L1 fusion.

[0120] In the context of the present disclosure, two or more checkpoint inhibitors can be used, and the two or more checkpoint inhibitors target distinct checkpoint pathways or the same checkpoint pathway. Preferably, the two or more checkpoint inhibitors are distinct checkpoint inhibitors. Preferably, when two or more distinct checkpoint inhibitors are used, in particular at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 distinct checkpoint inhibitors are used, preferably 2, 3, 4 or 5 distinct checkpoint inhibitors are used. A pesticidal agent is used, more preferably 2, 3 or 4 different checkpoint inhibitors are used, even more preferably 2 or 3 different checkpoint inhibitors are used, and most preferably 2 different checkpoint inhibitors are used. Preferred examples of combinations of different checkpoint inhibitors include a combination of an inhibitor of PD-1 signaling and an inhibitor of CTLA-4 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of TIGIT signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of B7-H3 and / or B7-H4 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of BTLA signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of KIR signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of LAG-3 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of TIM-3 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of CD94 / NKG2A signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of IDO signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of adenosine signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of VISTA signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of Siglec signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of CD20 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of GARP signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of CD47 signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of PVRIG signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of CSF1R signaling, a combination of an inhibitor of PD-1 signaling and an inhibitor of NOX signaling, and a combination of an inhibitor of PD-1 signaling and an inhibitor of TDO signaling.

[0121] In certain embodiments, the suppressive immunomodulatory agent (immune checkpoint blocker) is a component of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the PD-1 signaling pathway. In certain embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In certain embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor such as a PD-L1 inhibitor or a PD-L2 inhibitor. In preferred embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody or an antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and one or more of its ligands are known in the art. In certain embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-1. In certain embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1, inhibits the interaction with PD-1, thereby increasing immune activity. In certain embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L2, inhibits the interaction with PD-1, thereby increasing immune activity.

[0122] In certain embodiments, the suppressive immunomodulatory agent is a component of the CTLA-4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the CTLA-4 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 inhibitor. In certain embodiments, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 ligand inhibitor.

[0123] In certain embodiments, the inhibitory immunomodulatory agent is a component of the TIGIT signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the TIGIT signaling pathway. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor.

[0124] In certain embodiments, the inhibitory immunomodulatory agent is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of B7-H3 and / or B7-H4. Accordingly, certain embodiments of the present disclosure provide for administration to a subject an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. The B7 family does not have defined receptors, but their ligands are upregulated in tumor cells or tumor infiltrating cells. Preclinical mouse models have shown that blockade of these ligands can enhance anti-tumor immunity.

[0125] In certain embodiments, the inhibitory immunomodulatory agent is a component of the BTLA signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the BTLA signaling pathway. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is a BTLA inhibitor. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is an HVEM inhibitor.

[0126] In certain embodiments, the inhibitory immunomodulator is a component of one or more KIR signaling pathways. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of one or more KIR signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR inhibitor. In certain embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR ligand inhibitor. For example, a KIR inhibitor according to the present disclosure can be an anti-KIR antibody that binds to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0127] In certain embodiments, the inhibitory immunomodulator is a component of the LAG-3 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the LAG-3 signaling pathway. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 inhibitor. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 ligand inhibitor.

[0128] In certain embodiments, the inhibitory immunomodulator is a component of the TIM-3 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the TIM-3 signaling pathway. In certain embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 inhibitor. In certain embodiments, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 ligand inhibitor.

[0129] In certain embodiments, the suppressive immunomodulator is a component of the CD94 / NKG2A signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the CD94 / NKG2A signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A inhibitor. In certain embodiments, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A ligand inhibitor.

[0130] In certain embodiments, the suppressive immunomodulator is a component of the IDO signaling pathway . Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the IDO signaling pathway, such as an IDO inhibitor.

[0131] In certain embodiments, the suppressive immunomodulator is a component of the adenosine signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the adenosine signaling pathway. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD39 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD73 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2AR inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2BR inhibitor.

[0132] In certain embodiments, the suppressive immunomodulator is a component of the VISTA signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the VISTA signaling pathway. In certain embodiments, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor.

[0133] In certain embodiments, the suppressive immunomodulatory agent is a component of one or more Siglec signaling pathways. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of one or more Siglec signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec inhibitor. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec ligand inhibitor.

[0134] In certain embodiments, the suppressive immunomodulatory agent is a component of the CD20 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the CD20 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor.

[0135] In certain embodiments, the suppressive immunomodulatory agent is a component of the GARP signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the GARP signaling pathway. In certain embodiments, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor.

[0136] In certain embodiments, the suppressive immunomodulatory agent is a component of the CD47 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of a checkpoint inhibitor of the CD47 signaling pathway. In certain embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a CD47 inhibitor. In certain embodiments, the checkpoint inhibitor of the CD47 signaling pathway is a SIRPα inhibitor.

[0137] In certain embodiments, the suppressive immunomodulatory agent is a component of the PVRIG signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the PVRIG signaling pathway. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG inhibitor. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG ligand inhibitor.

[0138] In certain embodiments, the suppressive immunomodulatory agent is a component of the CSF1R signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the CSF1R signaling pathway. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1R inhibitor. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1 inhibitor.

[0139] In certain embodiments, the suppressive immunomodulatory agent is a component of the NOX signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the NOX signaling pathway, such as a NOX inhibitor.

[0140] In certain embodiments, the suppressive immunomodulatory agent is a component of the TDO signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject a checkpoint inhibitor of the TDO signaling pathway, such as a TDO inhibitor.

[0141] Exemplary PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see U.S. Patent No. 8,735,553, International Publication No. 2015 / 35606, and U.S. Patent Application Publication No. 2015 / 0079109), semiprimab (Regeneron; see International Publication No. 2015 / 112800), and lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409A1, h409A16, and h409A17 in International Publication No. 2008 / 156712), AB137132 (Abcam), EH12.2H7, and RMP1-14 (Catalog No. BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; International Publication No. 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; International Publication No. 2008 / 156712), pidilizumab (CT-011; CureTech; see Hardy et al., 1994, Cancer Res., 54(22):5793-6 and International Publication No. 2009 / 101611), PDR001 (Novartis; see International Publication No. 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see International Publication No. 2012 / 145493), TSR-042 (see International Publication No. 2014 / 179664), REGN-2810 (H4H7798N; see U.S. Patent Application Publication No. 2015 / 0203579), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al., 2007, J. Hematol. Oncol.70:136 (see, e.g., Li et al., 2016, Int J Mol Sci 17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), BGB-A317 (BeiGene; see WO 2015 / 35606 and US Patent Application Publication No. 2015 / 0079109), BI754091, SHR-1210 (see WO 2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 described in WO 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al., 2017, J. Hematol. Oncol. 70:136), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), e.g., U.S. Patent No. 7,48... U.S. Patent No. 8,802, U.S. Patent No. 8,008,449, U.S. Patent No. 8,168,757, International Publication No. 03 / 042402, International Publication No. 2010 / 089411 (which further discloses anti-PD-L1 antibodies), International Publication No. 2010 / 036959, International Publication No. 2011 / 159877 (which further discloses antibodies against TIM-3), International Publication No. 2011 / 082400, International Publication No. 2011 / 161699, International Publication No. 2009 / 014708, International Publication No. 03 / 099196, International Publication No. 2009 / 114335, International Publication No. 2012 / 145493 (which further discloses antibodies against PD-L1), International Publication No. 2015 / 035606, International Publication No. 2014 / 055648 (which further discloses anti-KIR antibodies), U.S. Patent Application Publication No. 2018 / 0185482 (which further discloses anti-PD-L1 and anti-TIGIT antibodies), U.S. Patent No. 8,008,449, U.S. Patent No. 8,779,105, U.S. Patent No. 6,808,710, U.S. Patent No. 8,168,757, anti-PD-1 antibodies described in U.S. Patent Application Publication No. 2016 / 0272708 and U.S. Patent No. 8,354,509, for example, low molecular weight antagonists against the PD-1 signaling pathway disclosed in Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, for example, siRNAs directed against PD-1 disclosed in International Publication No. 2019 / 000146 and International Publication No. 2018 / 103501, soluble PD-1 proteins disclosed in International Publication No. 2018 / 222711, and oncolytic viruses containing soluble forms of PD-1 described, for example, in International Publication No. 2018 / 022831.

[0142] In certain embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI754091, or SHR-1210.

[0143] Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors, including but not limited to anti-PD-L1 antibodies such as MEDI4736 (durvalumab; AstraZeneca; see International Publication No. WO 2011 / 066389), MSB-0010718C (see U.S. Patent Application Publication No. US 2014 / 0341917), YW243.55.S70 (see International Publication No. WO 2010 / 077634 and SEQ ID NO: 20 of U.S. Patent No. 8,217,149), MIH1 (Affymetrix eBioscience; see European Patent No. EP 3 230 319), MDX-1105 (Roche / Genentech; see International Publication No. WO 2013019906 and U.S. Patent No. 8,217,149), STI-1014 (Sorrento; see International Publication No. WO 2013 / 181634), CK-301 (Checkpoint Therapeutics), KN035 (3D Med / Alphamab; see Zhang et al., 2017, Cell Discov. 3:17004), atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see U.S. Patent No. 9,724,413), BMS-936559 (Bristol Myers Squibb; see U.S. Patent No. 7,943,743, International Publication No. WO 2013 / 173223), avelumab (bavencio; see U.S. Patent Application Publication No. US 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (also called Proclaim-CX-072; CytomX; see International Publication No. WO 2016 / 149201), FAZ053, KN035 (see International Publication No. WO 2017020801 and International Publication No. WO 2017020802), MDX-1105 (see U.S. Patent Application Publication No. US 2015 / 0320859), 3G10, 12A4 (also called BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, and anti-P disclosed in U.S. Patent No. 7,943,743 It includes anti-PD-L1 antibodies described in WO 2010 / 077634, US Patent No. 8,217,149, WO 2010 / 036959, WO 2010 / 077634, WO 2011 / 066342, US Patent No. 8,217,149, US Patent No. 7,943,743, WO 2010 / 089411, US Patent No. 7,635,757, US Patent No. 8,217,149, US Patent Application Publication No. 2009 / 0317368, WO 2011 / 066389, WO 2017 / 034916, WO 2017 / 020291, WO 2017 / 020858, WO 2017 / 020801, WO 2016 / 111645, WO 2016 / 197367, WO 2016 / 061142, WO 2016 / 149201, WO 2016 / 000619, WO 2016 / 160792, WO 2016 / 022630, WO 2016 / 007235, WO 2015 / 179654, WO 2015 / 173267, WO 2015 / 181342, WO 2015 / 109124, WO 2018 / 222711, WO 2015 / 112805, WO 2015 / 061668, WO 2014 / 159562, WO 2014 / 165082, WO 2014 / 100079.

[0144] Exemplary CTLA-4 inhibitors include, but are not limited to, the monoclonal antibodies ipilimumab (Yervoy; Bristol Myers Squibb) and tremelimumab (Pfizer / Medlmmune), toripalimab, AGEN-1884 (Agenus) and ATOR-1015, International Publication No. WO 2001 / 014424, U.S. Patent Application Publication No. 2005 / 0201994, European Patent No. 1212422, U.S. Patent No. 5,811,097, U.S. Patent No. 5,855,887, U.S. Patent No. 6,051,227, U.S. Patent No. 6,682,736, U.S. Patent No. 6,984,720, International Publication No. 01 / 14424, International Publication No. 00 / 37504, U.S. Patent Application Publication No. 2002 / 0039581, U.S. Patent Application Publication No. 2002 / 086014, International Publication No. 98 / 42752, U.S. Patent No. 6,207,156, U.S. Patent No. 5,977,318, U.S. Patent No. 7,109,003 and U.S. Patent No. 7,132,281; the dominant negative protein abatacept (Orencia; see European Patent No. 2855533) containing the Fc region of IgG1 fused to CTLA-4 ECD, and belatacept (Nulojix; see International Publication No. 2014 / 207748); second generation high affinity CTLA-4-Ig variants having two amino acid substitutions in CTLA-4 ECD relative to abatacept; soluble CTLA-4 polypeptides such as RG2077 and CTLA4-IgG4m (see U.S. Patent No. 6,750,334); anti-CTLA-4 aptamers; and siRNAs directed against CTLA-4 such as those disclosed in U.S. Patent Application Publication No. 2015 / 203848. Exemplary CTLA-4 ligand inhibitors are described in Pile et al., 2015 (Encyclopedia of Inflammatory Diseases, M. Parnham (ed.), doi:10.1007 / 978-3-0348-0620-6_20).

[0145] Exemplary checkpoint inhibitors of the TIGIT signaling pathway include, but are not limited to, anti-TIGIT antibodies such as BMS-986207, COM902 (CGEN-15137; Compugen), AB154 (Arcus Biosciences) or etigilimab (OMP-313M32; OncoMed Pharmaceuticals), or the antibodies disclosed in International Publication No. WO 2017 / 059095, particularly "MAB10", the antibodies disclosed in US Patent Application Publication No. US 2018 / 0185482, International Publication No. WO 2015 / 009856 and US Patent Application Publication No. US 2019 / 0077864.

[0146] Exemplary checkpoint inhibitors of B7-H3 include, but are not limited to, the Fc-optimized monoclonal antibody enoblituzumab (MGA271; Macrogenics; US Patent Application Publication No. US 2012 / 0294796) and the anti-B7-H3 antibodies MGD009 (Macrogenics) and pidilizumab (see US Patent No. 7332582).

[0147] Exemplary B7-H4 inhibitors include, but are not limited to, the antibodies described in Dangaj et al., 2013 (Cancer Research 73:4820-9) and Smith et al., 2014 (Gynecol Oncol, 134:181-189), International Publication No. WO 2013 / 025779 (e.g., 2D1 encoded by SEQ ID NOs: 3 and 4, 2H9 encoded by SEQ ID NOs: 37 and 39, and 2E11 encoded by SEQ ID NOs: 41 and 43), and International Publication No. WO 2013 / 067492 (e.g., antibodies having an amino acid sequence selected from SEQ ID NOs: 1-8), such as the morpholino antisense oligonucleotides described by Kryczek et al., 2006 (J Exp Med, 203:871-81), or the soluble recombinant form of B7-H4 disclosed, for example, in US Patent Application Publication No. US 2012 / 0177645.

[0148] Exemplary BTLA inhibitors include, but are not limited to, Crawford and Wherry, 2009 (J Leukocyte Biol 86:5-8), International Publication No. WO 2011 / 014438 (e.g., 4C7 or an antibody comprising heavy and light chains according to SEQ ID NOs: 8 and 15, and / or SEQ ID NOs: 11 and 18), International Publication No. WO 2014 / 183885 (e.g., the antibody deposited under number CNCM I-4752), and anti-BTLA antibodies described in U.S. Patent Application Publication No. 2018 / 155428.

