Therapies Comprising Antibodies Against Claudin 18.2 for the Treatment of Cancer - Patent application

Abstract

To provide a therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, in particular cancer diseases such as gastroesophageal cancer.SOLUTION: A method comprises administering an antibody having the ability of binding to CLDN18.2 so as to provide a serum level of at least 40 μg / ml.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cancer of the stomach and esophagus (gastroesophageal: GE) is one of the malignancies with the greatest unmet medical need. Gastric cancer is the second leading cause of cancer death worldwide. The incidence of esophageal cancer has increased over the past few decades, consistent with changes in histological type and primary tumor site. Esophageal adenocarcinoma is now more common than squamous cell carcinoma in the United States and Western Europe, with most tumors located in the distal esophagus. The overall 5-year survival rate for GE cancer is 20-25%, despite aggressive implementation of established standard treatments associated with substantial side effects.

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

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

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

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

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

[0007] IMAB362, a chimeric IgG1 antibody against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and combines four independent and highly potent mechanisms of action. After target binding, IMAB362 mediates cell death through ADCC, CDC, and induction of apoptosis induced by target cross-linking on the tumor cell surface, as well as direct inhibition of proliferation. Accordingly, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. When treated with IMAB362, mice bearing CLDN18.2-positive cancer cell lines experience a survival benefit, with up to 40% of mice showing tumor regression.

[0008] The toxicity and PK / TK profile of IMAB362 have been thoroughly investigated in mice and cynomolgus monkeys, including a dose-ranging study, a 28-day repeat-dose toxicity study in cynomolgus monkeys, and a 3-month repeat-dose toxicity study in mice. Repeated administration of IMAB362 iv is well tolerated in both mice (maximum treatment duration of 3 months, once weekly, at a maximum dose level of 400 mg / kg) and cynomolgus monkeys (up to 5 weeks, once weekly, at a maximum dose of 100 mg / kg). No signs of systemic or local toxicity are induced. In particular, gastric toxicity has not been observed in any toxicity study. IMAB362 does not induce immune activation or cytokine release. No adverse effects on male or female reproductive organs were recorded. IMAB362 does not bind to target-deprived tissues. Biodistribution in mice indicates that the absence of gastric toxicity is likely due to compartmentalization of tight junctions at the luminal site of healthy gastric epithelium, which appears to greatly reduce the accessibility of the IMAB362 epitope. This compartmentalization is lost upon malignant transformation, making this epitope druggable by IMAB362. Summary of the Invention [Problem to be solved by the invention]

[0009] Herein, the inventors demonstrate that administration of an anti-CLDN18.2 antibody, such as IMAB362, to a human patient with gastroesophageal cancer results in a dose of at least 1000 mg / m 2 Data are presented demonstrating that the antibody is safe and well tolerated up to doses of 100 mg / kg / day. Furthermore, the data presented herein demonstrate that the antibody is fully functional to exert anti-tumor cell effects in these patients and provides evidence of anti-tumor activity. [Means for solving the problem]

[0010] The present invention generally provides a therapeutic method 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 (e.g., 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 metastasis, e.g., Krukenberg tumor, peritoneal metastasis, and lymph node metastasis. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary.

[0011] In a first aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2, wherein the antibody is administered to provide a serum level of at least 40 μg / ml. In different embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In different embodiments, the antibody is administered to provide a serum level of 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml or less. In one embodiment, the serum level provided is between 40 μg / ml and 700 μg / ml, preferably between 40 μg / ml and 600 μg / ml, preferably between 50 μg / ml and 500 μg / ml, for example between 150 μg / ml and 500 μg / ml or between 300 μg / ml and 500 μg / ml. As used herein, the term "serum level" refers to the concentration of the substance in the serum. In one embodiment, the serum level is provided for at least 7 days or at least 14 days. In one embodiment, the method provides a serum level of at least 300 mg / m 2 , e.g., at least 600 mg / m 2 , preferably 1500 mg / m 2 Up to 1200 mg / m 2 or up to 1000 mg / m 2 The method comprises administering a dose of the antibody up to

[0012] In a second aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2, wherein said antibody is administered at a dose of at least 300 mg / m 2 , e.g., at least 600 mg / m 2 , preferably 1500 mg / m 2 Up to 1200 mg / m 2 or up to 1000 mg / m 2 Administer at doses up to

[0013] In a third aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2, wherein at least 50%, preferably 60%, 70%, 80%, or 90% of the patient's cancer cells are CLDN18.2-positive and / or at least 40%, preferably 50% or 60% of the patient's cancer cells are positive for surface expression of CLDN18.2. In this aspect, the present invention also provides a method for treating or preventing a cancer disease, comprising: a. identifying a patient exhibiting at least 50%, preferably 60%, 70%, 80%, or 90% CLDN18.2-positive cancer cells and / or exhibiting at least 40%, preferably 50% or 60% of cancer cells that are positive for surface expression of CLDN18.2; and b. administering to the patient an antibody capable of binding to CLDN18.2. In one embodiment, at least 95% or at least 98% of the patient's cancer cells are CLDN18.2-positive. In one embodiment, at least 70%, at least 80%, or at least 90% of the patient's cancer cells are positive for surface expression of CLDN18.2.

[0014] In one embodiment of the method of any of the aspects described herein, treating the cancer disease results in the achievement of stable disease, hi one embodiment, stable disease is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0015] In a fourth aspect, the present invention provides a method of achieving stable disease in a cancer patient, comprising administering to the patient an antibody capable of binding to CLDN18.2. In one embodiment, stable disease is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0016] In one embodiment of the method of any of the aspects described herein, the antibody is administered in a single dose or in multiple doses.

[0017] In a fifth aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2, wherein said antibody is administered in multiple doses.

[0018] According to the present invention, when the antibody is administered in multiple doses, the antibody is preferably administered in at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, or at least 10 doses, preferably 30, 25, 20, 15, or 10 doses. The antibody administrations are preferably separated by a time interval of at least 7 days, at least 10 days, at least 14 days, or at least 20 days. The antibody administrations are preferably separated by a time interval of 7 to 30 days, 10 to 20 days, preferably about 14 days.

[0019] In one embodiment of the method of the third, fourth, or fifth aspect, the antibody is administered to provide a serum level of at least 40 μg / ml. In different embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In different embodiments, the antibody is administered to provide a serum level of 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml or less. In one embodiment, the serum level provided is between 40 μg / ml and 700 μg / ml, preferably between 40 μg / ml and 600 μg / ml, preferably between 50 μg / ml and 500 μg / ml, for example between 150 μg / ml and 500 μg / ml or between 300 μg / ml and 500 μg / ml. In one embodiment, serum levels are provided for at least 7 days or at least 14 days. In one embodiment, the method provides a serum level of at least 300 mg / m 2 , e.g., at least 600 mg / m 2 , preferably 1500 mg / m 2 Up to 1200 mg / m 2 or up to 1000 mg / m 2 The method comprises administering a dose of the antibody up to

[0020] In one embodiment of the method of any of the above aspects, the method further comprises administering one or more selected from the group consisting of antiemetics, antispasmodics, parasympatholytics, and agents that protect the gastric mucosa.

[0021] In a sixth aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to CLDN18.2 and one or more selected from the group consisting of antiemetics, antispasmodics, parasympatholytics and agents that protect the gastric mucosa.

[0022] When the method of the present invention comprises administering one or more selected from the group consisting of an antiemetic, an antispasmodic, a parasympatholytic, and an agent that protects the gastric mucosa, the method in various embodiments comprises administering (i) an antiemetic and an antispasmodic, (ii) an antispasmodic and an agent that protects the gastric mucosa, (iii) an antiemetic and an agent that protects the gastric mucosa, or (iv) an antiemetic, an antispasmodic, and an agent that protects the gastric mucosa.

[0023] In one embodiment, an antiemetic drug is administered prior to administration of the antibody as an antiemetic drug. In one embodiment, an antiemetic drug is administered concurrently with or after administration of the antibody as an antiemetic treatment. In one embodiment, the antiemetic drug includes a 5-HT3 receptor antagonist and / or a neurokinin 1 (NK1) receptor antagonist. Preferably, the NK1 receptor antagonist includes aprepitant (e.g., Emend), and the 5-HT3 receptor antagonist includes ondansetron (e.g., Zofran), granisetron (e.g., Kytril, Sancuso), or palonosetron (e.g., Aloxi), or a combination of two or more thereof.

[0024] In one embodiment, antispasmodics include butylscopolamine (Buscopan).

[0025] In one embodiment, the agent that protects the gastric mucosa includes an agent that reduces gastric acid production. In one embodiment, the agent that protects the gastric mucosa includes an agent selected from the group consisting of a proton pump inhibitor, misoprostol, and omeprazole. In one embodiment, the agent that protects the gastric mucosa includes a combination of a proton pump inhibitor and misoprostol. In one embodiment, the proton pump inhibitor includes pantoprazole (e.g., Pantozol).

[0026] In one embodiment, the methods of the invention comprise administering to the patient an NK1 receptor antagonist such as aprepitant (e.g., Emend), a 5-HT3 receptor antagonist such as ondansetron (e.g., Zofran), granisetron (e.g., Kytril, Sancuso) or palonosetron (e.g., Aloxi) or a combination of two or more thereof, an anticonvulsant such as butylscopolamine (Buscopan), and a proton pump inhibitor such as pantoprazole (e.g., Pantozol).

[0027] In one embodiment of the method of any of the above aspects, the antibody is administered by iv infusion, hi one embodiment, the iv infusion is over a period of 1 to 4 hours, preferably over a period of about 2 hours.