[0149] Checkpoint inhibitors of KIR signaling include, but are not limited to, the monoclonal antibody lirilumab (1-7F9; IPH2102; see U.S. Patent No. 8,709,411), IPH4102 (Innate Pharma; see Marie-Cardine et al., 2014, Cancer 74(21):6060-70), and anti-KIR antibodies disclosed in, for example, U.S. Patent Application Publication No. 2018 / 208652, U.S. Patent Application Publication No. 2018 / 117147, U.S. Patent Application Publication No. 2015 / 344576, International Publication No. WO 2005 / 003168, International Publication No. WO 2005 / 009465, International Publication No. WO 2006 / 072625, International Publication No. WO 2006 / 072626, International Publication No. WO 2007 / 042573, International Publication No. WO 2008 / 084106 (e.g., an antibody comprising heavy and light chains according to SEQ ID NOs: 2 and 3), International Publication No. WO 2010 / 065939, International Publication No. WO 2012 / 071411, International Publication No. WO 2012 / 160448, and International Publication No. WO 2014 / 055648.

[0150] LAG-3 inhibitors include, but are not limited to, anti-LAG-3 antibodies BMS-986016 (Bristol-Myers Squibb; see International Publication No. WO 2014 / 008218 and International Publication No. WO 2015 / 116539), 25F7 (see U.S. Patent Application Publication No. 2011 / 0150892), IMP731 (see International Publication No. WO 2008 / 132601), H5L7BW (see International Publication No. WO 2014 / 140180), MK-4280 (28G-10; Merck; see International Publication No. WO 2016 / 028672), REGN3767 (Regeneron / Sanofi), BAP050 (see International Publication No. WO 2017 / 019894), IMP-701 (LAG-525; Novartis), Sym022 (Symphogen), TSR-033 (Tesaro), MGD013 (bispecific DART antibody targeting LAG-3 and PD-1 developed by MacroGenics), BI754111 (Boehringer Ingelheim), FS118 (bispecific antibody targeting LAG-3 and PD-1 developed by F-star), GSK2831781 (GSK), and International Publication No. WO 2009 / 044273, International Publication No. WO 2008 / 132601, International Publication No. WO 2015 / 042246, European Patent No. EP 2320940, U.S. Patent Application Publication No. 2019 / 169294, U.S. Patent Application Publication No. 2019 / 169292, International Publication No. WO 2016 / 028672, International Publication No. WO 2016 / Antibodies disclosed in JP 126858, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 220569, WO 2017 / 087589, WO 2017 / 219995, WO 2017 / 019846, WO 2017 / 106129, WO 2017 / 062888, WO 2018 / 071500, WO 2017 / 087901, US 2017 / 0260271, WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 015560, WO 2017 / 025498, WO 2017 / 149143, WO 2018 / 069500, WO 2018 / 083087, WO 2018 / 034227, WO 2014 / 140180, the LAG-3 antagonist protein AVA-017 (Avacta), the soluble LAG-3 fusion protein IMP321 (eftilagimod alpha; Immutep; see European Patent No. 2205257 and Brignone et al., 2007, J. Immunol., 179:4202-4211), and the soluble LAG-3 protein disclosed in WO 2018 / 222711 are included.

[0151] TIM-3 inhibitors include, but are not limited to, antibodies that target TIM-3, such as F38-2E2 (BioLegend), cobolimab (TSR-022; Tesaro), LY3321367 (Eli Lilly), MBG453 (Novartis), and antibodies disclosed in, for example, WO 2013 / 006490, WO 2018 / 085469 (e.g., antibodies comprising heavy and light chain sequences encoded by the nucleic acid sequences of SEQ ID NOs: 3 and 4), WO 2018 / 106588, WO 2018 / 106529 (e.g., antibodies comprising heavy and light chain sequences of SEQ ID NOs: 8-11).

[0152] TIM-3 ligand inhibitors include, but are not limited to, CEACAM inhibitors such as anti-CEACAM1 antibody CM10 (see cCAM Biotherapeutics; WO 2013 / 054331), antibodies disclosed in WO 2015 / 075725 (e.g., CM-24, 26H7, 5F4, TEC-11, 12-140-4, 4 / 3 / 17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, M8.7.7, D11-AD11, HEA81, Bl.l, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7, 12-140-5, scFv DIATHIS1, TET-2; cCAM Biotherapeutics), antibodies described in Watt et al., 2001 (Blood, 98:1469-1479) and WO 2010 / 12557, and PtdSer inhibitors such as bavituximab (Peregrine).

[0153] CD94 / NKG2A inhibitors include, but are not limited to, monalizumab (IPH2201; Innate Pharma), and antibodies and methods for their production disclosed in U.S. Patent No. 9,422,368 (e.g., humanized Z199; see European Patent No. 2628753), European Patent No. 3193929, and WO 2016 / 032334 (e.g., humanized Z270; see European Patent No. 2628753).

[0154] IDO inhibitors include, but are not limited to, exiguamine A, epacadostat (INCB024360; InCyte; see U.S. Patent No. 9624185), indoximod (Newlink Genetics; CAS No.: 110117-83-4), NLG919 (Newlink Genetics / Genentech; CAS No.: 1402836-58-1), GDC-0919 (Newlink Genetics / Genentech; CAS No. 1402836-58-1), F001287 (Flexus Biosciences / BMS; CAS No.: 2221034-29-1), KHK2455 (Cheong et al., 2018, Expert Opin Ther Pat. 28(4 ): 317-330), PF-06840003 (see International Publication No. 2016 / 181348), navoximod (RG6078, GDC-0919, NLG919; CAS No.: 1402837-78-8), linrodostat (BMS-986205; Bristol-Myers Squibb; CAS No.: 1923833-60-6), 1-methyl-tryptophan, small molecules such as pyrrolidine-2,5-dione derivatives (see International Publication No. 2015 / 173764), and IDO inhibitors disclosed by Sheridan, 2015, Nat Biotechnol 33: 321-322.

[0155] CD39 inhibitors include, but are not limited to, A001485 (Arcus Biosciences), PSB069 (CAS No.: 78510-31-3), and the anti-CD39 monoclonal antibody IPH5201 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8: 2411-2425.E9).

[0156] CD73 inhibitors include, but are not limited to, anti-CD73 antibodies such as CPI-006 (Corvus Pharmaceuticals), MEDI9447 (MedImmune; see International Publication No. 2016 / 075099), IPH5301 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9), anti-CD73 antibodies described in International Publication No. 2018 / 110555, small molecule inhibitor PBS12379 (Tocris Bioscience; CAS number: 1802226-78-3), A000830, A001190 and A001421 (Arcus Biosciences; see Becker et al., 2018, Cancer Research 78(13 Suppl):3691-3691, doi:10.1158 / 1538-7445.AM2018-3691), CB-708 (Calithera Biosciences) and diphosphonates based on purine cell cytotoxic nucleoside analogs described by Allard et al., 2018 (Immunol Rev., 276(1):121-144).

[0157] A2AR inhibitors include, but are not limited to, istradefylline (KW-6002; CAS No.: 155270-99-8), PBF-509 (Palobiopharma), cirazoline (CPI-444: Corvus Pharma / Genentech; CAS No.: 1202402-40-1), ST1535 ([2-butyl-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-ylamine]; CAS No.: 496955-42-1), ST4206 (see Stasi et al., 2015, Europ J Pharm 761:353-361; CAS No.: 1246018-36-9), tozadenant (SYN115; CAS No.: 870070-55-6), V81444 (see International Publication No. WO 2002 / 055082), preladenant (SCH420814; Merck; CAS No.: 377727-87-2), bipadenant (BIIB014; CAS No.: 442908-10-3), ST1535 (CAS No.: 496955-42-1), SCH412348 (CAS No.: 377727-26-9), SCH442416 (Axon 2283; Axon Medchem; CAS No.: 316173-57-6), ZM241385 (4-(2-(7-amino-2-(2-furyl)-(1,2,4)triazolo(2,3-a)-(1,3,5)triazin-5-yl-amino)ethyl)phenol; Cas No.: 139180-30-6), AZD4635 (AstraZeneca), AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), and SCH58261 (see Popoli et al., 2000, Neuropsychopharm 22:522-529; CAS No.: 160098-96-4), etc.

[0158] A2BR inhibitors include, but are not limited to, AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), MRS1706 (CAS No.: 26462 2-53-9), GS6201 (CAS No.: 752222-83-6), and PBS1115 (CAS No.: 152529-79-8).

[0159] VISTA inhibitors include, but are not limited to, anti-VISTA antibodies such as JNJ-61610588 (onartuzumab; Janssen Biotech) and small molecule inhibitor CA-170 (anti-PD-L1 / L2 and anti-VISTA small molecule; CAS number: 1673534-76-3).

[0160] Siglec inhibitors include, but are not limited to, anti-Sigle-7 antibodies disclosed in US Patent Application Publication No. 2019 / 023786 and International Publication No. 2018 / 027203 (for example, an antibody comprising a variable heavy chain region according to SEQ ID NO: 1 and a variable light chain region according to SEQ ID NO: 15), anti-Siglec-2 antibody inotuzumab ozogamicin (Besponsa; see US Patent No. 8153768 and US Patent No. 9642918), anti-Siglec-3 antibody gemtuzumab ozogamicin (Mylotarg; see US Patent No. 9359442) or antibodies comprising CDRs according to US Patent Application Publication No. 2019 / 062427, US Patent Application Publication No. 2019 / 023786, International Publication No. 2019 / 011855, International Publication No. 2019 / 011852 (for example, SEQ ID NOs: 171-176, or 3 and 4, or 5 and 6, or 7 and 8, or 9 and 10, or 11 and 12, or 13 and 14, or 15 and 16, or 17 and 18, or 19 and 20, or 21 and 22, or 23 and 24, or 25 and 26), anti-Siglec-9 antibodies disclosed in US Patent Application Publication No. 2017 / 306014 and European Patent No. 3146979.

[0161] CD20 inhibitors include, but are not limited to, anti-CD20 antibodies such as rituximab (RITUXAN; IDEC-102; IDEC-C2B8; see U.S. Patent No. 5,843,439), ABP798 (rituximab biosimilar), ofatumumab (2F2; see International Publication No. 2004 / 035607), obinutuzumab, ocrelizumab (2h7; see International Publication No. 2004 / 056312), ibritumomab tiuxetan (Zevalin), tositumomab, ublituximab (LFB-R603; LFB Biotechnologies), and the antibodies disclosed in U.S. Patent Application Publication No. 2018 / 0036306 (e.g., antibodies comprising light and heavy chains according to SEQ ID NOs: 1-3 and 4-6, or 7 and 8, or 9 and 10).

[0162] GARP inhibitors include, but are not limited to, anti-GARP antibodies such as ARGX-115 (arGEN-X), and the antibodies and methods for their production disclosed in U.S. Patent Application Publication No. 2019 / 127483, U.S. Patent Application Publication No. 2019 / 016811, U.S. Patent Application Publication No. 2018 / 327511, U.S. Patent Application Publication No. 2016 / 251438, and European Patent No. 3253796.

[0163] CD47 inhibitors include, but are not limited to, anti-CD47 antibodies such as HuF9-G4 (Stanford University / Forty Seven), CC-90002 / INBRX-103 (Celgene / Inhibrx), SRF231 (Surface Oncology), IBI188 (Innovent Biologics), AO-176 (Arch Oncology), TG-1801 (NI-1701; bispecific monoclonal antibody targeting CD47 and CD19; Novimmune / TG Bispecific antibodies targeting CD47, including NI-001 (Novimmune), NI-1701 (Novimmune), and NI-1801 (bispecific monoclonal antibody targeting CD47 and mesothelin; Novimmune), as well as CD47 fusion proteins, such as ALX148 (ALX Oncology; see Kauder et al., 2019, PLoS One, doi:10.1371 / journal.pone.0201832).

[0164] SIRPα inhibitors include, but are not limited to, anti-SIRPα antibodies, such as OSE-172 (Boehringer Ingelheim / OSE), FSI-189 (Forty Seven), anti-SIRPα fusion proteins, such as TTI-621 and TTI-662 (Trillium Therapeutics; see International Publication No. WO 2014 / 094122).

[0165] PVRIG inhibitors include, but are not limited to, anti-PVRIG antibodies such as COM701 (CGEN-15029) and antibodies such as those disclosed in International Publication No. WO 2018 / 033798 (e.g., CHA.7.518.1H4 (S241P), CHA.7.538.1.2.H4 (S241P), CPA.9.086H4 (S241P), CPA.9.083H4 (S241P), CHA.9.547.7.H4 (S241P), CHA.9.547.13.H4 (S241P), and antibodies comprising the variable heavy chain domain according to SEQ ID NO: 5 and the variable light chain domain according to SEQ ID NO: 10 of International Publication No. WO 2018 / 033798, or antibodies comprising the heavy chain according to SEQ ID NO: 9 and the light chain according to SEQ ID NO: 14; International Publication No. WO 2018 / 033798 further discloses anti-TIGIT antibodies and combination therapies with anti-TIGIT antibodies and anti-PVRIG antibodies), International Publication No. WO 2016 / 134333, International Publication No. WO 2018 / 017864 (e.g., antibodies comprising a heavy chain according to SEQ ID NOs: 5-7 having at least 90% sequence identity with SEQ ID NO: 11 and / or a light chain according to SEQ ID NOs: 8-10 having at least 90% sequence identity with SEQ ID NO: 12, or antibodies encoded by SEQ ID NO: 13 and / or 14, or SEQ ID NO: 24 and / or 29, or other antibodies disclosed in International Publication No. WO 2018 / 017864) and methods for their production, and anti-PVRIG antibodies and fusion peptides disclosed in International Publication No. WO 2016 / 134335.

[0166] CSF1R inhibitors include, but are not limited to, anti-CSF1R antibody cabiralizumab (FPA008; FivePrime; see International Publication No. 2011 / 140249, International Publication No. 2013 / 169264, and International Publication No. 2014 / 036357), IMC-CS4 (Eii Lilly), emactuzumab (R05509554; Roche), RG7155 (International Publication No. 2011 / 70024, International Publication No. 2011 / 107553, International Publication No. 2011 / 131407, International Publication No. 2013 / 87699, International Publication No. 2013 / 119716, International Publication No. 2013 / 132044), and small molecule inhibitors BLZ945 (CAS No.: 953769-46-5) and pexidartinib (PLX3397; Selleckchem; CAS No.: 1029044-16-3).

[0167] CSF1 inhibitors include, but are not limited to, anti-CSF1 antibodies disclosed in European Patent No. 1223980 and Weir et al., 1996 (J Bone Mineral Res 11:1474-1481), International Publication No. 2014 / 132072, and antisense DNA and RNA disclosed in International Publication No. 2001 / 030381.

[0168] Exemplary NOX inhibitors include, but are not limited to, NOX1 inhibitors such as small molecule ML171 (Gianni et al., 2010, ACS Chem Biol 5(10):981-93, NOS31 (Yamamoto et al., 2018, Biol Pharm Bull.41(3):419-426), NOX2 inhibitors such as small molecule ceplene (histamine dihydrochloride; CAS No.: 56-92-8), BJ-1301 (Gautam et al., 2017, Mol Cancer Ther 16(10):2144-2156; CAS No.: 1287234-48-3), and inhibitors described by Lu et al., 2017, Biochem Pharmacol 143:25-38, NOX4 inhibitors such as small molecule inhibitor VAS2870 (Altenhofer et al., 2012, Cell Mol Life Sciences 69(14):2327-2343), diphenylene It includes iodonium (CAS No.: 244-54-2) and GKT137831 (CAS No.: 1218942-37-0; see Tang et al., 2018, 19(10):578-585).

[0169] TDO inhibitors include, but are not limited to, 4-(indol-3-yl)-pyrazole derivatives (see U.S. Patent No. 9,126,984 and U.S. Patent Application Publication No. 2016 / 0263087), 3-indole substituted derivatives (see International Publication No. 2015 / 140717, International Publication No. 2017 / 025868, International Publication No. 2016 / 147144), 3-(indol-3-yl)-pyridine derivatives (see U.S. Patent Application Publication No. 2015 / 0225367 and International Publication No. 2015 / 121812), dual IDO / TDO antagonists, such as the small molecule dual IDO / TDO inhibitors disclosed in International Publication No. 2015 / 150097, International Publication No. 2015 / 082499, International Publication No. 2016 / 026772, International Publication No. 2016 / 071283, International Publication No. 2016 / 071293, International Publication No. 2017 / 007700, and the small molecule inhibitor CB548 (Kim, C et al., 2018, Annals Oncol 29(Suppl 8):viii400-viii441).

[0170] According to the present disclosure, an immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint protein, but preferably not an inhibitor of a stimulatory checkpoint protein. As described herein, inhibitors of several CTLA-4, PD-1, TIGIT, B7-H3, B7-H4, BTLA, KIR, LAG-3, TIM-3, CD94 / NKG2A, IDO, A2AR, A2BR, VISTA, Siglec, CD20, CD39, CD73, GARP, CD47, PVRIG, CSF1R, NOX and TDO, as well as inhibitors of their respective ligands, are known, and some of these are already in clinical trials or even approved. Based on these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors can be developed. In particular, known inhibitors of preferred immune checkpoint proteins can be used as such, or analogs thereof, in particular chimerized, humanized or human forms of antibodies and antibodies that cross-compete with any of the antibodies described herein can be used.

[0171] It will be understood by those skilled in the art that other immune checkpoint targets can also be targeted by an antagonist or antibody as long as the targeting results in stimulation of an immune response such as an anti-tumor immune response reflected by an increase in T cell proliferation, enhanced T cell activation and / or increased cytokine production (e.g., IFN-γ, IL-2).

[0172] Checkpoint inhibitors can be administered by any method and any route known in the art. The mode and route of administration will depend on the type of checkpoint inhibitor used.

[0173] Checkpoint inhibitors can be administered in the form of any suitable pharmaceutical composition described herein.

[0174] Checkpoint inhibitors can be administered in the form of nucleic acid molecules such as DNA or RNA molecules encoding immune checkpoint inhibitors, for example inhibitory nucleic acid molecules, or antibodies or fragments thereof. For example, antibodies can be encoded in an expression vector and delivered as described herein. Nucleic acid molecules can be delivered as such, for example in the form of plasmids or mRNA molecules, or complexed with delivery vehicles such as liposomes, lipoplexes or nucleic acid lipid particles. Checkpoint inhibitors can also be administered via oncolytic viruses containing an expression cassette encoding a checkpoint inhibitor. Checkpoint inhibitors can also be administered, for example in the form of cell therapy, by administration of autologous cells or allogeneic cells capable of expressing a checkpoint inhibitor. The term "cell based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells or stem cells) expressing an immune checkpoint inhibitor to a subject for the purpose of treating a disease or disorder (e.g., a cancer disease). In one embodiment, cell therapy includes genetically engineered cells. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor as described herein. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor that is a suppressive nucleic acid molecule, such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or a soluble immune checkpoint protein or fusion. The genetically engineered cells can also express additional agents that enhance T cell function. Such agents are known in the art. Cell therapy for use in inhibiting immune checkpoint signaling is disclosed, for example, in International Publication No. WO 2018 / 222711, which is hereby incorporated by reference in its entirety.

[0175]

[0176] ​As used herein, the term "oncolytic virus" refers to a virus that has no or minimal effect on normal cells, but can selectively replicate in cancerous or hyperproliferative cells in vitro or in vivo, and delay their growth or induce their death. Oncolytic viruses for delivering immune checkpoint inhibitors can contain an expression cassette encoding an immune checkpoint inhibitor that is a suppressive nucleic acid molecule, such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or a soluble immune checkpoint protein or fusion. The oncolytic virus is preferably replication-competent, and the expression cassette is under the control of a viral promoter, such as a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdovirus (e.g., picornavirus such as Seneca Valley virus; SVV-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retrovirus (e.g., influenza virus), measles virus, reovirus, Sindbis virus, vaccinia virus (including Copenhagen, Western Reserve, Wyeth strains) exemplified in International Publication No. WO 2017 / 209053, and adenovirus (e.g., delta-24, delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). The production and methods of use of recombinant oncolytic viruses containing soluble forms of immune checkpoint inhibitors are disclosed in International Publication No. WO 2018 / 022831, which is hereby incorporated by reference in its entirety. Oncolytic viruses can be used as attenuated viruses.

[0177] As described herein, an anti-CLDN18.2 antibody is administered to a subject, such as a patient, together with, i.e., co-administered with, a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor and the anti-CLDN18.2 antibody are administered to the subject as a single composition. In certain embodiments, the checkpoint inhibitor and the anti-CLDN18.2 antibody are administered to the subject concurrently (as separate compositions at the same time). In certain embodiments, the checkpoint inhibitor and the anti-CLDN18.2 antibody are administered to the subject separately. In certain embodiments, the checkpoint inhibitor is administered to the subject prior to the anti-CLDN18.2 antibody. In certain embodiments, the checkpoint inhibitor is administered to the subject after the anti-CLDN18.2 antibody. In certain embodiments, the checkpoint inhibitor and the anti-CLDN18.2 antibody are administered to the subject on the same day. In certain embodiments, the checkpoint inhibitor and the anti-CLDN18.2 antibody are administered to the subject on different days.

[0178] According to the present invention, the term "cytotoxic and / or cytostatic agent" includes chemotherapeutic agents or combinations of chemotherapeutic agents such as cytostatic agents. Chemotherapeutic agents can affect cells in one of the following ways: (1) damage the cell's DNA so that the cell can no longer replicate, (2) inhibit the synthesis of new DNA strands so that cell replication is no longer possible, (3) stop the cell's mitotic process so that the cell cannot divide into two cells. The cytotoxic and / or cytostatic agent can be an agent that stabilizes or increases the expression of CLDN18.2.

[0179] The term "agent stabilizing or increasing expression of CLDN18.2" refers to an agent or combination of agents that, upon its provision to a cell, results in increased RNA and / or protein levels of CLDN18.2, preferably increased levels of CLDN18.2 protein on the cell surface, compared to a situation where the cell is not provided with the agent or combination of agents. Preferably, the cell is a cancer cell, particularly a cancer cell expressing CLDN18.2, such as a cell of the cancer types described herein. The term "agent stabilizing or increasing expression of CLDN18.2" particularly refers to an agent or combination of agents that, upon its provision to a cell, results in CLDN18.2 on the surface of the cell at a high density compared to a situation where the cell is not provided with the agent or combination of agents. "Stabilizing expression of CLDN18.2" particularly includes situations where an agent or combination of agents prevents or reduces a decrease in the expression of CLDN18.2, e.g., the expression of CLDN18.2 decreases in the absence of the agent or combination of agents, or the provision of the agent or combination of agents prevents or reduces said decrease in CLDN18.2 expression. "Increasing expression of CLDN18.2" particularly includes situations where an agent or combination of agents increases the expression of CLDN18.2, e.g., the expression of CLDN18.2 decreases in the absence of the agent or combination of agents, remains essentially constant or increases, and the provision of the agent or combination of agents increases CLDN18.2 expression compared to a situation where the agent or combination of agents is not provided, and the resulting expression is higher compared to a situation where the expression of CLDN18.2 decreases, remains essentially constant or increases in the absence of the agent or combination of agents.

[0180] According to the present invention, the term "agent stabilizing or increasing expression of CLDN18.2" preferably relates to an agent or combination of agents, such as a cell growth inhibitory compound or a combination of cell growth inhibitory compounds, the provision of which to cells, particularly cancer cells, arrests or accumulates the cells in one or more phases of the cell cycle other than one or more phases of the cell cycle other than G1 and G0 phases, preferably other than the G1 phase, preferably one or more of the G2 or S phases of the cell cycle, for example the G1 / G2 phase, S / G2 phase, G2 phase or S phase of the cell cycle. The term "cells being arrested in or accumulating in one or more phases of the cell cycle" means that the percentage of cells in said 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 phase is called S, during which DNA synthesis occurs and the DNA is replicated. The next phase is the G2 phase where RNA and proteins are replicated . The last phase is the M phase, which is the stage of actual cell division. In this last phase, the replicated DNA and RNA divide and move to separate ends of the cell, and the cell actually divides into two identical functional cells. Chemotherapeutic agents that are DNA damaging agents usually result in the accumulation of cells in the G1 and / or G2 phases. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, usually result in the accumulation of cells in the S phase. Examples of these drugs include 6-mercaptopurine and 5-fluorouracil.

[0181] According to the present invention, the term "agent stabilizing or increasing expression of CLDN18.2" ​The term "such as epirubicin, an anthracycline such as epirubicin, a platinum compound such as oxaliplatin and cisplatin, a nucleoside analogue such as 5-fluorouracil or its prodrug, a taxane such as docetaxel, and a camptothecin analogue such as irinotecan and topotecan, and a combination of drugs comprising one or more of epirubicin, an anthracycline, oxaliplatin and 5-fluorouracil, for example, a combination of drugs comprising oxaliplatin and 5-fluorouracil or other drug combinations described herein."

[0182] In one preferred embodiment, the "cytotoxic and / or cytostatic agent" is an "agent inducing immunogenic cell death".

[0183] In certain situations, cancer cells can enter a lethal stress pathway associated with the spatio-temporal release of a 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 to release signals that are sensed by innate immune effectors such as dendritic cells, thereby inducing a cognate immune response involving CD8+ T cells and IFN-γ signaling, which can result in cell death and trigger a proliferative anti-cancer immune response. These signals include the pro-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, the pro-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, but cisplatin, for example, which is insufficient to induce processes that require the translocation of CRT from the ER to the surface of dying cells - ER stress, requires complementation with the ER stress inducer thapsigargin.

[0184] According to the present invention, the term "agent inducing immunogenic cell death" refers to an agent or combination of agents that, when provided to a cell, particularly a cancer cell, can induce the cell to enter a lethal stress pathway that ultimately results in a tumor-specific immune response. In particular, an agent that induces immunogenic cell death, when provided to a cell, induces the cell to release a defined spatio-temporal combination of signals, including the pro-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, the pro-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1.

[0185] According to the present invention, the term "agent inducing immunogenic cell death" includes anthracyclines and oxaliplatin.

[0186] Anthracyclines are a class of drugs commonly used in cancer chemotherapy that are also antibiotics. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[0187] Anthracyclines preferably result in one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by inserting between the base pairs of DNA / RNA strands, thus preventing the replication of rapidly proliferating cancer cells. 2. Inhibit the topoisomerase II enzyme, preventing the relaxation of supercoiled DNA, thus blocking DNA transcription and replication. 3. Create iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0188] According to the present invention, the term "anthracycline" preferably relates to an agent for inducing apoptosis, preferably an anticancer agent, by inhibiting the recombination of DNA of topoisomerase II.

[0189] Preferably, according to the present invention, the term "anthracycline" generally refers to the following ring structures, including its analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates, and prodrugs.

Chemical formula

[0190] 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), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and aclacinomycin A (aclarubicin). Mitoxantrone is a member of the anthracenedione class of compounds, an anthracycline analog that lacks the sugar moiety of anthracyclines but retains a planar polycyclic aromatic ring structure that permits insertion into DNA.

[0191] Particularly preferred as the anthracycline according to the present invention is a compound of the following formula:

Chemical formula

[0192] 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.

[0193] Specifically contemplated as an anthracycline in the context of the present invention is epirubicin. Epirubicin has the following formula:

Chemical formula

[0194] 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.

[0195] The term "cisplatin" or "cisplatinum" refers to the following formula: num) refers to the compound cis-diamminedichloroplatinum(II) (CDDP) of the following formula:

Chemical formula

[0196] The term "carboplatin" refers to the following formula:

Chemical formula

[0197] The term "oxaliplatin" refers to the following formula:

Chemical formula

[0198] 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 commercially available under the trade name Eloxatine.

[0199] The term "nucleoside analog" refers to a structural analog of a nucleoside, a category that includes both purine analogs and pyrimidine analogs. In particular, the term "nucleoside analog" refers to fluoropyrimidine derivatives including fluorouracil and its prodrugs.

[0200] The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the trade names Adrucil, Carac, Efudix, Efudex, and Fluoroplex) refers to the following formula:

Chem.

[0201] In particular, this term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0202] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted to 5-FU in tissues. Capecitabine, which can be administered orally, has the following formula:

Chem.

[0203] 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.

[0204] Taxanes are a class of diterpenoid compounds initially derived from natural sources such as plants of the genus Taxus, although some are synthetically produced. The main mechanism of action of drugs in the taxane class is to disrupt microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel and paclitaxel.

[0205] According to the present invention, the term "docetaxel" refers to the following formula:

Chem.

[0206] According to the present invention, the term "paclitaxel" refers to the following formula:

Chem.

[0207] 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:

Chem.

[0208] According to the present invention, preferred camptothecin analogs are inhibitors of DNA enzyme topoisomerase I (topo I). Preferred camptothecin analogs according to the present invention are irinotecan and topotecan.

[0209] Irinotecan is a drug that prevents DNA from unwinding by inhibiting topoisomerase I. In chemical terms, this is the following formula:

Chem.

[0210] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-lH-pyrano[3’,4’:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4’ bipiperidine]-1’-carboxylate.

[0211] Topotecan is a topoisomerase inhibitor of the formula:

Chem.