[0028] In a sixth aspect, the present invention provides a method for determining the responsiveness of a cancer patient to treatment or prevention of a cancer disease comprising administering an antibody capable of binding to CLDN18.2, the method comprising measuring the blood levels of one or more markers in the patient, where the one or more markers are selected from the group consisting of CA 125, CA 15-3, CA 19-9, CEA, IL-2, IL-15, IL-6, IFNγ, and TNFα. In this aspect, a biological sample, such as blood, can be collected from the patient before and after administration of the antibody capable of binding to CLDN18.2, e.g., after administration of a single dose of the antibody, to establish the levels of one or more markers. Multiple samples can be collected from the same tissue, and an average level can be determined to account for possible variations in the levels. The levels of one or more markers after administration of the antibody are compared with the levels measured before administration. Therefore, the effect of the antibody on the patient can be identified by a desirable change in the levels of the markers after administration of the antibody capable of binding to CLDN18.2. If the patient shows a desirable change in the level of the marker after administration of the antibody capable of binding to CLDN18.2, treatment with the antibody capable of binding to CLDN18.2 can be initiated.

[0029] In one embodiment, the levels are measured in blood, plasma or serum.

[0030] In one embodiment, the one or more markers are selected from the group consisting of CA 125, CA 15-3, CA 19-9, CEA, IL-2, IL-15, IFNγ and TNFα, and a decrease in the level of at least one of the markers following administration of the antibody indicates that the patient will be responsive to treatment or prevention of the cancer disease.

[0031] In one embodiment, the marker is IL-6, and an increase in the level of the marker after administration of the antibody indicates that the patient will be responsive to treatment or prevention of the cancer disease.

[0032] In an eighth aspect, the present invention provides a method for determining whether a cancer patient is suitable for treatment or prevention of a cancer disease comprising administering an antibody capable of binding to CLDN18.2, said method comprising a step of measuring the percentage of CLDN18.2-positive cancer cells.

[0033] In this embodiment, prior to administration of the antibody capable of binding to CLDN18.2, a biological sample, such as a tumor sample (e.g., a tumor biopsy), may be obtained from the patient to establish the level of CLDN18.2-positive cancer cells. Multiple samples may be obtained and an average level determined to account for possible variations in those levels. When the patient has a desired level of CLDN18.2-positive cancer cells, the antibody capable of binding to CLDN18.2 may be administered.

[0034] In one embodiment, a level of at least 50%, preferably 60%, 70%, 80%, or 90%, at least 95%, or at least 98% CLDN18.2-positive cancer cells indicates that the patient is suitable for treatment or prevention of a cancer disease. In one embodiment, a level of at least 40%, preferably at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of cancer cells that are positive for surface expression of CLDN18.2 indicates that the patient is suitable for treatment or prevention of a cancer disease.

[0035] An antibody capable of binding to CLDN18.2 can bind to a natural epitope of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, an antibody capable of binding to CLDN18.2 mediates cell death by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment, an antibody capable of binding to CLDN18.2 is a monoclonal, chimeric, or humanized antibody, or an antibody fragment. In one embodiment, the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of: (i) an antibody produced by and / or obtainable from the 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 chimeric or humanized form of an antibody included in (i); (iii) an antibody that has the specificity of an antibody included in (i); and (iv) an antibody that contains an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody included in (i) and preferably has the specificity of an antibody included in (i). In one embodiment, the antibody is linked to a therapeutic agent, such as a toxin, radioisotope, drug, or cytotoxic agent.

[0036] In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, cells of the cancer express CLDN18.2. In one embodiment, CLDN18.2 expression is on the cell surface. In one embodiment, at least 50%, preferably 60%, 70%, 80%, or 90% of the cancer cells are CLDN18.2 positive, and / or at least 40%, preferably at least 50% of the cancer cells are positive for surface expression of CLDN18.2. In one embodiment, at least 95% or at least 98% of the cancer cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80%, or at least 90% of the cancer cells are positive for surface expression of CLDN18.2.

[0037] In one embodiment, the cancer disease 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. The cancer disease may be Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly cancer of the lower esophagus, cancer of the gastroesophageal junction, and gastroesophageal cancer. In a particularly preferred embodiment, the cancer is gastroesophageal cancer, for example, metastatic, refractory, or recurrent advanced gastroesophageal cancer. The patient may be a HER2 / neu-negative patient or a patient with a HER2 / neu-positive status who is ineligible for trastuzumab treatment. In one embodiment, the patient has previously received treatment with at least one agent selected from the group consisting of pyrimidine analogs (e.g., fluorouracil and / or capecitabine), platinum compounds (e.g., cisplatin and / or oxaliplatin), epirubicin, docetaxel, and antidotes for antineoplastic drug treatment (e.g., calcium folinate and / or folinic acid). In one embodiment, the patient has an ECOG performance status of between 0 and 1 and / or a Karnofsky index of between 70 and 100%. In a particularly preferred embodiment, the patient is a human patient.

[0038] According to the present invention, CLDN18.2 preferably has an amino acid sequence according to SEQ ID NO:1.

[0039] The present invention also provides antibodies capable of binding to an agent described herein, such as CLDN18.2, for use in the methods described herein.

[0040] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0041] [Figure 1] Mean blood concentrations of IMAB362 during the study. [Figure 2A] (Figure 2) ADCC activity of patient PBMCs. (Figure 2A) PBMCs were purified from blood samples of six patients on day 7 (open squares) or day 14 (closed squares) after IMAB362 administration. Specific lysis of CLDN18.2-expressing NUGC-4 gastric cancer target cells obtained after 24 hours of addition of IMAB362 at 31.63 μg / ml and PBMCs from healthy donors or patient PBMCs (E:T = 20:1). [Figure 2B] IMAB362 concentration-dependent specific lysis of NUGC-4 cells obtained 24 hours after addition of PBMCs from different patients (graph shows mean ± standard deviation, p-values ​​calculated using unpaired t-test). [Figure 2C] ADCC response curve of healthy control PBMCs after addition of increasing concentrations of IMAB362. Assays were performed in parallel with each ADCC analysis on patient PBMCs. [Figure 2D] ADCC response curves of patient PBMCs after the addition of increasing concentrations of IMAB362 (not enough PBMCs were available to generate a curve for patient 0202). [Figure 2E] Half-maximal mortality rates for all patients and healthy donors were calculated in GraphPad Prism software using the built-in nonlinear regression analysis tool. [Figure 3]Ability of patient complement components to induce IMAB362-mediated CDC. CDC assays were performed using CLDN18.2 and luciferase-positive CHO-K1 target cells. Cells, serum (20% v / v), and antibodies were incubated at 37°C for 80 minutes. Patient samples were prepared by adding fresh 0.5 μg / ml IMAB362 to pre-infusion serum samples (gray bars). HSC: healthy human serum pool control spiked with 0.3–10 μg / ml IMAB362 (positive control). Hi: heat-inactivated human serum pool spiked with 10 μg / ml IMAB362 (negative control). Number of patients indicated. Error bars: ± standard deviation. [Figure 4A] (Figure 4) Ability of patient complement components to interact with IV-administered IMAB362 over time. Normalized CDC assays were performed using each patient's pre-infusion serum to adjust the IMAB362 concentration in each sample to 0.5 μg / ml (dilution factor 10-680). (Figure 4A) CDC assays were performed as shown in Figure 3. [Figure 4B] CDC assays were performed as shown in Figure 3 . [Figure 4C] Each dot represents the measurement result of one patient. Open squares: 0.5 μg / ml IMAB362 in human serum. p-values ​​obtained by paired t-test. Error bars: ± standard deviation. [Figure 5A] Kinetics of cytotoxicity induced by circulating IMAB362 administered intravenously. NUGC-4 target cells, healthy donor PBMCs (E:T = 40:1), and patient serum samples (25% v / v) were used in a total cytotoxicity assay to measure overall cytotoxic activity. For each patient, serum samples were collected 1, 7, 14, and 28-32 days after IMAB362 administration. Patients were treated with increasing doses of IMAB362 (33-1000 mg / m2). The antibody concentrations present in the assay are indicated below each bar. HSC: human serum pool control (EC80-100) spiked with 200.0 μg / ml fresh IMAB362. PSC: patient pre-infusion serum control spiked with 200.0 μg / ml fresh IMAB362. na: not available. [Figure 5B]Kinetics of cytotoxicity induced by circulating IMAB362 administered intravenously. NUGC-4 target cells, healthy donor PBMCs (E:T = 40:1), and patient serum samples (25% v / v) were used in a total cytotoxicity assay to measure overall cytotoxic activity. For each patient, serum samples were collected 1, 7, 14, and 28-32 days after IMAB362 administration. Patients were treated with increasing doses of IMAB362 (33-1000 mg / m2). The antibody concentrations present in the assay are indicated below each bar. HSC: human serum pool control (EC80-100) spiked with 200.0 μg / ml fresh IMAB362. PSC: patient pre-infusion serum control spiked with 200.0 μg / ml fresh IMAB362. na: not available. [Figure 6A] Kinetics of ADCC activity of IMAB362 in heat-inactivated patient serum. Assays were performed as described in previous figures, except that patient complement was heat-inactivated (56°C, 30 min), ADCC activity was detected (black and grey bars), and the additive effect of serum components (white bars) was calculated. [Figure 6B] Kinetics of ADCC activity of IMAB362 in heat-inactivated patient serum. Assays were performed as described in previous figures, except that patient complement was heat-inactivated (56°C, 30 min), ADCC activity was detected (black and grey bars), and the additive effect of serum components (white bars) was calculated. [Figure 7A] CDC activity induced by IMAB362 present in patient serum. CDC assays were performed using CLDN18.2 and luciferase-positive CHO-K1 target cells. These were incubated for 80 minutes with 20% v / v patient serum obtained 1, 7, 14, and 28-32 days after antibody infusion. Patients were treated with IMAB362 doses ranging from 33 to 1000 mg / m². The antibody concentration present in each assay is indicated below each bar. HSC: healthy human serum pool control spiked with decreasing concentrations of IMAB362 as indicated. PC: positive control (patient's pre-infusion serum spiked with 10 μg / ml IMAB362). [Figure 7B]CDC activity induced by IMAB362 present in patient serum. CDC assays were performed using CLDN18.2 and luciferase-positive CHO-K1 target cells. These were incubated for 80 minutes with 20% v / v patient serum obtained 1, 7, 14, and 28-32 days after antibody infusion. Patients were treated with IMAB362 doses ranging from 33 to 1000 mg / m². The antibody concentration present in each assay is indicated below each bar. HSC: healthy human serum pool control spiked with decreasing concentrations of IMAB362 as indicated. PC: positive control (patient's pre-infusion serum spiked with 10 μg / ml IMAB362). [Figure 8] Pharmacokinetic results of repeated infusions of IMAB362 in patients. Mean ± sd concentration of IMAB362 in serum (μg / ml) for 4 patients treated with repeated doses of 300 mg / m2 (Cohort 1, left panel) and up to 30 patients treated with repeated doses of 600 mg / m2 (1st infusion in 30 patients, 5th infusion in 12 patients) (Cohort 2 and Cohort 3 together, right panel). Arrows indicate IMAB362 infusions. The 1st infusion was performed on day 0. [Figure 9] Progression-free survival of patients in the full analysis set (FAS). [Figure 10] Progression-free survival of patients in the per-protocol (PP) set (n=20). DETAILED DESCRIPTION OF THE INVENTION

[0042] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, which may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are 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 those skilled in the art.