[0212] 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.

[0213] ​According to the present invention, the cytotoxic agent and / or cell growth inhibitor can be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, and can be 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 chemotherapeutic agents and can be a combination of drugs used in chemotherapeutic regimens selected from the group consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, CAPOX chemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCF chemotherapy, and FLOT chemotherapy.

[0214] The combination of drugs used in EOX chemotherapy includes epirubicin, oxaliplatin, and capecitabine. The combination of drugs used in ECF chemotherapy includes epirubicin, cisplatin, and 5-fluorouracil. The combination of drugs used in ECX chemotherapy includes epirubicin, cisplatin, and capecitabine. The combination of drugs used in EOF chemotherapy includes epirubicin, oxaliplatin, and 5-fluorouracil.

[0215] Epirubicin is usually administered at a dose of 50 mg / m2, cisplatin at 60 mg / m2, oxaliplatin at 130 mg / m2, the long-term intravenous infusion of 5-fluorouracil at 200 mg / m2 / day, and oral capecitabine at a dose of 625 mg / m2 twice a day over a total of 8 three-week cycles.

[0216] The combination of drugs used in FLO chemotherapy includes 5-fluorouracil, folinic acid, and oxaliplatin (usually 5-fluorouracil 2600 mg / m2 for 24-hour infusion, folinic acid 200 mg / m2, and oxaliplatin 85 mg / m2, every two weeks).

[0217] FOLFOX is a chemotherapy regimen composed of folic acid (leucovorin), 5-fluorouracil, and oxaliplatin. The recommended dosing schedule given every two weeks is as follows: Day 1: Oxaliplatin 85 mg / m as a 22-hour continuous infusion 2 IV infusion and leucovorin 200 mg / m 2 IV infusion, followed by 5-FU 400 mg / m 2 IV bolus, followed by 5-FU 600 mg / m 2 IV infusion; Day 2: Leucovorin 200 mg / m over 120 minutes as a 22-hour continuous infusion 2 IV infusion, followed by 5-FU 400 mg / m given over 2 - 4 minutes 2 IV, followed by 5-FU 600 mg / m 2 IV infusion.

[0218] The drug combination used in CAPOX chemotherapy includes capecitabine and oxaliplatin.

[0219] The drug combination used in FOLFIRI chemotherapy includes 5-fluorouracil, leucovorin, and irinotecan.

[0220] The drug combination used in DCF chemotherapy includes docetaxel, cisplatin, and 5-fluorouracil.

[0221] The drug combination used in FLOT chemotherapy includes docetaxel, oxaliplatin, 5-fluorouracil, and folic acid.

[0222] The term "folinic acid" or "leucovorin" refers to a compound useful in a synergistic combination with the chemotherapy agent 5-fluorouracil. Folinic acid has the following formula:

Chemical formula

[0223] 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.

[0224] 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 should 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, especially a surface antigen within the cell or on cancer cells, preferably an antigen that is produced in large quantities.

[0225] 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 developmental stage and is expressed or abnormally expressed in one or more tumors 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 very rarely expressed in normal tissues.

[0226] The term "epitope" refers to the antigenic determinant in a molecule, i.e., the portion of the molecule that is recognized by the immune system, for example, recognized by an antibody. For example, an epitope is a discrete three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that in the presence of a denaturing solvent, the binding to conformational epitopes is lost, but the binding to non-conformational epitopes is not lost. The epitopes of proteins such as CLDN18.2 are , preferably including a continuous or discontinuous portion of said protein, preferably having 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.

[0227] 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 the antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies and antibody fragments or derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies such as scFv, and antigen-binding antibody fragments such as Fab and Fab' fragments, and includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, as well as any antigen-binding antibody fragments and derivatives described herein. Each heavy chain is composed of a heavy-chain variable region (abbreviated as HV 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 and VL regions 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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. 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).

[0228] The antibodies described herein can be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or somatic mutations in vivo).

[0229] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin of 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 may comprise only a complete variable domain fused to a constant domain or complementarity-determining regions (CDRs) grafted onto a suitable framework region of a variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, e.g., modified to be more closely similar to a human immunoglobulin. Some forms of humanized antibodies conserve all CDR sequences (e.g., humanized mouse antibodies containing all six CDRs of a mouse antibody). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0230] The term "chimeric antibody" refers to an antibody in which certain portions of the amino acid sequences of the heavy and light chains are homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to the corresponding sequences of another. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from a particular mammalian species, and the constant portions are homologous to the sequences of antibodies derived from another. One distinct advantage of such a chimeric form is that, for example, in combination with constant regions derived from human cell preparations, the variable regions can be readily derived from currently known sources using B cells or hybridomas readily available from non-human host organisms. The variable regions have the advantage of ease of preparation and their specificity is not affected by the source, while the constant regions, which are human, are less likely to induce an immune response from a human subject than constant regions from non-human sources when the antibody is injected. However, the definition is not limited to this particular example.

[0231] The term "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") of an antibody, or similar terms, refers 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 a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bridges 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 the 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 may be linked by a synthetic linker. Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be joined using recombinant methods by a synthetic linker that enables them to pair to form a monovalent molecule (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 include a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding domain immunoglobulin fusion protein is further disclosed in US Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments can be 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.

[0232] The term "bispecific molecule" is intended to include any agent, such as a protein, peptide, or protein or peptide complex, having two different binding specificities. For example, the 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 terms "multispecific molecule" or "heterospecific molecule" are intended to include any agent, such as a protein, peptide, or protein or peptide complex, having three or more different binding specificities. For example, the 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, tetra-specific, and other multispecific molecules directed to CLDN18.2 and other targets, such as Fc receptors on effector cells. The term "bispecific antibody" also includes multivalent antibodies, such as trivalent antibodies having two different binding specificities, tetravalent antibodies having two or three different binding specificities, etc. "Two" antibody) also includes multivalent antibodies, such as trivalent antibodies having two different binding specificities, tetravalent antibodies having two or three different binding specificities, etc. "Two" The term "bispecific antibody" also includes diabodies. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but are too short to allow pairing between two domains on the same chain, thereby using a linker that pairs the domains with complementary domains of another chain to create two antigen-binding sites (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).

[0233] Antibodies can be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressive agent) or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells, particularly one that kills cells. Examples include maytansine (e.g., mertansine, labutansine or emtanside), auristatin (monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE)), dolastatin, calicheamicin (e.g., ozogamicin), pyrrolobenzodiazepine dimer (e.g., tesirine, tairine), duocarmycin (e.g., duocarmycin SA, CC-1065, duocarmazine) and α -amanitin, irinotecan or its derivative SN-38, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and its analogs or homologs, antimetabolites (e.g., methotrexate, 6 - mercaptopurine, 6 - thioguanine, cytarabine, fludarabine, 5 - fluorouracil decarbazine), 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 antimitotic agents (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.

[0234] The antibody can also be conjugated to a radioisotope, such as iodine-131, yttrium-90 or indium-111, to generate a cytotoxic radiopharmaceutical.

[0235] 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 can be a protein or polypeptide having the desired biological activity. Such proteins can include, for example, enzymatically active toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin or diphtheria toxin or active fragments thereof; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers such as, for example, 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.

[0236] Techniques for conjugating such therapeutic moieties to antibodies are well known, for example, see Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pages 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd Edition), Robinson et al. (eds.), pages 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.), pages 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.), pages 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0237] As used herein, when an antibody is obtained from a system by immunizing an animal or by screening an immunoglobulin gene library, the antibody is "derived from" a specific germline sequence, and 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. Typically, an antibody derived from a specific germline sequence exhibits no more than 10 amino acid differences, more preferably no more than 5, even more preferably no more than 4, 3, 2 or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0238] As used herein, the term "heteroantibody" refers to two or more antibodies, derivatives thereof, or linked antigen-binding regions, at least two of which 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.

[0239] The antibodies described herein can be monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma comprising B cells obtained from a non-human animal, such as a mouse, fused to immortalized cells.

[0240] The antibodies described herein can be recombinant antibodies. As used herein, the term "recombinant antibody" encompasses all antibodies prepared, expressed, produced, 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 a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express an antibody, such as a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of an immunoglobulin gene sequence to other DNA sequences.

[0241] The antibodies described herein may be derived from different species including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0242] The antibodies described herein include polyclonal and monoclonal antibodies and include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly 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).

[0243] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi including yeast cells.

[0244] As used herein, the term "heterologous antibody" is defined with respect to a transgenic organism that produces such an antibody. This term refers to an antibody found in an organism that is not a transgenic organism and that generally has an amino acid sequence or corresponding coding nucleic acid sequence derived from a species other than the transgenic organism.

[0245] As used herein, the term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain with a mouse light chain is a heterohybrid antibody.

[0246] 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 a conjugate of an antibody with another agent or an antibody or antibody fragment.

[0247] The antibodies described herein are preferably isolated. The term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell. As used herein, "isolated antibody" is intended to include an antibody that substantially does not contain other antibodies having different antigen specificities (for example, 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 have cross-reactivity to other related antigens of other species (for example, CLDN18.2 homologs). Furthermore, an isolated antibody may substantially not contain other cellular materials and / or may substantially not contain chemical substances. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies relates to antibodies that have different specificities and are combined in a specifically defined composition or mixture.

[0248] As used in the present invention, the term "binding" preferably relates to specific binding.

[0249] According to the present invention, an antibody has a significant affinity for a given target in a standard assay and can bind to the given target when binding to the given target. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant (K D ). Preferably, the term "significant affinity" means 10 -5 M or less, 10 -6 M or less, 10-7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, or 10 -12 Dissociation constant (K D ) that binds to a predetermined target.

[0250] An antibody that does not have a significant affinity for a target and does not significantly bind to the target, particularly not detectably bind, in a standard assay, cannot (substantially) bind to the target. Preferably, the antibody does not detectably bind to the target when present at a concentration of up to 2, preferably 10, more preferably 20, particularly 50 or 100 μg / ml or higher. Preferably, the antibody has a K D of at least 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold or 10 6 -fold higher K D when binding to the target and does not have a significant affinity for the target. For example, if the K D for the binding of the antibody to a target that the antibody can bind is 10 -7 M, the K D for the binding of the antibody to a target that does not have a significant affinity for the antibody will be at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M.

[0251] An antibody is specific for a given target if it can bind to that target but cannot bind to other targets, i.e., has no significant affinity for other targets and does not significantly bind 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 cannot (substantially) bind to other targets. Preferably, an antibody is specific for CLDN18.2 if its affinity and binding for such other antibodies are not significantly exceeded by its affinity or binding for 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. Preferably, an antibody is specific for a target if its K D is at least 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold or 10 6 -fold lower than its K D for binding to a non-specific target. For example, if the K D for binding of an antibody to a specific target is 10 -7 M, the K D for binding to a non-specific target will be at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 M.

[0252] The binding of an antibody to a target can be determined experimentally using any suitable method; for example, 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 See York, N.Y. (1992). Affinity can be determined readily using conventional techniques such as equilibrium dialysis; using the 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. 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, the measurement of affinity and other antigen-binding parameters, such as K , IC D , should preferably be done with standardized solutions of antibody and antigen, and a standardized buffer. 50

[0253] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0254] As used herein, "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to another.

[0255] As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including a virus) that has not been intentionally modified by man in the laboratory is naturally occurring.

[0256] ​As used herein, the term "rearranged" with respect to a V segment refers to the arrangement of the heavy or light chain immunoglobulin locus in which the V segment is positioned immediately adjacent to a D-J or J segment in a conformation that essentially fully encodes a VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA; rearranged loci have at least one recombined heptamer / nonamer homology element.

[0257] As used herein, the term "unrearranged" or "germline configuration" with respect to a V segment refers to an arrangement in which the V segment has not been rearranged to be immediately adjacent to a D or J segment.

[0258] According to the present invention, an anti-CLDN18.2 antibody is an antibody that can bind to an epitope present on CLDN18.2, preferably the extracellular domain of CLDN18.2, particularly the first extracellular domain, preferably an epitope located within amino acids 29 to 78 of CLDN18.2. In certain embodiments, the anti-CLDN18.2 antibody 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 located on CLDN18.2-loop1, preferably SEQ ID NO: 8, (iii) an epitope located on CLDN18.2-loop2, preferably SEQ ID NO: 10, (iv) an epitope located 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 located on CLDN18.2-loop D3, preferably SEQ ID NO: 9.

[0259] According to the present invention, the anti-CLDN18.2 antibody is preferably an antibody that binds to CLDN18.2 but does not bind to CLDN18.1. Preferably, the anti-CLDN18.2 antibody is specific for CLDN18.2. Preferably, the anti-CLDN18.2 antibody is an antibody that binds to CLDN18.2 expressed on the cell surface. In certain preferred embodiments, the anti- CLDN18.2 antibody binds to the native epitope of CLDN18.2 present on the surface of live cells. Preferably, the anti-CLDN18.2 antibody binds to one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, the anti-CLDN18.2 antibody is specific for the above protein, peptide, or its immunogenic fragment or derivative. The anti-CLDN18.2 antibody can be obtained by a method comprising 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 the protein or peptide. Preferably, the antibody binds to cancer cells, particularly cells of the above cancer types, and preferably does not substantially bind to non-cancerous cells.

[0260] Preferably, the binding of the anti-CLDN18.2 antibody to cells expressing CLDN18.2 induces or mediates the death of cells expressing CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells, and in particular are selected from the group consisting of tumoral gastric, esophageal, pancreatic, lung, ovarian, colon, liver, head and neck, and gallbladder cancer cells. Preferably, the antibody induces or mediates cell death 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, in certain embodiments, the ADCC-mediated lysis of cells occurs in the presence of effector cells 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 replace thymidine during DNA replication and be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle). For example, detection of the incorporated chemical using an antibody specific for BrdU indicates cells that were actively replicating their DNA.

[0261] In preferred embodiments, the antibodies described herein may be characterized by one or more of the following specificities: 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 in CLDN18.2-positive cells; d) Ability to induce or mediate ADCC in 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.

[0262] In certain preferred embodiments, the anti-CLDN18.2 antibody is produced by hybridomas deposited with DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and having the following designations 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, accession number DSM ACC2810, deposited on October 26, 2006.

[0263] Preferred antibodies according to the present invention are those produced by the above hybridomas and obtainable from the above hybridomas, i.e., in the case of 182-D1106-055, 37G11, in the case of 182-D1106-056, 37H8, in the case of 182-D1106-057, 38G5, in the case of 182-D1106-058, 38H3, in the case of 182-D1106-059, 39F11, in the case of 182-D1106-062, 43A11, in the case of 182-D1106-067, 61C2, in the case of 182-D758-035, 26B5, in the case of 182-D758-036, 26D12, in the case of 182-D758-040, 28D10, in the case of 182-D1106-061, 42E12, in the case of 182-D1106-279, 125E1, in the case of 182-D1106-294, 163E12, and in the case of 182-D1106-362, 175D10; and their chimeric and humanized forms.