[0043] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with many of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0044] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0045] The practice of the present invention is based on the teachings of the art (e.g., Molecular Cloning: A Laboratory Manual, 2002), unless otherwise indicated. nd Conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques are used, as described in The Genetics of the Invention (see, for example, J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0046] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to refer to the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not to the exclusion of any other member, integer, or step or group of members, integers, or steps; however, in some embodiments, such other member, integer, or step or group of members, integers, or steps may be excluded, i.e., the subject matter resides in the inclusion of the stated member, integer, or step or group of members, integers, or steps. As used in connection with the description of the invention (particularly in connection with the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to be a shorthand way of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not impose limitations on the scope of the invention or what is claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0047] Several documents are cited throughout the text of this specification. 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 herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0048] The term "CLDN18" refers to claudin 18 and encompasses any variants, including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0049] The term "CLDN18.2" preferably relates to human CLDN18.2, and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 of the Sequence Listing or a variant of said amino acid sequence.

[0050] The term "CLDN18.1" preferably relates to human CLDN18.1, and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 2 of the Sequence Listing or a variant of said amino acid sequence.

[0051] The term "variant" according to the present invention particularly refers to mutants, splice variants, conformational variants, isoforms, allelic variants, species variants and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unknown. Complete gene sequencing often identifies numerous allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences that originate from a different species than that of a given nucleic acid or amino acid sequence. The term "variant" encompasses any post-translationally modified variants and conformational variants.

[0052] According to the present invention, the term "CLDN18.2-positive cancer" means a cancer comprising cancer cells that express CLDN18.2, preferably cancer cells that express CLDN18.2 on the surface of said cancer cells.

[0053] "Cell surface" is used according to its ordinary meaning in the art and thus includes the outside of the cell that is accessible to binding by proteins and other molecules.

[0054] CLDN18.2 is expressed on the surface of a cell if it is located on the surface of the cell and accessible for binding by a CLDN18.2-specific antibody added to the cell.

[0055] According to the present invention, CLDN18.2 is not substantially expressed in cells when its expression level is lower than that in gastric cells or gastric tissues. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower than that in gastric cells or gastric tissues. Preferably, CLDN18.2 is not substantially expressed in cells when its expression level is only 2-fold, preferably only 1.5-fold, higher than that in non-cancerous tissues other than the stomach, and preferably does not exceed that in said non-cancerous tissues. Preferably, CLDN18.2 is not substantially expressed in cells when its expression level is lower than the detection limit and / or when its expression level is so low that it does not allow binding by a CLDN18.2-specific antibody added to the cells.

[0056] According to the present invention, CLDN18.2 is expressed in cells when the expression level is at least 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10,000-fold higher than the expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in cells when the expression level is higher than the detection limit and / or when the expression level is high enough to allow binding by a CLDN18.2-specific antibody added to the cells. Preferably, CLDN18.2 expressed in cells is expressed or exposed on the surface of the cells.

[0057] According to the present invention, the term "disease" refers to any pathological condition, including cancer, particularly the forms of cancer described herein. Reference herein to cancer or a particular form of cancer also encompasses cancer metastasis thereof. In a preferred embodiment, the disease to be treated according to the present application involves cells that express CLDN18.2.

[0058] According to the present invention, a "disease associated with cells expressing CLDN18.2" or similar expression means that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. Increased 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, expression is observed only in diseased tissue, while expression in healthy tissue is suppressed. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those in which cancer cells express CLDN18.2.

[0059] As used herein, "cancer disease" or "cancer" encompasses diseases characterized by dysregulated cell growth, proliferation, differentiation, adhesion, and / or migration. The three malignant characteristics of cancer—uncontrolled proliferation (division beyond normal limits), invasion (invasion of and destruction of adjacent tissues), and sometimes metastasis (spread to lymph nodes or elsewhere in the body)—distinguish cancer from benign tumors, which are self-limited and do not invade or metastasize. While most cancers form tumors, some, such as leukemia, do not. "Cancer cells" refer to abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow even after the stimulus that initiated new growth has ceased. Preferably, "cancer diseases" are characterized by cells that express CLDN18.2, and the cancer cells express CLDN18.2. The CLDN18.2-expressing cells are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0060] According to the present invention, the term "tumor" or "tumor disease" refers to the abnormal proliferation of cells (called neoplastic cells, tumorigenic cells, or tumor cells), preferably forming a swelling or lesion. By "tumor cells" is meant abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow even after the stimulus that initiated the new growth has ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue and usually form a distinct tissue mass, which may be either benign, premalignant, or malignant.

[0061] According to the present invention, the tumor is preferably a malignant tumor. "Malignant tumor" is used synonymously with cancer.

[0062] "Adenocarcinoma" is a cancer that arises from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues lining body cavities and organs. Epithelium is embryologically derived from 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 forms may not. By staining cells from a biopsy, a pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Due to the ubiquity of glands in the body, adenocarcinoma can arise in many tissues. Although not every gland secretes the same substances, as long as the cells have an exocrine function, they are considered glandular, and therefore their malignant forms are named adenocarcinoma. Malignant adenocarcinomas can invade other tissues and often metastasize if given enough time. Ovarian adenocarcinoma is the most common type of ovarian cancer and includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0063] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that relies 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 vessels, and then infiltration of the target organ after transport by blood. Finally, the growth of new tumors at the target site relies on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis," which refers to metastasis far from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the therapeutic methods of the present invention is metastasis arising from gastric cancer as the primary site. In a preferred embodiment, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis, and / or lymph node metastasis.

[0064] Krukenberg tumor is a rare metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. The prognosis for Krukenberg tumor is still very poor, and no established treatment exists for Krukenberg tumor. Krukenberg tumor is a metastatic signet-ring cell adenocarcinoma of the ovary. The stomach is the primary site in the majority of Krukenberg tumor cases (70%). Cancers of the colon, appendix, and breast (primarily invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from cancer of the gallbladder, bile duct, pancreas, small intestine, ampulla of Vater, cervix, and bladder / urachus have been reported.

[0065] Women with Krukenberg tumors are typically in their 50s, with a mean age of 45 years, making them quite young for patients with metastatic cancer. This young age distribution may be related, in part, to the higher incidence of gastric signet-ring cell adenocarcinoma in young women. Common presenting symptoms are usually related to ovarian involvement, most commonly abdominal pain and bloating (primarily due to usually bilateral, often large ovarian masses). The remaining patients have nonspecific gastrointestinal symptoms or are asymptomatic. In addition, Krukenberg tumors are reportedly associated with virilization resulting from hormone production by the ovarian stroma. Ascites is present in 50% of cases and usually reveals malignant cells.

[0066] Krukenberg tumors are bilateral in more than 80% of reported cases. The ovaries are usually asymmetrically enlarged and have a raised outline. The sections have a yellow or white surface; they are usually solid, but occasionally cystic. Importantly, the capsular surface of ovaries with Krukenberg tumors is typically smooth and free of adhesions or peritoneal deposits. Of note, other metastatic tumors to the ovary tend to be associated with surface implants. This may explain why the gross morphology of Krukenberg tumors can sometimes appear similar to primary ovarian tumors. However, the bilateral nature of Krukenberg tumors is consistent with their metastatic nature.

[0067] Patients with Krukenberg tumors have a significantly higher overall mortality rate. Most patients die within 2 years (median survival, 14 months). Several studies indicate that the prognosis is poor if the primary tumor is identified after ovarian metastasis is discovered, and even worse if the primary tumor remains undisclosed.

[0068] "Treating" means administering a compound or composition or combination of compounds or compositions to a subject to prevent or eliminate disease, including reducing tumor size or number of tumors in the subject; halting or slowing the progression of disease in the subject; preventing or slowing the onset of new disease in the subject; reducing the frequency or severity of symptoms and / or recurrences in a subject who currently has or has previously had the disease; and / or prolonging, i.e., increasing, the survival of the subject.

[0069] In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or arresting the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms.

[0070] The term "patient", according to the present invention, refers to a subject for treatment, in particular a diseased subject, including a human, a non-human primate or another animal, in particular a mammal, such as a cow, horse, pig, sheep, goat, dog, cat or rodent, such as a mouse or rat. In a particularly preferred embodiment, the patient is a human.

[0071] According to the present invention, an antibody capable of binding to CLDN18.2 may be administered in combination with an agent that stabilizes or increases the expression of CLDN18.2, i.e., simultaneously with said agent, before the agent and / or after the agent.