[0264] Preferred chimeric antibodies and their sequences are shown in the following table.

Table 1

[0265] In preferred embodiments, antibodies according to the present invention, particularly chimeric forms of the antibodies, include antibodies comprising a heavy chain constant region (CH) comprising 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 52 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. In a more preferred embodiment, antibodies according to the present invention, particularly chimeric forms of the antibodies, include antibodies comprising a light chain constant region (CL) comprising 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 functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0266] In certain preferred embodiments, the antibodies according to the invention, in particular chimeric antibodies of the antibodies, comprise a CH comprising an amino acid sequence derived from a human CH, such as the amino acid sequence represented by SEQ ID NO: 13 or 52 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and a CL comprising an amino acid sequence derived from a human CL, such as the amino acid sequence represented by SEQ ID NO: 12 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0267] In one embodiment, the anti-CLDN18.2 antibody is a chimeric mouse / human IgG1 monoclonal antibody comprising kappa, mouse variable light chain, human kappa light chain constant region allotype Km(3), mouse heavy chain variable region, human IgG1 constant region, allotype G1m(3).

[0268] In certain preferred embodiments, the chimeric form of the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 51 and functional variants thereof, or fragments of the amino acid sequence or functional variants, and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28 and functional variants thereof, or fragments of the amino acid sequence or functional variants.

[0269] In certain preferred embodiments, the chimeric form of the antibody comprises an antibody comprising a combination of heavy and light chains selected from the following possibilities (i) - (ix): (i) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 14 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 21 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, (ii) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 15 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 20 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (iii) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 16 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 22 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (iv) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 18 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 25 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (v) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 17 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 24 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (vi) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 19 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 23 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (vii) The heavy chain contains the amino acid sequence represented by SEQ ID NO: 19 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain contains the amino acid sequence represented by SEQ ID NO: 26 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (viii) The heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 27 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (ix) The heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 28 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and (x) The heavy chain comprises the amino acid sequence represented by SEQ ID NO: 51 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 24 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0270] In one particularly preferred embodiment, the anti-CLDN18.2 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 17 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0271] In one particularly preferred embodiment, the anti-CLDN18.2 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 51 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 24 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0272] A fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 51, 20, 21, 22, 23, 24, 25, 26, 27 and 28 preferably relates to the sequence from which 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus have been removed.

[0273] In a preferred embodiment, the anti-CLDN18.2 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and functional variants thereof, or a fragment of an amino acid sequence or functional variant.

[0274] In a preferred embodiment, the anti-CLDN18.2 antibody comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43 and functional variants thereof, or a fragment of an amino acid sequence or functional variant.

[0275] In certain preferred embodiments, the anti-CLDN18.2 antibody 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 comprises an amino acid sequence represented by SEQ ID NO: 29 or a functional variant thereof, or a fragment of an amino acid sequence or functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 36 or a functional variant thereof, or a fragment of an amino acid sequence or functional variant. (ii) VH comprises an amino acid sequence represented by SEQ ID NO: 30 or a functional variant thereof, or a fragment of an amino acid sequence or functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 35 or a functional variant thereof, or a fragment of an amino acid sequence or functional variant. (iii) The VH contains the amino acid sequence represented by SEQ ID NO: 31 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 37 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (iv) The VH contains the amino acid sequence represented by SEQ ID NO: 33 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 40 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (v) The VH contains the amino acid sequence represented by SEQ ID NO: 32 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (vi) The VH contains the amino acid sequence represented by SEQ ID NO: 34 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 38 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (vii) The VH contains the amino acid sequence represented by SEQ ID NO: 34 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 41 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (viii) The VH contains the amino acid sequence represented by SEQ ID NO: 34 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 42 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. (ix) The VH contains the amino acid sequence represented by SEQ ID NO: 34 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL contains the amino acid sequence represented by SEQ ID NO: 43 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant.

[0276] In one particularly preferred embodiment, the anti-CLDN18.2 antibody comprises a VH containing the amino acid sequence represented by SEQ ID NO: 32 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and a VL containing the amino acid sequence represented by SEQ ID NO: 39 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant. In a more preferred embodiment, the anti-CLDN18.2 antibody comprises a VH containing the amino acid sequence represented by SEQ ID NO: 32, and the VL contains the amino acid sequence represented by SEQ ID NO: 39, for example, IMAB362 (Zolbetuximab).

[0277] The term "fragment" particularly refers to one or more complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL), preferably the CDR3 sequence, optionally in combination with the CDR1 sequence and / or CDR2 sequence. In one embodiment, the one or more complementarity-determining regions (CDRs) are selected from the set of complementarity-determining regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL).

[0278] In a preferred embodiment, the anti-CLDN18.2 antibody comprises a VH containing a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i)-(vi): (i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, (ii) CDR1: positions 45 - 52 of SEQ ID NO: 15, CDR2: positions 70 - 77 of SEQ ID NO: 15, CDR3: positions 116 - 126 of SEQ ID NO: 15, (iii) CDR1: positions 45 - 52 of SEQ ID NO: 16, CDR2: positions 70 - 77 of SEQ ID NO: 16, CDR3: positions 116 - 124 of SEQ ID NO: 16, (iv) CDR1: positions 45 - 52 of SEQ ID NO: 17, CDR2: positions 70 - 77 of SEQ ID NO: 17, CDR3: positions 116 - 126 of SEQ ID NO: 17, (v) CDR1: positions 44 - 51 of SEQ ID NO: 18, CDR2: positions 69 - 76 of SEQ ID NO: 18, CDR3: positions 115 - 125 of SEQ ID NO: 18, and, (vi) CDR1: positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID NO: 19, CDR3: positions 117 - 128 of SEQ ID NO: 19.

[0279] In a preferred embodiment, the anti - CLDN18.2 antibody comprises a VH comprising at least one, preferably two, more preferably all three of the CDR sequences of the set of complementarity - determining regions CDR1, CDR2, and CDR3 selected from the above embodiments (i) - (vi).

[0280] In a preferred embodiment, the anti - CLDN18.2 antibody comprises a VL comprising a set of complementarity - determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) - (ix): (i) CDR1: positions 47 - 58 of SEQ ID NO: 20, CDR2: positions 76 - 78 of SEQ ID NO: 20, CDR3: positions 115 - 123 of SEQ ID NO: 20, (ii) CDR1: positions 49 - 53 of SEQ ID NO: 21, CDR2: positions 71 - 73 of SEQ ID NO: 21, CDR3: positions 110 - 118 of SEQ ID NO: 21, (iii) CDR1: positions 47 - 52 of SEQ ID NO: 22, CDR2: positions 70 - 72 of SEQ ID NO: 22, CDR3: positions 109 - 117 of SEQ ID NO: 22, (iv) CDR1: positions 47 - 58 of SEQ ID NO: 23, CDR2: positions 76 - 78 of SEQ ID NO: 23, CDR3: positions 115 - 123 of SEQ ID NO: 23, (v) CDR1: positions 47 - 58 of SEQ ID NO: 24, CDR2: positions 76 - 78 of SEQ ID NO: 24, CDR3: positions 115 - 123 of SEQ ID NO: 24, (vi) CDR1: positions 47 - 58 of SEQ ID NO: 25, CDR2: positions 76 - 78 of SEQ ID NO: 25, CDR3: positions 115 - 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 - 58 of SEQ ID NO: 26, CDR2: positions 76 - 78 of SEQ ID NO: 26, CDR3: positions 115 - 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 - 58 of SEQ ID NO: 27, CDR2: positions 76 - 78 of SEQ ID NO: 27, CDR3: positions 115 - 123 of SEQ ID NO: 27, and, (ix) CDR1: positions 47 - 52 of SEQ ID NO: 28, CDR2: positions 70 - 72 of SEQ ID NO: 28, CDR3: positions 109 - 117 of SEQ ID NO: 28.

[0281] In a preferred embodiment, the anti - CLDN18.2 antibody comprises a VL comprising at least one, preferably two, more preferably all three of the CDR sequences of a set of complementarity - determining regions CDR1, CDR2, and CDR3 selected from the above embodiments (i) - (ix).

[0282] In a preferred embodiment, the anti - CLDN18.2 antibody comprises a combination of VH and VL each comprising a set of complementarity - determining regions CDR1, CDR2, and CDR3 selected from the following embodiments (i) - (ix): (i) VH: CDR1: positions 45 - 52 of SEQ ID NO: 14, CDR2: positions 70 - 77 of SEQ ID NO: 14, CDR3: positions 116 - 125 of SEQ ID NO: 14, VL: CDR1: positions 49 - 53 of SEQ ID NO: 21, CDR2: positions 71 - 73 of SEQ ID NO: 21, CDR3: positions 110 - 118 of SEQ ID NO: 21, (ii) VH: CDR1: positions 45 - 52 of SEQ ID NO: 15, CDR2: positions 70 - 77 of SEQ ID NO: 15, CDR3: positions 116 - 126 of SEQ ID NO: 15, VL: CDR1: positions 47 - 58 of SEQ ID NO: 20, CDR2: positions 76 - 78 of SEQ ID NO: 20, CDR3: positions 115 - 123 of SEQ ID NO: 20, (iii) VH: CDR1: positions 45 - 52 of SEQ ID NO: 16, CDR2: positions 70 - 77 of SEQ ID NO: 16, CDR3: positions 116 - 124 of SEQ ID NO: 16, VL: CDR1: positions 47 - 52 of SEQ ID NO: 22, CDR2: positions 70 - 72 of SEQ ID NO: 22, CDR3: positions 109 - 117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44 - 51 of SEQ ID NO: 18, CDR2: positions 69 - 76 of SEQ ID NO: 18, CDR3: positions 115 - 125 of SEQ ID NO: 18, VL: CDR1: positions 47 - 58 of SEQ ID NO: 25, CDR2: positions 76 - 78 of SEQ ID NO: 25, CDR3: positions 115 - 122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45 - 52 of SEQ ID NO: 17, CDR2: positions 70 - 77 of SEQ ID NO: 17, CDR3: positions 116 - 126 of SEQ ID NO: 17, VL: CDR1: positions 47 - 58 of SEQ ID NO: 24, CDR2: positions 76 - 78 of SEQ ID NO: 24, CDR3: positions 115 - 123 of SEQ ID NO: 24, (vi) VH: CDR1: positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID NO: 19, CDR3: positions 117 - 128 of SEQ ID NO: 19, VL: CDR1: positions 47 - 58 of SEQ ID NO: 23, CDR2: positions 76 - 78 of SEQ ID NO: 23, CDR3: positions 115 - 123 of SEQ ID NO: 23, (vii) VH: CDR1: positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID NO: 19, CDR3: positions 117 - 128 of SEQ ID NO: 19, VL: CDR1: positions 47 - 58 of SEQ ID NO: 26, CDR2: positions 76 - 78 of SEQ ID NO: 26, CDR3: positions 115 - 123 of SEQ ID NO: 26, (viii) VH: CDR1: positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID NO: 19, CDR3: positions 117 - 128 of SEQ ID NO: 19, VL: CDR1: positions 47 - 58 of SEQ ID NO: 27, CDR2: positions 76 - 78 of SEQ ID NO: 27, CDR3: positions 115 - 123 of SEQ ID NO: 27, and, (ix) VH: CDR1: positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID NO: 19, CDR3: positions 117 - 128 of SEQ ID NO: 19, VL: CDR1: positions 47 - 52 of SEQ ID NO: 28, CDR2: positions 70 - 72 of SEQ ID NO: 28, CDR3: positions 109 - 117 of SEQ ID NO: 28.

[0283] In a preferred embodiment, the anti-CLDN18.2 antibody comprises a VH comprising at least one, preferably two, more preferably all three of the VH CDR sequences of the set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the above embodiments (i) to (ix), and a VL comprising at least one, preferably two, more preferably all three of the VL CDR sequences of the set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the above embodiments (i) to (ix).

[0284] The term "at least one, preferably two, more preferably all three of the CDR sequences" preferably relates to at least the CDR3 sequence, optionally in combination with the CDR1 sequence and / or the CDR2 sequence.

[0285] In one particularly preferred embodiment, the anti-CLDN18.2 antibody comprises a combination of VH and VL each comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 as follows: VH: CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17, VL: CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24.

[0286] In a more preferred embodiment, the anti-CLDN18.2 antibody preferably comprises one or more complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, preferably comprising one or more complementarity-determining regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or light chain variable region (VL) described herein. In one embodiment, the one or more complementarity-determining regions (CDRs) are selected from the set of complementarity-determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, the anti-CLDN18.2 antibody preferably comprises complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, preferably comprises complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) described herein.

[0287] 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 the CDRs together with its intervening framework regions. Preferably, the portion comprises at least about 50% of either or both of the first and fourth framework regions, where 50% is the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies made by recombinant DNA techniques may result in the introduction of N-terminal or C-terminal residues relative to the variable regions, including the introduction of linkers to facilitate cloning or other manipulation steps, including the introduction of linkers that bind variable regions or bind variable regions to additional protein sequences comprising the sequences described herein.

[0288] 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 in a human antibody framework.

[0289] With respect to its heavy chain, a reference herein to an antibody comprising a particular chain or particular region or sequence preferably relates to a situation in which all heavy chains of said antibody comprise said particular chain, region or sequence. This applies mutatis mutandis to the light chains of the antibody.

[0290] In one embodiment, an anti-CLDN18.2 antibody competes with the anti-CLDN18.2 antibodies described herein for CLDN18.2 binding and / or has specificity for CLDN18.2 of the anti-CLDN18.2 antibodies described herein. In these and other embodiments, the anti-CLDN18.2 antibody may be highly homologous to the anti-CLDN18.2 antibodies described herein. It is contemplated that preferred anti-CLDN18.2 antibodies have CDR regions that are identical or highly homologous to the CDR regions of the anti-CLDN18.2 antibodies described herein. By "highly homologous", it is contemplated that 1 to 5, preferably 1 to 4, for example 1 to 3 or 1 or 2 substitutions may be made in each CDR region.

[0291] The term "compete" refers to the competition between two binding molecules, such as antibodies, for binding to a target antigen. If two binding molecules do not block each other for binding to the target antigen, such binding molecules are non-competing, which indicates that the binding molecules do not bind to the same portion of the target antigen, i.e., the epitope. Methods for testing the competition of binding molecules such as antibodies for binding to a target antigen are well known to those skilled in the art. Examples of such methods are so-called cross-competition assays, which can be carried out, for example, as ELISA or by flow cytometry. For example, an ELISA-based assay coats an ELISA plate well with one of the antibodies; adds a competing antibody and a His-tagged antigen / target, and detects whether the added antibody inhibits the binding of the His-tagged antigen to the coated antibody by adding, for example, a biotinylated anti-His antibody, followed by streptavidin-polyHRP, further developing the reaction with ABTS, and measuring the absorbance at 405 nm. For example, a flow cytometry assay can be carried out by incubating cells expressing the antigen / target with an excess of unlabeled antibody, incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin, and analyzing by flow cytometry.