[0072] The term "agent that stabilizes or increases CLDN18.2 expression" refers to an agent or combination of agents, the provision of which to a cell results in increased CLDN18.2 RNA and / or protein levels, preferably increased CLDN18.2 protein levels on the cell surface, compared to when the cell is not provided with the agent or combination of agents. Preferably, the cell is a cancer cell, particularly a cancer cell that expresses CLDN18.2, such as a cell of a cancer type described herein. The term "agent that stabilizes or increases CLDN18.2 expression" particularly refers to an agent or combination of agents, the provision of which to a cell results in a higher density of CLDN18.2 on the surface of the cell, compared to when the cell is not provided with the agent or combination of agents. "Stabilizing the expression of CLDN18.2" particularly includes a situation in which an agent or combination of agents prevents or reduces the decrease in CLDN18.2 expression, e.g., when an agent or combination of agents is not provided, CLDN18.2 expression would be decreased and providing the agent or combination of agents prevents or reduces the decrease in CLDN18.2 expression. "Increasing the expression of CLDN18.2" particularly includes a situation in which an agent or combination of agents increases CLDN18.2 expression, e.g., when an agent or combination of agents is not provided, CLDN18.2 expression would be decreased, remain essentially constant, or increase and providing the agent or combination of agents increases CLDN18.2 expression compared to a situation in which the agent or combination of agents is not provided, such that the resulting expression is higher compared to a situation in which CLDN18.2 expression would be decreased, remain essentially constant, or increase when the agent or combination of agents is not provided.

[0073] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" includes a chemotherapeutic agent or combination of chemotherapeutic agents, such as cytostatic agents. Chemotherapeutic agents can affect cells in one of the following ways: (1) by damaging the cell's DNA so that the cell can no longer replicate, (2) by inhibiting the synthesis of new DNA strands so that cell replication is not possible, or (3) by stopping the cell's mitotic process so that the cell cannot divide into two cells.

[0074] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" preferably relates to an agent or combination of agents, such as a cytostatic compound or combination of cytostatic compounds, that, when provided to cells, particularly cancer cells, causes the cells to arrest or accumulate in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the G1 and G0 phases, preferably other than the G1 phase, preferably in the G2 or S phase of the cell cycle, such as one or more of the G1 / G2, S / G2, G2 or S phases of the cell cycle. The term "cells arresting 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 goes through a cycle containing four phases to replicate itself. The first phase, called G1, is the stage in which the cell prepares to replicate its chromosomes. The second phase, called S, is the phase in which DNA synthesis occurs and DNA is replicated. The next phase is G2, where RNA and proteins replicate. The final phase is M, which is the actual cell division phase. In this final phase, the replicated DNA and RNA separate 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 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 S phase. Examples of these agents are 6-mercaptopurine and 5-fluorouracil.

[0075] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" includes anthracyclines such as epirubicin, platinum compounds such as oxaliplatin and cisplatin, nucleoside analogues such as 5-fluorouracil or its prodrugs, taxanes such as docetaxel, and camptothecin analogues such as irinotecan and topotecan, as well as drug combinations, for example, drug combinations comprising one or more of anthracyclines such as epirubicin, oxaliplatin and 5-fluorouracil, for example, drug combinations comprising oxaliplatin and 5-fluorouracil, or other drug combinations described herein.

[0076] In one preferred embodiment, the "agent that stabilizes or increases the expression of CLDN18.2" is an "agent that induces immunogenic cell death."

[0077] In certain circumstances, cancer cells can enter a lethal stress pathway that leads to the release of a spatiotemporally defined combination of signals that are decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:798-804). In such situations, cancer cells trigger the release of signals that are sensed by innate immune effectors such as dendritic cells and trigger cognate immune responses, including CD8+ T cells and IFN-γ signaling, so that tumor cell death can elicit an effective anticancer immune response. These signals include preapoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) at the cell surface, preapoptotic secretion of ATP, and postapoptotic release of the nuclear protein HMGB1. Taken together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Although anthracyclines, oxaliplatin, and gamma irradiation can induce all of the signals that define ICD, cisplatin, for example, lacks the ability to induce CRT translocation from the ER to the surface of dying cells, a step that requires ER stress, and requires complementation with the ER stress inducer thapsigargin.

[0078] According to the present invention, the term "agent that induces immunogenic cell death" refers to an agent or combination of agents that, when provided to cells, particularly cancer cells, can induce the cells to enter a lethal stress pathway that ultimately results in a tumor-specific immune response. In particular, when provided to cells, an agent that induces immunogenic cell death induces the cells to release a combination of spatiotemporally defined signals, including, inter alia, pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) at the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1.

[0079] According to the present invention, the term "agent that induces immunogenic cell death" encompasses anthracyclines and oxaliplatin.

[0080] Anthracyclines are a class of drugs that are also antibiotics and are commonly used in cancer chemotherapy. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[0081] Anthracyclines preferably exert one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by intercalating between the base pairs of DNA / RNA strands, thus preventing replication of rapidly proliferating cancer cells; 2. Inhibit topoisomerase II enzymes, preventing the relaxation of supercoiled DNA and thus blocking DNA transcription and replication; 3. Generate iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0082] According to the present invention, the term "anthracycline" relates to an agent, preferably an anti-cancer agent, for inducing apoptosis, preferably by inhibiting the DNA rebinding of topoisomerase II.

[0083] Preferably, according to the present invention, the term "anthracycline" generally refers to a compound having the following ring structure: [ka] and includes analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof.

[0084] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pirarubicin, valrubicin, N-trifluoroacetyldoxorubicin-14-valerate, aclacinomycin, morpholinodoxorubicin (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, which are anthracycline analogs that lack the sugar moiety of anthracyclines but retain a planar polycyclic aromatic ring structure that allows for intercalation into DNA.

[0085] Particularly preferred as anthracyclines according to the present invention are compounds of the following formula: [ka] wherein R1 is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and OMe, R3 is selected from the group consisting of H and OH, and R4 is selected from the group consisting of H and OH.

[0086] 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.

[0087] Particularly contemplated as an anthracycline in the context of the present invention is epirubicin, which has the following formula: [ka] It is an anthracycline drug having the formula: Ellence (Ellence) in the United States and Pharmorubicin (Ebewe) elsewhere. In particular, the term "epirubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. Epirubicin is preferred over doxorubicin, the most common anthracycline, in some chemotherapy regimens because it is believed to cause fewer side effects.

[0088] According to the present invention, the term "platinum compound" refers to a compound containing platinum in its structure, such as a platinum complex, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

[0089] The term "cisplatin" or "cisplatinum" refers to the compound of the formula: [ka] This refers to the compound cis-diamminedichloroplatinum(II) (CDDP).

[0090] The term "carboplatin" refers to the compound of the formula: [ka] This refers to the compound cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II).

[0091] The term "oxaliplatin" refers to the compound of the formula: [ka] refers to a compound that is a platinum compound complexed to a diaminocyclohexane carrier ligand of the formula:

[0092] In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II). Injectable oxaliplatin is also commercially available under the trade name Eloxatine.

[0093] The term "nucleoside analog" refers to structural analogs of nucleosides, a category that encompasses both purine and pyrimidine analogs. In particular, the term "nucleoside analog" refers to fluoropyrimidine derivatives, including fluorouracil and its prodrugs.

[0094] The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (commercially available under the trade names Adrucil, Carac, Efudix, Efudex, and Fluoroplex) has the following formula: [ka] It is a compound that is a pyrimidine analogue of

[0095] In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0096] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapy agent that is a prodrug that is converted to 5-FU in tissues. Orally administered capecitabine has the following formula: [ka] It has.

[0097] 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.

[0098] Taxanes are a class of diterpene compounds originally derived from natural sources such as the yew plant, although some have been synthetically synthesized. The primary mechanism of action of the taxane class of drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0099] According to the present invention, the term "docetaxel" means a compound of the formula: [ka] It refers to a compound having the formula:

[0100] According to the present invention, the term "paclitaxel" means a compound of the formula: [ka] It refers to a compound having the formula:

[0101] 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 derivative having the following structure: [ka] It refers to a compound containing

[0102] According to the present invention, preferred camptothecin analogs are inhibitors of the DNA enzyme topoisomerase I (topo I). Preferred camptothecin analogs according to the present invention are irinotecan and topotecan.

[0103] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. In chemical terms, it has the following formula: [ka] Camptothecin is a semisynthetic analogue of the natural alkaloid camptothecin, having the formula:

[0104] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0105] Topotecan has the formula: [ka] It is a topoisomerase inhibitor.

[0106] 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.

[0107] According to the present invention, the agent that stabilizes or increases the expression of CLDN18.2 may be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, or may be part of a drug combination, for example, a drug combination established for use in cancer treatment. Such a drug combination may be a drug combination used in chemotherapy, or may be a drug combination used in a chemotherapy regimen selected from the group consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCF chemotherapy, and FLOT chemotherapy.

[0108] The drug combination used in EOX chemotherapy includes epirubicin, oxaliplatin, and capecitabine. The drug combination used in ECF chemotherapy includes epirubicin, cisplatin, and 5-fluorouracil. The drug combination used in ECX chemotherapy includes epirubicin, cisplatin, and capecitabine. The drug combination used in EOF chemotherapy includes epirubicin, oxaliplatin, and 5-fluorouracil.

[0109] Epirubicin is usually 50 mg / m 2 , cisplatin 60 mg / m 2 , oxaliplatin 130 mg / m 2 and 5-fluorouracil at a dose of 200 mg / m 2 / day continuous intravenous infusion and oral capecitabine 625 mg / m 2 It is administered twice daily over a three-week cycle for a total of eight doses.

[0110] The drug combination used in FLO chemotherapy is 5-fluorouracil, folinic acid, and oxaliplatin (usually 5-fluorouracil 2,600 mg / m 2 24-hour infusion of folinic acid 200 mg / m 2 and oxaliplatin 85 mg / m 2 every two weeks).

[0111] FOLFOX is a chemotherapy regimen consisting of folinic acid (leucovorin), 5-fluorouracil, and oxaliplatin. The recommended dose schedule, administered every 2 weeks, is as follows: Day 1: Oxaliplatin 85 mg / m 2 and leucovorin 200 mg / m 2 IV infusion of 5-FU 400 mg / m 2 IV bolus of 5-FU 600 mg / m 2 Day 1: leucovorin 200 mg / m IV infusion as a 22-hour continuous infusion; Day 2: leucovorin 200 mg / m 2 IV infusion over 120 minutes, followed by 5-FU 400 mg / m 2IV bolus administered over 2-4 minutes, followed by 5-FU 600 mg / m 2 IV infusion as a 22-hour continuous infusion.