[0292] Two binding molecules have "same specificity" if they bind to the same antigen and the same epitope. The molecule to be tested has the same epitope as a certain binding molecule Whether a binding molecule recognizes a epitope, i.e., whether the binding molecules bind to the same epitope, can be tested by various methods known to those skilled in the art, for example, based on the competition of binding molecules such as antibodies for the same epitope. The competition between binding molecules can be detected by a cross-blocking assay. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the wells of a microtiter plate, and an antigen-binding antibody and a candidate competitive test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the well is indirectly correlated with the binding ability of the candidate competitive test antibody that competes with it for binding to the same epitope. Specifically, the greater the affinity of the candidate competitive test antibody for the same epitope, the less the amount of antigen-binding antibody bound to the antigen-coated well. The amount of antigen-binding antibody bound to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0293] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When the positions in both of the two sequences being compared are occupied by the same base or amino acid monomer subunit, these molecules are homologous at that position. The percentage of homology between two sequences is a function obtained by dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and multiplying by 100. For example, if 6 out of 10 positions of two sequences match or are homologous, the two sequences are 60% homologous. Generally, the comparison is made when the two sequences are aligned to give the maximum homology. According to the present disclosure, homologous sequences exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98% or at least 99% identity of amino acid or nucleotide residues.

[0294] A "fragment" related to an amino acid sequence (peptide or protein) refers to a part of the amino acid sequence, that is, a sequence representing an amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame as long as the truncated open reading frame contains a start codon that helps initiate translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the amino acid residues of the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids of the amino acid sequence.

[0295] A "fragment" of an antibody sequence, when it replaces the antibody sequence of the antibody, preferably retains the binding of the antibody to CLDN18.2 and preferably the function of the antibody described herein, such as CDC-mediated lysis or ADCC-mediated lysis.

[0296] As used herein, "variant", "variant protein" or "variant polypeptide" means a protein that differs from the parent protein by at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide, or a modified version of a wild-type polypeptide. Preferably, the variant polypeptide has at least one amino has amino acid modifications, e.g., 1 to about 20 amino acid modifications, preferably 1 to about 10 or 1 to about 5 amino acid modifications compared to the parent.

[0297] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide" or "precursor protein" means an unmodified polypeptide that is subsequently modified to generate a variant. The parent polypeptide can be a wild-type polypeptide, or a variant or engineered version of a wild-type polypeptide.

[0298] As used herein, "wild type" or "WT" or "native" means an amino acid sequence found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0299] For the purposes of the present disclosure, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those that occur naturally.

[0300] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of an amino acid sequence variant having an insertion, one or more amino acid residues are inserted at a specific site in the amino acid sequence, but random insertion by appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants that include deletions at the N-terminal and / or C-terminal of the protein are also called N-terminal and / C-terminal truncation variants. Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. It is preferred that the modification is at a position in the amino acid sequence that is not conserved among homologous proteins or peptides, and / or that the amino acid is substituted with another amino acid having similar properties. Preferably, the amino acid changes in the peptide and protein variants are conservative amino acid changes, i.e., substitutions of amino acids with similar charge or lack of charge. Conservative amino acid changes involve the substitution of one member of a family of amino acids related by their side chains. Naturally occurring amino acids are generally divided into four families: 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. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; and, Phenylalanine, tyrosine.

[0301] Preferably, 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. The alignment for determining sequence similarity, preferably sequence identity, can preferably be performed 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, by tools known in the art.

[0302] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0303] The term "percentage identity" represents the percentage of amino acid residues that are identical between two sequences being compared, obtained after the best alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two amino acid sequences is customarily performed by comparing these sequences after optimally aligning them, and the comparison is done by segments or "windows of comparison" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be created by, in addition to manually, the local homology algorithms of Smith and Waterman, 1981, Adv. Appl. Math. 2, 482; Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444; or similarity search methods, or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0304] Percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the resulting value by 100 to obtain the percentage identity between these two sequences.

[0305] The teachings provided herein in connection with a specific amino acid sequence, such as those set forth in the Sequence Listing, should be interpreted as relating to variants of said specific sequence that result in sequences that are functionally equivalent to the specific sequence, e.g., amino acid sequences that exhibit the same or similar properties as the properties of the specific amino acid sequence. One important property is to retain the binding of the antibody to its target or to maintain the effector function of the antibody. Preferably, a sequence that is a variant with respect to a specific sequence, when it replaces the specific sequence of the antibody, retains the binding of the antibody to CLDN18.2 and preferably the function of the antibody described herein, e.g., CDC-mediated lysis or ADCC-mediated lysis. In particular, it will be understood by those skilled in the art that the sequences of the CDRs, hypervariable regions and variable regions can be modified without loss of the ability to bind to CLDN18.2. For example, the CDR regions are identical or highly homologous to the regions of the antibody specified herein. By "highly homologous", it is contemplated that 1 to 5, preferably 1 to 4, e.g., 1 to 3 or 1 or 2 substitutions can be made in the CDRs. Furthermore, the hypervariable regions and variable regions can be modified to exhibit substantial homology to the regions of the antibodies specifically disclosed herein.

[0306]

[0307] As used herein, the term "functional variant" includes an amino acid sequence that has been altered by one or more amino acids as compared to the amino acid sequence of a parent molecule or sequence and still performs one or more of the functions of the parent molecule or sequence, such as being able to bind to a target molecule or contribute to binding to a target molecule. When the parent molecule or sequence is an antibody molecule or sequence, the alteration is preferably not in the variable region of the antibody and more preferably not in the CDR region of the antibody. In one embodiment, the functional variant competes with the parent molecule or sequence for binding to the target molecule, either alone or in combination with other elements. In other words, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect and does not change the binding properties of the molecule or sequence. In different embodiments, the binding of the functional variant may be reduced but still significantly present, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the parent molecule or sequence. However, in other embodiments, the binding of the functional variant may be enhanced as compared to the parent molecule or sequence.

[0308] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof.

[0309] As used herein, the term "nucleic acid" is intended to include DNA and RNA. The nucleic acid can be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0310] 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 term also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable.

[0311] The term "transgenic animal" has a genome that contains one or more transgenes, preferably heavy / light chain transgenes, or a transchromosome (integrated or not integrated into the animal's natural genomic DNA), and preferably is capable of expressing the transgene. For example, a transgenic mouse can have either a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, such that when the mouse is immunized with the CLDN18.2 antigen and / or cells expressing CLDN18.2, it produces human anti-CLDN18.2 antibodies. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as in the case of transgenic mice such as HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene can be maintained extrachromosomally, as in the case of transchromosomal (e.g., KM) mice described in WO 02 / 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.

[0312] As used herein, "reduce," "decrease," or "inhibit" means an overall decrease of preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, or the ability to cause an overall decrease, in a level, e.g., an expression level or a cell proliferation level.

[0313] 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 more.

[0314] When "inducing" is used with respect to certain activities or functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), it may mean that such activities or functions did not exist prior to induction, but it may also mean that a certain level of such activity or function existed prior to induction and that after induction, said activity or function is enhanced. Thus, the term "inducing" includes "enhancing". mAb mechanism of action

[0315] The following provides considerations regarding the mechanisms underlying the therapeutic effectiveness of the antibodies of the present invention, but should not be construed as limiting the present invention in any way.

[0316] The antibodies described herein preferably interact with components of the immune system, preferably through ADCC or CDC. The antibodies described herein can also be used to target a payload (e.g., a radioisotope, drug or toxin) to directly kill tumor cells, or can be used in combination with traditional chemotherapeutic agents to attack tumors through additional mechanisms of action that may include anti-tumor immune responses that could have been impaired by the cytotoxic side effects of chemotherapeutic agents on T lymphocytes. However, the antibodies described herein exert their effect simply by binding to CLDN18.2 on the cell surface and can thus, for example, also block cell proliferation.

[0317] Antibody-dependent cell-mediated cytotoxicity ADCC describes the cell killing capacity of effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cell be marked by an antibody.

[0318] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain binds to the antigen. This occurs when it engages with Fc receptors (FcR) on the surface of effector cells. Several families of Fc receptors have been identified, and certain cell populations characteristically express certain Fc receptors. ADCC can be considered as a mechanism that directly induces various degrees of immediate tumor destruction, which leads to antigen presentation and tumor-directed T cell responses. Preferably, the in vivo induction of ADCC leads to tumor-directed T cell responses and host-induced antibody responses.

[0319] Complement-dependent cytotoxicity CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex leads to the C-terminal activation of the participating antibody molecules, such as IgG molecules. H This results in the uncloaking of multiple closely spaced C1q binding sites on the 2 domain (C1q is one of three subcomponents of complement C1). Preferably, these uncloaked C1q binding sites convert the previously low affinity C1q-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade culminates in the formation of a membrane attack complex, which creates a pore in the cell membrane that facilitates the free passage of water and solutes in and out of the cell.

[0320] The antibodies described herein can be made by a variety of techniques including conventional monoclonal antibody methodologies, such as the standard somatic cell hybridization techniques of Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are generally preferred, other techniques for making monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of antibody genes, can also be used.

[0321] A preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0322] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (see, e.g., Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), also see Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0323] In yet another preferred embodiment, human monoclonal antibodies can be made using transgenic or translchromosomal mice that possess a part of the human immune system instead of the mouse system. These transgenic and translchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mouse". The production of human antibodies in such transgenic mice can be carried out as described in detail for CD20 in WO 2004 / 035607.

[0324] 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; see also a novel strategy for generating monoclonal antibodies from a single isolated lymphocyte that produces antibodies of defined specificity. For details of recombinant antibody manipulation For details, see also Welschof and Kraus, Recombinant antibody for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN1-58829-092-1.

[0325] To produce antibodies, mice can be immunized with antigen sequences, i.e., carrier-conjugated peptides derived from the antigen sequence, the sequence to which the antibody is targeted, recombinantly expressed antigens or fragments thereof, and / or enriched preparations of cells expressing the antigen, as described. Alternatively, mice can be immunized with DNA encoding the antigen or a fragment thereof. If immunization with a purified or enriched preparation of the antigen does not result in antibodies, mice can also be immunized with cells expressing the antigen, such as cell lines, to enhance the immune response.

[0326] The immune response can be monitored over the course of the immunization protocol by plasma and serum samples obtained by tail vein or retro-orbital bleeding. Mice with a sufficient titer of immunoglobulins can be used for fusion. Three days after boosting the mice intraperitoneally or intravenously with antigen-expressing cells, the mice are sacrificed and the spleens are removed to increase the proportion of specific antibody-secreting hybridomas.

[0327] 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 for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies with specificity for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Antibody-secreting hybridomas can be reseeded and rescreened, and if still positive for monoclonal antibodies, can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to produce antibodies in tissue culture medium for characterization.

[0328] For example, as is well known in the art (see Morrison, S. (1985) Science 229:1202), antibodies can also be produced in host cell transfectomas using a combination of recombinant DNA technology and gene transfer methods.

[0329] For example, in one embodiment, the gene(s) of interest, such as 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 WO 87 / 04462, WO 89 / 01036, and EP 338841 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 such as a CHO cell, NS / 0 cell, HEK293T cell, or HEK293 cell, or alternatively, other eukaryotic cells such as plant-derived cells, fungi, or yeast cells. The methods used to introduce these genes can be methods described in the art such as electroporation, lipofectin, lipofectamine, etc. After introducing these antibody genes into the host cells, cells expressing the antibody can be identified and selected. These cells represent transfectomas that can then be amplified for expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0330] Alternatively, the cloned antibody gene can be expressed in other expression systems containing prokaryotic cells such as microorganisms, for example, E. coli. Furthermore, the antibody can be produced in transgenic non-human animals such as goats and cows, or in the milk of goats and cows, or in the eggs of hens, or in transgenic plants; 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.

[0331] Chimerization When labeled with a toxin or radioisotope, mouse monoclonal antibodies can be used as therapeutic antibodies in humans. Unlabeled mouse antibodies are highly immunogenic in humans upon repeated application, leading to a decrease in therapeutic efficacy. The major immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimerizing or humanizing each antibody. Chimeric antibodies are antibodies in which different parts are derived from different animal species, for example, antibodies having variable regions derived from mouse antibodies and human immunoglobulin constant regions. Chimerization of an antibody is achieved by linking the variable regions of the mouse heavy and light chains to the constant regions of the human heavy and light chains (for example, as described in Kraus et al., Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by linking the human kappa light chain constant region to the mouse light chain variable region. Similarly, in a preferred embodiment, a chimeric antibody can be produced by linking the human lambda light chain constant region to the mouse light chain variable region. Preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0332] Humanization Antibodies interact with target antigens mainly through amino acid residues located in six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than the sequences outside CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector containing the CDR sequences of a particular naturally occurring antibody grafted onto the framework sequences of 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 are different from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B cell maturation. Germline gene sequences also differ from the sequences of individual high-affinity secondary repertoire antibodies uniformly across the variable region.

[0333] The ability of an antibody to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blotting, immunofluorescence, and flow cytometry analysis).

[0334] 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. The supernatant can be filtered and concentrated, if necessary, prior to affinity chromatography using protein G sepharose or protein A sepharose. The eluted IgG can be confirmed by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be exchanged for PBS. It is possible to determine the concentration by OD280 using a decay coefficient of 1.43. The monoclonal antibody can be divided into a certain amount and stored at -80°C.

[0335] Site-directed mutagenesis or multiple site-directed mutagenesis can be used to determine whether the selected monoclonal antibody binds to a specific epitope.

[0336] 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 the microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with the monoclonal antibody or purified isotype control at ambient temperature for 2 hours. Then, the wells can 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 ABTS substrate (1 mg / ml) and analyzed at OD405-650. Alternatively, IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, catalog number 1493027) can be used as described by the manufacturer.

[0337] To demonstrate the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to live cells expressing an antigen, flow cytometry can be used. Cell lines expressing 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 supernatant or PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, APC- or Alexa647-labeled anti-IgG antibodies can bind to the antigen-binding monoclonal antibodies under the same conditions as the primary antibody staining. Samples can be analyzed by flow cytometry using an FACS instrument, using light and side scatter characteristics to gate single live cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, the cotransfection method can be used. 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 antibodies preferentially bind to the fluorescent marker-expressing cells, and non-specific antibodies bind to non-transfected cells at an equivalent ratio. An alternative assay using fluorescence microscopy can be used in addition to or instead of the flow cytometry assay. Cells can be stained as described above and examined by fluorescence microscopy.

[0338] Immunofluorescence microscopy 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 expressing an antigen spontaneously or after transfection, and negative controls lacking antigen expression, are 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 against the antigen for 30 minutes at 25°C. After washing, the cells can be reacted with an Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0339] Cell extracts can be prepared from cells expressing the antigen and appropriate negative controls and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Electroph After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with an FCL substrate.

[0340] The antibody can be further tested for reactivity with the antigen by immunohistochemistry in a manner well known to those skilled in the art using, for example, non-cancerous or cancerous tissue samples obtained from a patient during routine surgical procedures or paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from mice bearing xenograft tumors inoculated with cell lines expressing an antigen spontaneously or after transfection. For immunostaining, the antibody reactive with the antigen can be incubated, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (DAKO) according to the vendor's instructions.

[0341] Antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CLDN18.2. Testing of monoclonal antibody activity in vitro provides an initial screening prior to testing in in vivo models.

[0342] 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 subsequently contaminating red blood cells can be lysed. The washed effector cells can be resuspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum, or alternatively 5% heat-inactivated human serum, and mixed with Cr-labeled target cells expressing CLDN18.2 at various ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence-enhancing ligand (BATDA). The highly fluorescent chelate of europium and the enhancing ligand released from dead cells can be measured by a fluorometer. Another alternative technique can utilize transfection of target cells with luciferase. Subsequently, added luciferin can be oxidized only by live cells. Then, purified anti-CLDN18.2 IgG can be added at various concentrations. Irrelevant human IgG can be used as a negative control. Depending on the effector cell type used, the assay can be performed at 37 °C for 4 - 20 hours. 51 Cells can be assayed for cell lysis by measuring the release of Cr or the presence of EuTDA chelate in the culture supernatant. Alternatively, luminescence resulting from the oxidation of luciferin can serve as a measure of live cells. 51 Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine whether cell lysis is enhanced with multiple monoclonal antibodies. Anti-CLDN18.2 monoclonal antibodies can be tested in various combinations to determine whether cell lysis is enhanced with multiple monoclonal antibodies.

[0343] Complement-dependent cytotoxicity (CDC): Using various known techniques, the ability of monoclonal anti-CLDN18.2 antibodies to mediate CDC can be tested. For example, serum for complement can be obtained from blood by methods known to those skilled in the art. Various methods can be used to determine the CDC activity of the mAb. For example, 51 Cr can be measured, or increased membrane permeability can be evaluated using a propidium iodide (PI) exclusion assay. Briefly, target cells are washed and can be incubated with 5×10 5 / ml of the mAb at various concentrations for 10 - 30 minutes at room temperature or 37°C. Then, serum or plasma can 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 of each sample can be added to the PI solution in a FACS tube. Then, the mixture can be analyzed immediately by flow cytometry using a FACSArray.

[0344] In an alternative assay, induction of CDC can be determined in adherent cells. In one embodiment of this assay, cells are seeded at a density of 3×104 / well in tissue culture flat-bottom microtiter plates 24 hours before the assay. The next day, the growth medium is removed, and the cells are incubated in triplicate with the antibody. Control cells are incubated with growth medium or growth medium containing 0.2% saponin to determine background lysis and maximum lysis, respectively. After a 20-minute incubation at room temperature, the supernatant is removed, 20% (v / v) human plasma or serum in DMEM (pre-warmed to 37°C) is added to the cells, and the cells are incubated for an additional 20 minutes at 37°C. All cells of each sample are added to a propidium iodide solution (10 μg / ml). Then, the supernatant is replaced with PBS containing 2.5 μg / ml ethidium bromide, and fluorescence emission at 520 nm excitation is measured at 600 nm using a Tecan Safire. The percentage specific lysis is calculated as follows: % specific lysis = (fluorescent sample - fluorescent background) / (fluorescent maximum lysis - fluorescent background) × 100.

[0345] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies: To test the ability to initiate apoptosis, for example, a monoclonal anti-CLDN18.2 antibody can be incubated with CLDN18.2-positive tumor cells, such as SNU-16, DAN-G, KATO-III or CLDN18.2 transfected tumor cells, at 37 °C for about 20 hours. The cells can be harvested, washed with Annexin-V binding buffer (BD biosciences), and incubated with Annexin V conjugated to FITC or APC (BD biosciences) for 15 minutes in the dark. All cells of 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, the overall inhibition of cell proliferation by monoclonal antibodies can be detected with a commercially available kit. 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, a Fix solution is used to fix the cells and denature the DNA. Unbound antibody is washed away and a DELFIA inducer is added to dissociate europium ions from the labeled antibody into the solution, where the europium ions form a highly fluorescent chelate with a component of the DELFIA inducer. Fluorescence measured using time-resolved fluorescence measurement in detection is proportional to DNA analysis in the cells of each well.

[0346] Preclinical trials The monoclonal antibody that binds to CLDN18.2 can also be tested in an in vivo model (e.g., cell lines expressing CLDN18.2, such as DAN-G, SNU-16 or KATO-III, or immunodeficient mice bearing xenograft tumors inoculated with, for example, HEK293 after transfection) to determine its effectiveness in controlling the growth of CLDN18.2-expressing tumor cells.

[0347] Using the antibodies described herein, in vivo tests can be performed after xenotransplanting CLDN18.2-expressing tumor cells into immunodeficient mice or other animals. The antibody can be administered to mice without tumors, followed by injection of tumor cells to measure the effect of the antibody in preventing tumor formation or tumor-related symptoms. The antibody can be administered to mice with tumors to determine the therapeutic effectiveness of each antibody in reducing tumor growth, metastasis or tumor-related symptoms. Antibody application can be combined with the application of immune checkpoint inhibitors, cell growth inhibitors, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other substances as other antibodies to determine the effectiveness and potential toxicity of the combination. To analyze the toxic side effects mediated by the antibody, animals can be inoculated with the antibody or a control reagent to thoroughly investigate the symptoms possibly associated with CLDN18.2 antibody therapy. CL Possible side effects of in vivo application of CLDN18.2 antibodies include toxicity, especially in CLDN18.2-expressing tissues including the stomach. Antibodies that recognize CLDN18.2 in humans and other species, such as mice, are particularly useful for predicting possible side effects mediated by the application of monoclonal CLDN18.2 antibodies in humans.

[0348] Mapping of epitopes recognized by 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.

[0349] The compounds and agents described herein can be administered in the form of any suitable pharmaceutical composition.

[0350] The term "pharmaceutical composition" preferably relates to a formulation containing a therapeutically effective agent together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administration of the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0351] Pharmaceutical compositions are usually provided in a uniform dosage form and can be prepared by methods known per se. The pharmaceutical composition can be in the form of, for example, a solution or a suspension.

[0352] The pharmaceutical compositions according to the present disclosure are generally applied as a "pharmaceutically effective amount" and as a "pharmaceutically acceptable preparation".

[0353] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active components of the pharmaceutical composition.

[0354] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that, alone or in conjunction with additional dosages, achieves a desired response or desired effect. In the case of treating a particular disease, the desired response preferably relates to inhibiting the progression of the disease. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in treating a disease can also be a delay in the onset or prevention of the onset of the disease or the condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, size and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Accordingly, the dosage administered of the compositions described herein can depend on various such parameters. If the response in a patient is inadequate with an initial dosage, higher dosages (or equivalently high dosages achieved by a different, more localized route of administration) can be used.

[0355] The pharmaceutical compositions of the present disclosure can contain salts, buffers, preservatives and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0356] Preservatives suitable for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0357] The term "excipient" as used herein refers to a substance that can be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents or coloring agents.

[0358] The term "diluent" relates to a diluting and / or thinning agent. Further, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and / or a mixed medium. Examples of suitable diluents include ethanol, glycerol and water.

[0359] The term "carrier" refers to a component, which may be natural, synthetic, organic or inorganic, with which the active ingredients are combined to facilitate, enhance or enable the administration of a pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene and, in particular, biocompatible polylactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0360] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (ed. A.R Gennaro 1985).

[0361] A pharmaceutical carrier, excipient or diluent may be selected with respect to the intended route of administration and standard pharmaceutical practice.

[0362] In one embodiment, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration can include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any method other than through the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical compositions are formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration. The composition can be injected directly into a tumor or lymph node.

[0363] As used herein, the term "co-administering" means the process by which different compounds or compositions are administered to the same patient. For example, the anti-CLDN18.2 antibody and the immune checkpoint inhibitor described herein can be administered together, essentially simultaneously, or sequentially. When administration is sequential, the anti-CLDN18.2 antibody can be administered before or after the immune checkpoint inhibitor. When administration is together, the anti-CLDN18.2 antibody and the immune checkpoint inhibitor need not be administered within the same composition. The anti-CLDN18.2 antibody and the immune checkpoint inhibitor can be administered one or more times, and the number of administrations of each component can be the same or different. Further, the anti-CLDN18.2 antibody and the immune checkpoint inhibitor need not be administered simultaneously. is not required.

[0364] The agents and compositions described herein can be administered to a patient, for example, in vivo, to treat or prevent various disorders, such as those described herein. Preferred patients include human patients having a disorder that can be corrected or improved by administration of the agents and compositions described herein. This includes disorders involving cells characterized by the expression of CLDN18.2.

[0365] For example, in one embodiment, the agents described herein can be used to treat a patient having a cancer disease as described herein, such as a cancer disease characterized by the presence of cancer cells expressing CLDN18.2.

[0366] The pharmaceutical compositions and treatment methods described by the present invention can also be used for immunization or vaccination to prevent the diseases described herein.

[0367] As used herein, "instructional material" or "instruction" includes publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kit of the present invention can be attached, for example, to a container containing the composition of the present invention, or can be shipped together with the container containing the composition. Alternatively, the instructional material can be shipped separately from the container, intending that the instructional material and the composition be used cooperatively by the recipient.

[0368] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention.

Example

[0369] Example 1 Efficacy test of the combination of anti-CLDN18.2 antibody and immune checkpoint inhibitor in vivo To determine whether the combination of anti-CLDN18.2 antibody and immune checkpoint inhibitor provides improved antitumor activity in vivo compared to single agent alone, the antitumor activity of IMAB362 in combination with anti-mPD-1 antibody was examined in a subcutaneous syngeneic gastric cancer model in immunocompetent outbred Crl:NMRI(Han) mice using CLS-103 cells transduced with lentivirus expressing murine CLDN18.2 (CLS-103 LVT-mouse CLDN18.2). Rituximab was used as an isotype control for IMAB362.

[0370] Test antibody · Anti-CLDN18.2 antibody: IMAB362 (Astellas Pharma Inc.) · Control antibody: Rituximab BS intravenous injection [KHK] 500 mg (Kyowa Hakko Kirin Co., Ltd., catalog number 22900AMX00971000) · Anti-mPD-1 antibody: InVivoMAb anti-mouse PD-1, clone RMP1-14 (BioXCell, catalog number BE0146) · Isotype control antibody: InVivoMAb rat IgG2a isotype control, anti-trinitrophenol, clone 2A3 (BioXCell, catalog number BE0089) CLS-103 LVT-mouse CLDN18.2 gastric cancer mouse model

[0371] Lentivirus-transduced mouse CLDN18.2-expressing tumor cells (CLS-103 LV T-mouse CLDN18.2) were subcutaneously transplanted into the right flank of female Crl:NMRI(Han) mice (10 weeks old) at 2×10 6 cells / mouse. Based on the tumor volume measured 2 days after transplantation, the mice were randomized into 4 groups (n = 12 / group). The day of randomization was defined as day 0. IMAB362 or the control antibody rituximab was administered at 800 μg / mouse. The anti-mPD-1 antibody or the isotype control antibody was administered at 100 μg / mouse. All antibodies were administered by intraperitoneal injection twice a week starting on day 0. The tumors were measured twice a week. The test endpoint was defined as day 14. The tumor volume was determined by length × width × width × 0.5. The tumor growth inhibition (TGI [%]) of each group was calculated using the equation described below. TGI [%]=100×(1 - increase in the average tumor volume of each group ※ ÷ increase in the average tumor volume of the control group ※ ) ※ : increase in tumor volume [mm 3 = average tumor volume at the last measurement of each group - average tumor volume at randomization #, ##: p < 0.05, p < 0.01, compared with each single-agent group (Student's t-test).

[0372] As shown in Figure 1, in the mouse CLS-103 LVT-mouse CLDN18.2 tumor test, the combination treatment of IMAB362 and anti-mPD-1 antibody inhibited tumor growth to a much greater extent than the single-agent controls. On day 14, treatment with 800 μg of IMAB362 or 100 μg of anti-mPD-1 antibody resulted in 50% or 60% TGI, respectively, while the combination treatment containing 800 μg of IMAB362 + 100 μg of anti-mPD-1 antibody resulted in 91% TGI. On day 14, the mean tumor volume of the combination group was significantly smaller compared to each single-agent group (Student's t-test). Individual CLS-103 LVT-mouse CLDN18.2 tumor volumes for each of 12 mice / group are shown for all treatment groups (Figure 2). This spider plot analysis of individual tumor growth for all treated mice showed a significant delay and / or inhibition of tumor growth in mice treated with the combination of IMAB362 and anti-mPD-1 antibody.

[0373] Example 2 Long-term Efficacy Study of the Combination of Anti-CLDN18.2 Antibody and Immune Checkpoint Inhibitor In Vivo To determine whether the combination of anti-CLDN18.2 antibody and immune checkpoint inhibitor improves antitumor activity over a long period in vivo compared to single-agent alone, the antitumor activity of IMAB362 in combination with anti-mPD-1 antibody was examined up to day 28 in a subcutaneous syngeneic gastric cancer model in immune-responsive outbred Crl:NMRI(Han) mice using CLS-103 cells transduced with lentivirus expressing mouse CLDN18.2 (CLS-103 LVT-mouse CLDN18.2). Rituximab was used as an isotype control for IMAB362.