[0112] The drug combination used in FOLFIRI chemotherapy includes 5-fluorouracil, leucovorin, and irinotecan.

[0113] The drug combination used in DCF chemotherapy includes docetaxel, cisplatin, and 5-fluorouracil.

[0114] The drug combination used in FLOT chemotherapy includes docetaxel, oxaliplatin, 5-fluorouracil and folinic acid.

[0115] The term "folinic acid" or "leucovorin" refers to a compound useful in synergistic combination with the chemotherapy agent 5-fluorouracil. Folinic acid has the following formula: [ka] It has.

[0116] In particular, the 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.

[0117] According to the present invention, antibodies having the ability to bind to CLDN18.2 may be administered in combination with, i.e. simultaneously with, before and / or after an agent that stimulates γδ T cells.

[0118] γδ T cells (gamma delta T cells) are a small subset of T cells that possess a unique T cell receptor (TCR) on their surface. The majority of T cells possess a TCR consisting of two glycoprotein chains, termed the α-TCR chain and the β-TCR chain. In contrast, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is typically much rarer than αβ T cells. Human γδ T cells play an important role in stress surveillance responses, such as infection and autoimmunity. It has been suggested that transformation-induced changes in tumors also generate a stress surveillance response mediated by γδ T cells, enhancing antitumor immunity. Importantly, after antigen engagement, activated γδ T cells at the lesion site provide cytokines (e.g., INFγ, TNFα) and / or chemokines that mediate the recruitment of other effector cells, exhibiting immediate effector functions such as cytotoxicity (via death receptor and cytolytic granule pathways) and ADCC.

[0119] The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cell receptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates and are predicted to play an early and essential role in sensing "danger" from invading pathogens, as they expand dramatically during many acute infections, including tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria, and can outnumber all other lymphocytes within days.

[0120] γδ T cells respond to small, non-peptide phosphoantigens (phosphoantigens), such as pyrophosphate synthesized in bacteria and isopentenyl pyrophosphate (IPP), produced in mammalian cells via the mevalonate pathway. While IPP production in normal cells is insufficient for γδ T cell activation, dysregulation of the mevalonate pathway in tumor cells leads to IPP accumulation and γδ T cell activation. IPP can also be therapeutically increased by aminobisphosphonates, which inhibit farnesyl pyrophosphate synthase (FPPS), an enzyme in the mevalonate pathway. Zoledronic acid (ZA, zoledronate, Zometa™, Novartis), among others, is a representative such aminobisphosphonate and has already been administered clinically to patients for the treatment of osteoporosis and metastatic bone disease. Following in vitro treatment of PBMCs, ZA is specifically taken up by monocytes. IPP accumulates in monocytes and differentiates into antigen-presenting cells, which stimulate the development of γδ T cells. In this situation, the addition of interleukin 2 (IL-2) as a growth and survival factor for activated γδ T cells is preferred. Finally, certain alkylating amines have been described to activate Vγ9Vδ2 T cells in vitro, but only at millimolar concentrations.

[0121] According to the present invention, the term "agent that stimulates γδ T cells" relates to a compound that stimulates the expression of γδ T cells, in particular Vγ9Vδ2 T cells, in vitro and / or in vivo, in particular by inducing γδ T cell activation and proliferation. Preferably, the term relates to a compound that increases isopentenyl pyrophosphate (IPP) production in mammalian cells in vitro and / or in vivo, preferably by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS).

[0122] One particular group of compounds that stimulate γδ T cells are the bisphosphonates, particularly the nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

[0123] For example, suitable bisphosphonates for use in the present invention may include one or more of the following compounds, including analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof: [1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, e.g., zoledronate; (Dichloro-phosphono-methyl)phosphonic acids, such as clodronate; {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronic acid, e.g., ibandronate; (3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic acid, e.g., pamidronate; (1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acids, risedronate, for example; (1-hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, minodronic acid; [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid; [4-amino-1-hydroxy-1-(hydroxy-oxide-phosphoryl)-butyl]phosphonic acid, alendronic acid, e.g., alendronate; [(cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid; (1-hydroxyethane-1,1-diyl)bis(phosphonic acid), etidronic acid, e.g., etidronate; and {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic acid.

[0124] According to the present invention, zoledronic acid (INN) or zoledronate (marketed by Novartis under the trade names Zometa, Zomera, Aclasta, and Reclast) is a particularly preferred bisphosphonate. Zometa is used to prevent fractures in patients with cancer, such as multiple myeloma and prostate cancer, and to treat osteoporosis. It can also be used to treat hypercalcemia of malignancy and may be useful in treating pain from bone metastases.

[0125] In one particularly preferred embodiment, the γδ T cell stimulating agent according to the invention is administered in combination with IL-2, such a combination has been shown to be particularly effective in mediating the proliferation and activation of γ9δ2 T cells.

[0126] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. It is a lymphocyte-attracting protein and is part of the body's natural response to microbial infection and in distinguishing self from foreign (non-self) substances. IL-2 mediates its effects by binding to the IL-2 receptor, which is expressed by lymphocytes.

[0127] The IL-2 used in accordance with the present invention may be any IL-2 that supports or enables stimulation of γδ T cells and may be derived from any species, preferably human. The IL-2 may be isolated, recombinantly produced, or synthetic, and may be naturally occurring or modified IL-2.

[0128] According to the present invention, the term "antiemetic drug" relates to a compound, composition or agent that is effective against vomiting and / or nausea. In one embodiment, antiemetic drugs include 5-HT3 receptor antagonists and / or neurokinin 1 (NK1) receptor antagonists.

[0129] 5-HT3 receptor antagonist blocks serotonin receptor in central nervous system and gastrointestinal tract.Examples include but are not limited to: ondansetron (Zofran), which can be administered in oral tablet form, oral dissolving tablet form or in injection; dolasetron (Anzemet), which can be administered in tablet form or in injection; granisetron (Kytril, Sancuso), which can be administered in tablet form (Kytril), oral solution (Kytril), injection (Kytril) or single transdermal patch (Sancuso) on the upper arm; tropisetron (Navoban), which can be administered in oral capsule or injection; palonosetron (Aloxi) and mirtazapine (Remeron), which can be administered in injection or oral capsule form.

[0130] NK1 receptor antagonists include, but are not limited to, aprepitant (Emend).

[0131] A preferred combination of a 5-HT3 receptor antagonist and an NK1 receptor antagonist is the combination of ondansetron (Zofran) and aprepitant (Emend).

[0132] Additional antiemetic drugs that may be used in accordance with the present invention, particularly in combination with a 5-HT3 receptor antagonist and / or an NK1 receptor antagonist, include, but are not limited to, metoclopramide (Reglan), lorazepam, atropine, alizapride (Litican, Plitican, Superan, Vergentan) and dimenhydrinate (Dramamine, Driminate, Gravol, Gravamin, Vomex, Vertirosan), which act on the GI tract as prokinetic agents.

[0133] According to the present invention, an antispasmodic (synonym: antispasmodic) can be administered. According to the present invention, the term "antispasmodic" relates to a compound, composition, or agent that inhibits muscle spasms. Preferably, the antispasmodic is useful for smooth muscle contraction. Antispasmodic agents that are effective in treating spastic effects in the digestive system are preferred according to the present invention. Thus, preferred antispasmodic agents are effective in relieving gastrointestinal spasms.

[0134] Antispasmodic drugs include, but are not limited to, butylscopolamine, also known as butylscopolamine bromide, butylhyoscine, and butylhyoscine bromide, which is marketed under the trade name Buscopan by Boehringer Ingelheim GmbH, Germany.

[0135] According to the present invention, a parasympatholytic drug can be administered. According to the present invention, the term "parasympatholytic drug" refers to a compound, composition, or agent that reduces the activity of the parasympathetic nervous system. Parasympatholytic drugs include, but are not limited to, atropine.

[0136] According to the present invention, the term "proton pump inhibitor" relates to compounds, compositions or agents whose primary action is a significant and long-lasting reduction of gastric acid production.

[0137] Proton pump inhibitors include benzimidazole derivatives and imidazopyridine derivatives. Examples of proton pump inhibitors include, but are not limited to, omeprazole (trade names: Gasec, Losec, Prilosec, Zegerid, ocid, Lomac, Omepral, Omez), lansoprazole (trade names: Prevacid, Zoton, Monolitum, Inhibitol, Levant, Lupizole), dexlansoprazole (trade names: Kapidex, Dexilant), esomeprazole (trade names: Nexium, Esotrex, esso), pantoprazole (trade names: Protonix, Somac, Pantoloc, Pantozol, Zurcal, Zentro, Pan, Controloc, Tecta), rabeprazole (trade names: AcipHex, Pariet, Erraz, Zechin, Rabecid, Nzole-D, Rabeloc, Razo), and ilaprazole (trade names: Ilapro, Lupilla, Adiza).

[0138] According to the present invention, other compounds, compositions or agents that have a protective effect on the gastric mucosa may be administered, particularly when nonsteroidal anti-inflammatory drugs (NSAIDs) are administered.

[0139] For example, other compounds, compositions or drugs can be administered to prevent the general adverse effect of NSAIDs on gastric ulcer formation, and particularly to prevent NSAID-induced gastric ulcers.In one embodiment, misoprostol, a synthetic prostaglandin E1 (PGE1) analogue used to prevent NSAID-induced gastric ulcers, can be administered.Misoprostol acts on gastric parietal cells to inhibit the secretion of gastric acid through G protein-coupled receptor-mediated inhibition of adenylate cyclase, which leads to a decrease in intracellular cyclic AMP level and a decrease in the proton pump activity at the apical surface of gastric parietal cells.

[0140] Furthermore, omeprazole has been shown to be at least as effective as misoprostol in treating NSAID-induced ulcers, but is significantly better tolerated.

[0141] Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs that provide analgesic and antipyretic (fever-reducing) effects and, at higher doses, anti-inflammatory effects. The term "nonsteroidal" distinguishes these drugs from steroids. The most prominent members of this group of drugs are aspirin, ibuprofen, and naproxen.

[0142] One of the major adverse drug reactions (ADRs) associated with NSAIDs is their gastrointestinal (GI) effects. These effects are severe enough in many cases to pose a risk of ulcer perforation and upper GI bleeding. NSAID-administered patients experience dyspepsia, NSAID-related upper GI adverse events, GI tract irritation, and GI ulcer formation. NSAIDs cause a dual attack on the GI tract: acidic molecules directly irritate the gastric mucosa, and inhibition of COX-1 and COX-2 reduces the levels of protective prostaglandins. Inhibition of prostaglandin synthesis in the GI tract results in increased gastric acid secretion, decreased bicarbonate secretion, decreased mucus secretion, and decreased trophic effects on the epithelial mucosa. Therefore, NSAIDs are preferably not administered according to the present invention. Placetamol or "acetaminophen," which is not classified as an NSAID because it exerts only weak anti-inflammatory effects, can be administered as an analgesic in accordance with the present invention, but may not be effective for pain management and therefore may require administration of an NSAID, particularly to avoid administration of opioids.

[0143] Generally, adverse effects in the stomach (but not necessarily the intestines) can be reduced through suppression of acid production by the concomitant use of proton pump inhibitors such as omeprazole, esomeprazole, or the prostaglandin analogue misoprostol.

[0144] The term "antigen" relates to an agent such as a protein or peptide containing an epitope against which an immune response is 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 originate from the cytoplasm, cell surface and cell nucleus, in particular an antigen that is produced intracellularly or as a surface antigen on cancer cells, preferably in large amounts.

[0145] In the context of the present invention, the term "tumor-associated antigen" preferably relates to a protein which, under normal conditions, is specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage, and which is expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed at all or only rarely expressed in normal tissues.

[0146] The term "epitope" refers to an antigenic determinant in a molecule, i.e., a portion of a molecule that is recognized by the immune system, e.g., 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 groups 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 and nonconformational epitopes are distinguished by the fact that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein such as CLDN18.2 preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length, for example the epitope may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

[0147] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and encompasses any molecule containing an antigen-binding portion thereof. The term "antibody" encompasses monoclonal antibodies and antibody fragments or derivatives, including, but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, e.g., scFvs, and antigen-binding antibody fragments such as Fab and Fab' fragments, as well as all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, aglycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an 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 (Clq) of the classical complement system.

[0148] The antibodies described herein may be human antibodies. The term "human antibody," as used herein, is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0149] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise complete variable domains fused to constant domains, or may comprise only the complementarity-determining regions (CDRs) grafted into appropriate framework regions within the variable domains. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions, e.g., to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more CDRs that are altered compared to the original antibody.

[0150] The term "chimeric antibody" refers to an antibody in which portions of the amino acid sequences of the heavy and light chains are homologous to corresponding sequences in antibodies from a particular species or class, while the remaining segments of the chains are homologous to corresponding sequences in another antibody. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammalian species, while the constant regions are homologous to sequences in antibodies from another species. One obvious advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, for example, using B cells or hybridomas from readily available non-human host organisms, in combination with constant regions derived from human cell preparations. While the variable regions have the advantage of ease of preparation and their specificity is independent of their source, human constant regions are less likely to provoke an immune response from a human subject when the antibody is injected than constant regions from non-human sources. However, the definition is not limited to this specific example.

[0151] The terms "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") of an antibody or similar terms refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of the VH and CH domains; (iv) an Fv fragment, which consists of the VL and VH domains of one arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain; (vi) an isolated complementarity-determining region (CDR), and (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be recombinantly linked by a synthetic linker that allows them to be produced as a single-chain protein in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., 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 encompassed by the term "antigen-binding fragment" of an antibody. A further example is 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.Binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0152] The term "bispecific molecule" is intended to encompass any agent, e.g., 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 term "multispecific molecule" or "heterospecific molecule" is intended to encompass any agent, e.g., 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 encompasses, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules directed against CLDN18.2 and other targets, e.g., Fc receptors on effector cells. The term "bispecific antibody" also encompasses diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing them to pair with the complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0153] The antibody may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells, and in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Suitable therapeutic agents for forming antibody conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), and steroids (e.g., steroids ... sorbicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic or 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.

[0154] Antibodies can also be conjugated to radioactive isotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals.

[0155] The antibody conjugates of the present invention can be used to modulate 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 possessing a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon gamma; or biological response modifiers, such as lymphokines, interleukin 1 ("IL-1"), interleukin 2 ("IL-2"), interleukin 6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other growth factors.

[0156] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospect of The Therapeutic Use Of Radiolabeled Antibodies" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); See "In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0157] As used herein, an antibody is "derived" from a particular germline sequence if it is obtained by immunizing an animal or by screening an immunoglobulin gene library, and the antibody selected in said screen is at least 90%, more preferably at least 95%, and even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a particular germline sequence displays no more than 10 amino acid differences, more preferably no more than 5, or 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.

[0158] As used herein, the term "heteroantibody" refers to two or more antibodies, derivatives thereof, or antigen-binding regions thereof linked together, at least two of which have different specificities, including binding specificity for an Fc receptor on an effector cell and binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

[0159] The antibody described herein may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal, such as a mouse, fused to an immortalized cell.

[0160] The antibodies described herein may be recombinant antibodies. The term "recombinant antibody," as used herein, encompasses all antibodies made, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a hybridoma made therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies made, expressed, created, or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences.

[0161] The antibodies described herein may be derived from various species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0162] Antibodies as referred to herein encompass 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).

[0163] The term "transfectoma" as used herein includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungal cells, including yeast cells, that express an antibody.

[0164] As used herein, a "heterologous antibody" is defined with respect to the transgenic organism producing such an antibody. The term refers to an antibody that is not constructed from the transgenic organism and generally has an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism derived from a species other than the transgenic organism.

[0165] As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain combined with a murine light chain is a heterohybrid antibody.

[0166] The present invention includes all antibodies and antibody derivatives described herein that are encompassed by the term "antibody" for purposes of the present invention. The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another agent or antibody, or an antibody fragment.

[0167] The antibodies described herein are preferably isolated. As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind to antigens other than CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may, however, have cross-reactivity with other related antigens, such as related antigens from other species (e.g., CLDN18.2 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, an "isolated" monoclonal antibody combination relates to antibodies with different specificities that are combined in a well-defined composition or mixture.

[0168] The term "binding" according to the present invention preferably relates to specific binding.

[0169] According to the present invention, an antibody is capable of binding to a predetermined target if it has significant affinity for and binds to the predetermined target in a standard assay. "Affinity" or "binding affinity" is often measured in terms of the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or a lower dissociation constant (K D ) to bind to a predetermined target.

[0170] If an antibody does not have significant affinity for a target in a standard assay and does not significantly bind to the target, in particular does not detectably bind, the antibody is (substantially) unable to bind to the target. Preferably, the antibody does not detectably bind to the target when present at a concentration of up to 2 μg / ml, preferably up to 10 μg / ml, more preferably up to 20 μg / ml, in particular up to 50 μg / ml or 100 μg / ml or more. Preferably, the antibody has a K D At least 10 times, 100 times, or 10 times 3 double, 10 4 double, 10 5 double or ten 6 Twice as high as K D If the antibody binds to the target at a K D is 10 -7 M, the K for binding to a target for which the antibody does not have significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3M, 10 -2 M or 10 -1 I am M.

[0171] An antibody is specific for a predetermined target if it can bind to the predetermined target but cannot bind to other targets, i.e., it has no significant affinity for other targets and does not significantly bind to other targets in standard assays. 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 to such other targets do not significantly exceed its affinity or binding to proteins unrelated to CLDN18.2, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins, such as MHC molecules or transferrin receptors, or any other specific polypeptides. Preferably, an antibody has a K for binding to a target for which the antibody is not specific. D At least 10 times, 100 times, or 10 times 3 double, 10 4 double, 10 5 double or ten 6 Twice as low as K D An antibody is specific for a given target if it binds to the given target at a K D is 10 -7 M, the K D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 I am M.

[0172] Antibody binding to a target can be experimentally determined using any suitable method, see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press, New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company, New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, for example, by equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NYAcad.ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may differ if measured under different conditions, for example, different salt concentrations, pH. Thus, affinity and other antigen binding parameters, such as K D ,I C 50 The determination is preferably carried out using standard solutions of antibody and antigen and standard buffers.

[0173] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0174] As used herein, "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0175] As used herein, the term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0176] As used herein, the term "rearranged" refers to a configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned immediately adjacent to a DJ or J segment in a conformation that essentially encodes a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA, and rearranged loci have at least one recombined heptamer / 9amer homology element.

[0177] The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to a configuration in which the V segment has not recombined so that it is immediately adjacent to a D or J segment.

[0178] According to the present invention, an antibody capable of binding to CLDN18.2 is an antibody that can bind to an epitope present in CLDN18.2, preferably an epitope located within the extracellular domain of CLDN18.2, particularly within the first extracellular domain, preferably within amino acid positions 29 to 78 of CLDN18.2. In certain embodiments, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NO: 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-loop 1 and CLDN18.2-loop D3, or (vi) a non-glycosylated epitope located on CLDN18.2-loop D3, preferably SEQ ID NO: 9.