[0374] Test Antibodies · Anti-CLDN18.2 antibody: IMAB362 (Astellas Pharma Inc.) · Control antibody: Rituximab BS intravenous injection [KHK] 500 mg (Kyowa Hakko Kirin Co., Ltd., catalog number 22900AMX00971000) · Anti-mPD-1 antibody: InVivoMAb anti-mouse PD-1, clone RMP1-14 (BioXCell, catalog number BE0146) · Isotype control antibody: InVivoMAb rat IgG2a isotype control, anti-trinitrophenol, clone 2A3 (BioXCell, catalog number BE0089) CLS-103 LVT-mouse CLDN18.2 gastric cancer mouse model

[0375] Lentivirus-transduced mouse CLDN18.2-expressing tumor cells (CLS-103 LVT-mouse CLDN18.2) were subcutaneously transplanted into the right flank of female Crl:NMRI(Han) mice (11 weeks old) at 2 × 10 6 cells / mouse. Based on the tumor volume measured 2 days after transplantation, the mice were randomized into 4 groups (n = 12 / group). The day of randomization was defined as day 0. IMAB362 or the control antibody rituximab was administered at 800 μg / mouse. The anti-mPD-1 antibody or the isotype control antibody was administered at 100 μg / mouse. All antibodies were administered by intraperitoneal injection twice a week starting on day 0. The tumors were measured twice a week. The study endpoint was defined as day 28. The tumor volume was determined by length × width × width × 0.5. One of the 12 mice in the rituximab / isotype treatment group and the anti-mPD-1 antibody monotherapy group was sacrificed due to a tumor size exceeding 2000 mm 3 so the mean tumor volume was calculated until day 21. Using the equation described below, the tumor growth inhibition (TGI [%]) of each group was calculated based on the measurements obtained until day 21. Complete regression (CR) was determined until day 28 as the tumor volume of the individuals that regressed to zero. TGI [%]=100 × (1 - increase in the mean tumor volume of each group # ÷ increase in the mean tumor volume of the control group # ) # : increase in the mean tumor volume [mm 3= Mean tumor volume at day 21 of each group - Mean tumor volume at randomization (day 0) * p < 0.05, ** p < 0.01, compared to each single-agent group (Student's t-test).

[0376] Results In this mouse CLS-103 LVT - mouse CLDN18.2 tumor test, IMAB362 in combination with an anti-mPD-1 antibody synergistically improved the long-term antitumor effect determined by the number of CRs up to day 28. As shown in Figure 3, the individual tumor growth and the number of CR mice for each of 12 mice / group are shown for all treatment groups. At day 28, treatment with 800 μg of IMAB362 or 100 μg of the anti-mPD-1 antibody resulted in 2 or 1 CR, respectively, among the groups of 12 mice, while the combination treatment containing 800 μg of IMAB362 + 100 μg of the anti-mPD-1 antibody resulted in 6 CRs among the groups of 12 mice. The results of the present inventors showed the number of mice with CR in the treatment group of the combination of IMAB362 and the anti-mPD-1 antibody, which was increased synergistically compared to the single-agent groups. As shown in Figure 4, at day 21, treatment with 800 μg of IMAB362 or 100 μg of the anti-mPD-1 antibody resulted in 40% TGI or no tumor growth inhibition, respectively, while the combination treatment containing 800 μg of IMAB362 + 100 μg of the anti-mPD-1 antibody resulted in 87% TGI. The mean tumor volume of the combination group was significantly smaller compared to each single-agent group, demonstrating that IMAB362 and the anti-mPD-1 antibody act synergistically.

[0377] Example 3 Efficacy test of the combination of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor in vivo To determine whether the combination of an anti-CLDN18.2 antibody and an immune checkpoint inhibitor improves antitumor activity in vivo compared to single-agent monotherapy, the antitumor activity of IMAB362 in combination with an anti-mCTLA-4 antibody was examined in a subcutaneous syngeneic gastric cancer model in immunocompetent outbred Crl:NMRI(Han) mice using CLS-103 cells transduced with lentiviral mouse CLDN18.2 (CLS-103 LVT-mouse CLDN18.2). Rituximab was used as an isotype control for IMAB362.

[0378] Test antibody · Anti-CLDN18.2 antibody: IMAB362 (Astellas Pharma Inc .) · Control antibody: Rituximab BS intravenous injection [KHK] 500 mg (Kyowa Hakko Kirin Co., Ltd., catalog number 22900AMX00971000) · Anti-mCTLA-4 antibody: InVivoMAb anti-mouse CTLA-4, clone 9D9 (BioXCell, catalog number BE0164) · Isotype control antibody: InVivoMAb mouse IgG2b isotype control, unknown specificity, clone MPC-11 (BioXCell, catalog number BE0086) CLS-103 LVT-mouse CLDN18.2 gastric cancer mouse model

[0379] Lentivirus-transduced mouse CLDN18.2-expressing tumor cells (CLS-103 LVT-mouse CLDN18.2) were administered at 2×10 6In cells / mice, it was subcutaneously transplanted into the right abdomen of female Crl:NMRI(Han) mice (11 weeks old). Based on the tumor volume measured 2 days after transplantation, the mice were randomized into 4 groups (n = 12 / group). The day of randomization was defined as day 0. IMAB362 or the control antibody rituximab was administered at 800 μg / mouse. The anti-mCTLA-4 antibody or the isotype control antibody was administered at 300 μg / mouse. All antibodies were administered by intraperitoneal injection twice a week starting on day 0. The tumors were measured twice a week. The test endpoint was defined as day 14. The tumor volume was determined by length × width × width × 0.5. The tumor growth inhibition (TGI [%]) or tumor regression rate (TRR [%]) of each group was calculated using the equations described below. TGI [%]=100×(1 - increase in the average tumor volume of each group # ÷ increase in the average tumor volume of the control group # ) # : increase in the average tumor volume [mm 3 = average tumor volume of the measurement on day 14 of each group - average tumor volume at the time of randomization (day 0) TRR [%]=100×(1 - average tumor volume of the measurement on day 14 of each group ÷ average tumor volume of each group at the time of randomization) * : p < 0.05, compared with each single-agent group (Student's t-test).

[0380] Results As shown in Figure 5, in the mouse CLS-103 LVT-mouse CLDN18.2 tumor test, the combined treatment of IMAB362 and anti-mCTLA-4 antibody inhibited tumor growth to a greater extent than the single-agent group. On day 14, treatment with 800 μg of IMAB362 or 300 μg of anti-mCTLA-4 antibody resulted in 72% or 87% TGI, respectively, while the combined treatment containing 800 μg of IMAB362 + 300 μg of anti-mCTLA-4 antibody not only inhibited the tumor but also regressed the tumor to 3% TRR. The individual CLS-103 LVT-mouse CLDN18.2 tumor volumes for each of 12 mice / group are shown for all treatment groups (Figure 6). In mice treated with the combination of IMAB362 and anti-mCTLA-4 antibody, individual tumor growth for all treated mice showed inhibition or even regression of tumor growth.

[0381] Example 4 Efficacy study of the combination of anti-CLDN18.2 antibody and immune checkpoint inhibitor in vivo To determine whether the combination of anti-CLDN18.2 antibody and immune checkpoint inhibitor improves antitumor activity in vivo compared to single-agent alone, the antitumor activity of IMAB362 in combination with anti-mPD-L1 antibody was examined in a subcutaneous syngeneic gastric cancer model in immunocompetent outbred Crl:NMRI(Han) mice using CLS-103 cells transduced with lentivirus of mouse CLDN18.2 (CLS-103 LVT-mouse CLDN18.2). Rituximab was used as an isotype control for IMAB362.

[0382] Test antibodies · Anti-CLDN18.2 antibody: IMAB362 (Astellas Pharma Inc.) · Control antibody: Rituximab BS intravenous injection [KHK] 500 mg (Kyowa Hakko Kirin Co., Ltd., catalog number 22900AMX00971000) · Anti-mPD-L1 antibody: InVivoMAb anti-mouse PD-L1, clone 10F.9G2 (BioXCell, catalog number BE0101) · Isotype control antibody: InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin, clone LTF-2 (BioXCell, catalog number BE0090) CLS-103 LVT - Mouse CLDN18.2 Gastric Cancer Mouse Model

[0383] Lentivirus-transduced mouse CLDN18.2-expressing tumor cells (CLS-103 LVT - Mouse CLDN18.2) were subcutaneously implanted into the right flank of female Crl:NMRI(Han) mice (11 weeks old) at 2×10 6 cells / mouse. Based on the tumor volume measured 2 days after implantation, the mice were randomized into 4 groups (n = 12 / group). The rituximab / isotype control group consisted of data from 11 out of 12 mice due to the death of 1 mouse that occurred on day 11. The day of randomization was defined as day 0. IMAB362 or the control antibody rituximab was administered at 800 μg / mouse. The anti-mPD-L1 antibody or isotype control antibody was administered at 300 μg / mouse. All antibodies were administered by intraperitoneal injection twice a week starting on day 0. The tumors were measured twice a week. The test endpoint was defined as day 14. The tumor volume was determined by length × width × width × 0.5. The tumor growth inhibition (TGI [%]) or tumor regression rate (TRR [%]) of each group was calculated using the equations described below. TGI [%]=100×(1 - increase in the average tumor volume of each group # ÷ increase in the average tumor volume of the control group # ) # : increase in the average tumor volume [mm 3 = average tumor volume at the measurement on day 14 of each group - average tumor volume at randomization (day 0) TRR [%]=100×(1 - average tumor volume at the measurement on day 14 of each group ÷ average tumor volume at randomization of each group) * , ** : p < 0.05, p < 0.01, compared with each single-agent group (Student's t-test).

[0384] Results In Figure 7, in the mouse CLS-103 LVT-mouse CLDN18.2 tumor test, the combination treatment of IMAB362 and anti-mPD-L1 antibody inhibited tumor growth to a much greater extent than the single-agent groups. On day 14, treatment with 800 μg of IMAB362 or 300 μg of anti-mPD-L1 antibody resulted in 60% or 62% TGI, respectively, while the combination treatment containing 800 μg of IMAB362 + 300 μg of anti-mPD-L1 antibody not only inhibited tumors but also regressed tumors to 13% TRR. On day 14, the mean tumor volume of the combination group was significantly smaller compared to each single-agent group, demonstrating that IMAB362 and anti-mPD-L1 antibody act synergistically. The individual CLS-103 LVT-mouse CLDN18.2 tumor volumes for each mouse / group are shown for all treatment groups (Figure 8). In mice treated with the combination of IMAB362 and anti-mPD-L1 antibody, individual tumor growth for all treated mice showed synergistic inhibition or even regression of tumor growth.

Claims

1. A method for treating or preventing cancer in a patient, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

2. A method for inhibiting tumor growth in a patient having cancer, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

3. A method for inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells in a patient having cancer, the method comprising administering to the patient an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

4. The method according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor and a PD-L1 inhibitor.

5. The method according to any one of claims 1 to 4, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody and an anti-PD-L1 antibody.

6. The method according to any one of claims 1 to 5, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

7. The method according to claim 6, wherein the anti-PD-1 antibody is nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN2810), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ63723283), toripalimab (JS001), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI754091, or SHR-1210.

8. The method according to any one of claims 1 to 5, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

9. The method according to claim 8, wherein the anti-PD-L1 antibody is atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (bavencio), rodaplimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, or MDX-1105.

10. The method according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor.

11. The method according to any one of claims 1 to 3 and 10, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

12. The method according to claim 11, wherein the anti-CTLA-4 antibody is ipilimumab (Yervoy; Bristol Myers Squibb), tremelimumab (Pfizer / MedImmune), toripalimab, AGEN-1884 (Agenus) or ATR-1015.

13. The method according to any one of claims 1 to 12, wherein the anti-CLDN18.2 antibody binds to the first extracellular loop of CLDN18.

2.

14. The method according to any one of claims 1 to 13, wherein the anti-CLDN18.2 antibody mediates cell death 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.

15. The anti-CLDN18.2 antibody is (i) an antibody produced by and / or obtained from a clone deposited under accession number 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 of (i), (iii) an antibody having the specificity of the antibody of (i), and (iv) an antigen-binding portion or antigen-binding site of the antibody of (i), particularly an antibody comprising a variable region and preferably having the specificity of the antibody of (i), The method according to any one of claims 1 to 14, which is an antibody selected from the group consisting of.

16. The method according to any one of claims 1 to 15, wherein the anti-CLDN18.2 antibody comprises a heavy chain variable region CDR1 comprising the sequence at positions 45 to 52 of the sequence shown in SEQ ID NO: 17, a heavy chain variable region CDR2 comprising the sequence at positions 70 to 77 of the sequence shown in SEQ ID NO: 17, a heavy chain variable region CDR3 comprising the sequence at positions 116 to 126 of the sequence shown in SEQ ID NO: 17, a light chain variable region CDR1 comprising the sequence at positions 47 to 58 of the sequence shown in SEQ ID NO: 24, a light chain variable region CDR2 comprising the sequence at positions 76 to 78 of the sequence shown in SEQ ID NO: 24, and a light chain variable region CDR3 comprising the sequence at positions 115 to 123 of the sequence shown in SEQ ID NO:

24.

17. The method according to any one of claims 1 to 16, wherein the anti-CLDN18.2 antibody comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 32 or a functional variant thereof, or a fragment of said amino acid sequence or functional variant.

18. The method according to any one of claims 1 to 17, wherein the anti-CLDN18.2 antibody comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 39 or a functional variant thereof, or a fragment of said amino acid sequence or functional variant.

19. The method according to any one of claims 1 to 18, wherein the anti-CLDN18.2 antibody comprises a heavy chain constant region comprising the sequence shown in SEQ ID NO: 13 or 52, or a functional variant thereof, or a fragment of said amino acid sequence or functional variant.

20. The method according to any one of claims 1 to 19, wherein the anti-CLDN18.2 antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO: 17 or 51, or a functional variant thereof, or a fragment of said amino acid sequence or functional variant.

21. The method according to any one of claims 1 to 20, wherein the anti-CLDN18.2 antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 24 or a functional variant thereof, or a fragment of said amino acid sequence or functional variant.

22. At a dose of up to 1000 mg / m ２ The method according to any one of claims 1 to 21, comprising the step of administering the anti-CLDN18.2 antibody at the dose.

23. Administering the anti-CLDN18.2 antibody at a dose of 300 to 600 mg / m ２ The method according to any one of claims 1 to 22, comprising the step of administering the anti-CLDN18.2 antibody at a dose of

24. The method according to any one of claims 1 to 23, wherein the cancer is CLDN18.2 positive.

25. The method according to any one of claims 1 to 24, wherein the cancer is adenocarcinoma, particularly advanced adenocarcinoma.

26. The method according to any one of claims 1 to 25, wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly cancer of the lower esophagus, cancer of the esophagogastric junction, and gastroesophageal cancer.

27. The method according to any one of claims 1 to 26, wherein CLDN18.2 has the amino acid sequence according to SEQ ID NO:

1.

28. A pharmaceutical preparation comprising an anti-CLDN18.2 antibody and an immune checkpoint inhibitor.

29. The pharmaceutical preparation according to claim 28, comprising a first container containing the anti-CLDN18.2 antibody and a second container containing the immune checkpoint inhibitor.

30. The pharmaceutical preparation according to claim 28 or 29, further comprising printed instructions for using the preparation for treating cancer.

31. The pharmaceutical preparation according to claim 28, which is a composition comprising the anti-CLDN18.2 antibody and the immune checkpoint inhibitor.

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