[0179] According to the present invention, an antibody capable of binding to CLDN18.2 is preferably an antibody capable of binding to CLDN18.2 but not to CLDN18.1. Preferably, an antibody capable of binding to CLDN18.2 is specific to CLDN18.2. Preferably, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to CLDN18.2 expressed on the cell surface. In a particularly preferred embodiment, an antibody capable of binding to CLDN18.2 binds to a native epitope of CLDN18.2 present on the surface of living cells. Preferably, an antibody capable of binding to CLDN18.2 binds to one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, an antibody capable of binding to CLDN18.2 is specific to the aforementioned proteins, peptides, or immunogenic fragments or derivatives thereof. An antibody capable of binding to CLDN18.2 can be obtained by a method comprising the step of immunizing an animal with a protein or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50, or a nucleic acid or host cell expressing said protein or peptide. Preferably, the antibody binds to cancer cells, particularly cells of the aforementioned cancer types, and preferably does not substantially bind to non-cancerous cells.

[0180] Preferably, binding of an antibody capable of binding to CLDN18.2 to a cell expressing CLDN18.2 induces or mediates the death of the cell expressing CLDN18.2. The cell expressing CLDN18.2 is preferably a cancer cell, particularly selected from the group consisting of tumorigenic gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, 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 cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation of the cell expressing CLDN18.2. Preferably, ADCC-mediated lysis of the cell occurs in the presence of effector cells, which, in a specific embodiment, are selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs. Inhibition of cell proliferation can be measured in vitro by quantifying cell proliferation in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside, an analog of thymidine, that can be incorporated into newly synthesized DNA of replicating cells (during the S phase of the cell cycle), replacing thymidine during DNA replication. Detecting the incorporated chemical, for example, using antibodies specific for BrdU, indicates cells that were actively replicating their DNA.

[0181] In preferred embodiments, the antibodies described herein have the following properties: a) specificity for CLDN18.2; b) a 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) the ability to induce or mediate CDC on CLDN18.2-positive cells; d) the ability to induce or mediate ADCC on CLDN18.2-positive cells; e) the ability to inhibit the proliferation of CLDN18.2-positive cells; f) Ability to induce apoptosis in CLDN18.2-positive cells The compound may be characterized by one or more of the following:

[0182] In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 is produced by a hybridoma deposited at DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) with the following name and accession number: a.182-D1106-055, accession number DSM ACC2737, deposited October 19, 2005 b.182-D1106-056, accession number DSM ACC2738, deposited October 19, 2005 c.182-D1106-057, accession number DSM ACC2739, deposited October 19, 2005 d.182-D1106-058, accession number DSM ACC2740, deposited October 19, 2005 e.182-D1106-059, accession number DSM ACC2741, deposited October 19, 2005 f.182-D1106-062, accession number DSM ACC2742, deposited October 19, 2005 g.182-D1106-067, accession number DSM ACC2743, deposited October 19, 2005 h.182-D758-035, accession number DSM ACC2745, deposited November 17, 2005 i.182-D758-036, accession number DSM ACC2746, deposited November 17, 2005 j.182-D758-040, accession number DSM ACC2747, deposited November 17, 2005 k.182-D1106-061, accession number DSM ACC2748, deposited November 17, 2005 l.182-D1106-279, accession number DSM ACC2808, deposited October 26, 2006 m.182-D1106-294, accession number DSM ACC2809, deposited October 26, 2006 n.182-D1106-362, accession number DSM ACC2810, deposited October 26, 2006.

[0183] Preferred antibodies according to the invention are those produced by and obtainable from the above mentioned hybridomas, namely 37G11 for 182-D1106-055, 37H8 for 182-D1106-056, 38G5 for 182-D1106-057, 38H3 for 182-D1106-058, 39F11 for 182-D1106-059, 43A11 for 182-D1106-062, 43A11 for 182-D1106-063, 43A12 for 182-D1106-064, 43A13 for 182-D1106-065, 43A14 for 182-D1106-066, 43A15 for 182-D1106-067, 43A16 for 182-D1106-068, 43A17 for 182-D1106-069, 43A18 for 182-D1106-070, 43A19 for 182-D1106-071, 43A20 for 182-D1106-072, 43A21 for 182-D1106-073, 43A22 for 182-D1106-074, 43A23 for 182-D1106-075, 43A24 for 182-D1106-076, 43A25 for 182-D1106-077, 43A26 for 182-D1106-078, 43A27 for 61C2 for D1106-067, 26B5 for 182-D758-035, 26D12 for 182-D758-036, 28D10 for 182-D758-040, 42E12 for 182-D1106-061, 125E1 for 182-D1106-279, 163E12 for 182-D1106-294, and 175D10 for 182-D1106-362; and chimeric and humanized forms thereof.

[0184] Preferred chimeric antibodies and their sequences are shown in the table below. [Table 1-1]

[0185] In preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a heavy chain constant region (CH) having an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof. In further preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a light chain constant region (CL) having an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In particularly preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a CH having an amino acid sequence derived from a human CH, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof, and a CL having an amino acid sequence derived from a human CL, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.

[0186] In one embodiment, the antibody capable of binding to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising a mouse kappa variable light chain, a human kappa light chain constant region allotype Km(3), a mouse heavy chain variable region, a human IgG1 constant region, allotype G1m(3).

[0187] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and fragments thereof, and / or a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28 and fragments thereof.

[0188] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a heavy and light chain combination selected from the following possibilities (i) to (ix): (i) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof; (ii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof; (iii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof; (iv) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof; (v) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof; (vi) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof; (vii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof; (viii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof; and (ix) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 28 or a fragment thereof.

[0189] Antibodies according to (v) are particularly preferred.

[0190] The terms "fragment" or "fragment of an amino acid sequence" as used above refer to a portion of an antibody sequence, i.e., an antibody sequence truncated at the N-terminus and / or C-terminus, which, when substituted for said antibody sequence in an antibody, retains the binding of said antibody to CLDN18.2 and preferably the function of said antibody as described herein, e.g., CDC-mediated lysis or ADCC-mediated lysis. Preferably, a fragment of an amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues from said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 preferably refers to said sequences from which the N-terminal 17, 18, 19, 20, 21, 22, or 23 amino acids have been removed.

[0191] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a heavy chain variable region (VH) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and fragments thereof.

[0192] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a light chain variable region (VL) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, and fragments thereof.

[0193] In certain preferred embodiments, the antibody having the ability of binding to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (ix): (i) the VH comprises the amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof; (ii) VH comprises the amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof; (iii) VH comprises the amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof; (iv) VH comprises the amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof; (v) VH comprises the amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof; (vi) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof; (vii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof; (viii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof; (ix) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

[0194] Antibodies according to (v) are particularly preferred.

[0195] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VH containing a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi): (i) CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, (ii) CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, (iii) CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, (iv) CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, (v) CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, and (vi) CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19.

[0196] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VL containing a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix): (i) CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (ii) CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (iii) CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (v) CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0197] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a combination of a VH and a VL, each containing a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix): (i) VH: CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, VL: CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (ii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, VL: CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (iii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, VL: CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, VL: CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, VL: CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (vii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0198] In a further preferred embodiment, the antibody capable of binding to CLDN18.2 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, and 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) described herein. In one embodiment, one or more of said 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 antibody capable of binding to CLDN18.2 preferably comprises the 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, and preferably comprises the 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 described herein.

[0199] In one embodiment, an antibody comprising one or more CDRs, sets of CDRs, or combinations of sets of CDRs described herein comprises the CDRs together with their intervening framework regions. Preferably, the portions comprise at least about 50% of either or both of the first and fourth framework regions, said 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable region encoded by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers for linking the variable regions of the invention to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the generation of diabodies), or protein tags.

[0200] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs described herein comprises said CDRs within a human antibody framework.

[0201] Reference herein to an antibody comprising a particular chain or a particular region or sequence in relation to its heavy chain preferably relates to the situation where all heavy chains of said antibody comprise said particular chain, region or sequence, and this applies correspondingly to the light chains of the antibody.

[0202] The term "nucleic acid," as used herein, is intended to encompass DNA and RNA. Nucleic acids can be single-stranded or double-stranded, but preferably are double-stranded DNA.

[0203] 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 a nucleic acid. Furthermore, expression can be performed transiently or stably.

[0204] Any teachings given herein regarding specific amino acid sequences, e.g., those shown in the Sequence Listing, should also be construed as relating to sequences that are functionally equivalent to the specific sequence, e.g., variants of the specific sequence that result in amino acid sequences that exhibit the same or similar properties as 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 variant with respect to a specific sequence, when it replaces the specific sequence in an 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.

[0205] Those skilled in the art will recognize that the sequences of the CDRs, hypervariable regions, and variable regions, in particular, can be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions can be identical or highly homologous to regions of the antibodies identified herein. By "highly homologous," it is contemplated that one to five, preferably one to four, e.g., one to three, or one or two substitutions can be made within the CDRs. In addition, the hypervariable and variable regions can be modified to exhibit substantial homology with regions of the antibodies specifically disclosed herein.

[0206] For purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0207] Amino acid insertion variants include the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites within the amino acid sequence, although random insertions with appropriate screening of the resulting products are also possible.

[0208] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0209] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be in any position in the protein.

[0210] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the modification occurs at a position in the amino acid sequence that is not conserved among homologous proteins or peptides and / or the amino acid is replaced with another amino acid with similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids whose side chains are related. 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 classified together as aromatic amino acids.

[0211] Preferably, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence that is a variant of said 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 over 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 entire 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 consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::Needle, matrix:Blosum62, cap open 10.0, gap extension 0.5.

[0212] "Sequence similarity" indicates the percentage of amino acids that are identical or that are conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0213] The term "percent identity" is intended to refer to the percentage of amino acid residues that are identical between the two sequences to be compared, obtained after the best alignment, and this percentage is purely statistical, with the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is conventionally carried out by comparing these sequences after optimal alignment, said comparison being carried out segment by segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be performed manually or by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0214] The percent 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 to obtain the percent identity between the two sequences, and multiplying the result by 100.

[0215] The term "transgenic animal" refers to an animal having a genome containing one or more transgenes, preferably heavy and / or light chain transgenes, or transchromosomes (integrated or not integrated into the animal's native genomic DNA), and preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with a CLDN18.2 antigen and / or cells expressing CLDN18.2. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as in the case of transgenic mice such as HCo7 or HCol2 mice, e.g., HuMAb 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 and transchromosomal mice are capable of producing multiple isotypes (eg, IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN18.2 by undergoing VDJ recombination and isotype switching.

[0216] As used herein, "reduce" or "inhibit" refers to an overall decrease or ability to cause an overall decrease in a level, e.g., a level of expression or level of proliferation of a cell, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0217] Terms such as "increase" or "enhance" relate to an increase or enhancement of preferably 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%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

[0218] Mechanism of action of mAbs The following provides a discussion of the mechanisms underlying the therapeutic effects of the antibodies of the present invention, but should not be construed as a limitation on the present invention in any way.

[0219] The antibodies described herein preferably interact with components of the immune system, preferably via ADCC or CDC. The antibodies described herein can also be used to target payloads (e.g., radioisotopes, drugs, or toxins) to directly kill tumor cells, or can be used synergistically with traditional chemotherapeutic agents to attack tumors through a complementary mechanism of action, which may involve an anti-tumor immune response that may be impaired due to the cytotoxic side effects of the chemotherapeutic agent on T lymphocytes. However, the antibodies described herein can also act simply by binding to CLDN18.2 on the cell surface, thus, for example, blocking cell proliferation.

[0220] Antibody-dependent cell-mediated cytotoxicity ADCC refers to the cell killing capacity of effector cells, particularly lymphocytes, as described herein, which preferably requires that target cells be marked by antibodies.

[0221] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain engages with an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be considered a mechanism that directly induces various degrees of immediate tumor destruction, resulting in antigen presentation and the induction of T cell responses against the tumor. Preferably, in vivo induction of ADCC results in a T cell response and a host-derived antibody response against the tumor.

[0222] 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 also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is initiated by the C-terminal end of the participating antibody molecules, such as IgG molecules. H This results in the exposure of multiple C1q binding sites in close proximity on the two domains (C1q is one of three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-avidity interaction, which initiates a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector cell chemotactic / activators C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0223] The antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes.

[0224] The preferred animal system for producing hybridomas secreting monoclonal antibodies is the murine system. The production of hybridomas in mice is a very well-established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0225] Other preferred animal systems for producing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0226] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Production of human antibodies in such transgenic mice can be performed as detailed for CD20 in WO 2004 / 035607.

[0227] Yet another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding genes from lymphocytes that produce antibodies of defined specificities; see, e.g., Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo, Antibody Engineering ISBN 1-58829-092-1.

[0228] To generate antibodies, mice can be immunized with carrier-bound peptides derived from the antigen sequence, i.e., the sequence against which the antibody is to be directed, an enriched preparation of recombinantly expressed antigen or its fragment, and / or cells expressing the antigen, as described above. Alternatively, mice can be immunized with DNA encoding the antigen or its fragment. If immunization with a purified or enriched preparation of antigen does not produce antibodies, mice can also be immunized with cells, e.g., a cell line, expressing the antigen to stimulate an immune response.

[0229] During the course of the immunization protocol, immune responses can be monitored with plasma and serum samples obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. To enhance the proportion of hybridomas secreting specific antibodies, mice can be boosted intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and splenectomy.

[0230] To generate hybridomas that produce monoclonal antibodies, spleen cells 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 for antibody-secreting hybridomas by ELISA. Antibodies with specificity for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Antibody-secreting hybridomas can be replated, screened again, 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.

[0231] Antibodies can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0232] For example, in one embodiment, a gene of interest, e.g., an antibody gene, can be ligated into an expression vector, such as a eukaryotic expression plasmid, as used by the GS gene expression system disclosed in International Publication Nos. WO 87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems known in the art. The purified plasmid containing the cloned antibody gene can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells, such as plant-derived cells, fungi, or yeast cells. The method used to introduce these genes can be any method described in the art, such as electroporation, lipofectin, lipofectamine, or others. After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomas, which can then be amplified for expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from the culture supernatant and / or cells.

[0233] Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli. Furthermore, antibodies can be produced in transgenic non-human animals, e.g., milk from sheep and rabbits or eggs from hens, or in transgenic plants; see, e.g., 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.

[0234] Chimerization Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with toxins or radioisotopes. Unlabeled murine antibodies are highly immunogenic in humans when administered repeatedly, resulting in reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of murine antibodies in humans can be reduced or completely avoided by chimerizing or humanizing the respective antibodies. Chimeric antibodies are antibodies whose different portions are derived from different animal species, such as those with variable regions derived from a murine antibody and human immunoglobulin constant regions. Antibody chimerization is achieved by linking the heavy and light chain variable regions of a murine antibody with human heavy and light chain constant regions (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by linking a human kappa light chain constant region to a murine light chain variable region. In an equally preferred embodiment, chimeric antibodies can be produced by linking a human lambda light chain constant region to a 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.

[0235] Humanization Antibodies interact with target antigens primarily through amino acid residues located within the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because 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 from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., 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. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation. Germline gene sequences also differ individually from the sequences of high affinity secondary repertoire antibodies uniformly throughout the variable regions.

[0236] The ability of the antibody to bind the antigen can be determined using standard binding assays, such as ELISA, Western blot, immunofluorescence and flow cytometry analysis.

[0237] To purify the antibody, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysis-based bioreactors. The supernatant can be filtered and concentrated as needed before being subjected to affinity chromatography with protein G-Sepharose or protein A-Sepharose. The eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80°C.

[0238] To determine whether a selected monoclonal antibody binds to a unique epitope, site-directed or multi-site directed mutagenesis can be used.

[0239] To determine the antibody isotype, isotype ELISA can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics). The wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with a monoclonal antibody or purified isotype control for 2 hours at ambient temperature. The wells can then be reacted with either mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or mouse IgM-specific peroxidase-conjugated probes. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at an OD of 405–650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, catalog number 1493027) can be used as described by the manufacturer.

[0240] Flow cytometry can be used to determine the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection, as well as negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, APC- or Alexa647-labeled anti-IgG antibodies can be bound to the antigen-bound monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry on a FACS instrument using side light scatter characteristics to gate on single live cells. Cotransfection methods can be used to distinguish antigen-specific monoclonal antibodies from nonspecific binders in a single measurement. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. Because the majority of transfected cells express both transgenes, antigen-specific monoclonal antibodies will selectively bind to cells expressing the fluorescent marker, while nonspecific antibodies will bind to non-transfected cells in equal proportions. A selective assay using fluorescence microscopy can be used in addition to or instead of a flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

[0241] Immunofluorescence microscopy can be used to reveal the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. For example, cell lines expressing the antigen naturally or after transfection, as well as 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. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against the antigen for 30 minutes at 25°C. After washing, cells can be reacted with Alexa555-conjugated anti-mouse IgG secondary antibodies (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

[0242] Cell extracts from cells expressing the antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

[0243] The reactivity of the antibody with the antigen can be further tested by immunohistochemistry using methods well known to those skilled in the art, for example, using frozen sections fixed with paraformaldehyde or acetone or paraffin-embedded tissue sections fixed with paraformaldehyde from non-cancerous or cancerous tissue samples obtained from patients during routine surgery or from mice bearing xenograft tumors inoculated with cell lines expressing the antigen, either naturally or after transfection. For immunostaining, antibodies reactive with the antigen can be incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the supplier's instructions.

[0244] Antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CLDN18.2. Testing monoclonal antibody activity in vitro provides an initial screen prior to testing in in vivo models.

[0245] 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 gradient centrifugation followed by lysis of contaminating red blood cells. Washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum and purified to express CLDN18.2. 51 Cr-labeled target cells can be mixed with various ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence-enhancing ligand (BATDA). Highly fluorescent chelates of europium with the enhancing ligand, released from dead cells, can be measured by fluorometer. Another alternative technique utilizes transfection of target cells with luciferase. Added lucifer yellow can then be oxidized only by live cells. Purified anti-CLDN18.2 IgG can then be added at various concentrations. An irrelevant human IgG can be used as a negative control. The assay can be performed at 37°C for 4 to 20 hours, depending on the effector cell type used. The elution of the culture supernatant 51 Samples can be assayed for cell lysis by measuring Cr release or the presence of the EuTDA chelate. Alternatively, luminescence resulting from the oxidation of Lucifer Yellow can be a measure of viable cells.

[0246] Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine whether cytolysis is enhanced with multiple monoclonal antibodies.

[0247] Complement-dependent cytotoxicity (CDC) Monoclonal anti-CLDN18.2 antibodies can be tested for their ability to mediate CDC using various known techniques. For example, serum for complement can be obtained from blood using methods known to those skilled in the art. Various methods can be used to measure the CDC activity of mAbs. For example, 51 Cr release can be measured, or a propidium iodide (PI) exclusion assay can be used to assess membrane permeabilization. Briefly, target cells were washed and 5×10 5 1 / ml can be incubated with various concentrations of mAb for 10-30 minutes at room temperature or 37°C. Serum or plasma can then be added to a final concentration of 20% (v / v), and the cells can be incubated for 20-30 minutes at 37°C. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be analyzed immediately by flow cytometry using a FACSArray.

[0248] In an alternative assay, induction of CDC can be measured in adherent cells. In one embodiment of this assay, cells are plated at 3x10 in tissue culture flat-bottom microtiter plates 24 hours prior to the assay. 4 Cells are seeded at a density of 1000 μg / well. The next day, the growth medium is removed, and the cells are incubated with the antibody in triplicate. Control cells are incubated with growth medium or growth medium containing 0.2% saponin for measurements of background lysis and maximum lysis, respectively. After 20 minutes of incubation at room temperature, the supernatant is removed, and 20% (v / v) human plasma or serum in DMEM (prewarmed to 37°C) is added to the cells and incubated for an additional 20 minutes at 37°C. All cells from each sample are added to a propidium iodide solution (10 μg / ml). The supernatant is then 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 of specific lysis is calculated as follows: % specific lysis = (sample fluorescence - background fluorescence) / (maximum lysis fluorescence - background fluorescence) × 100.

[0249] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies To test for the ability to initiate apoptosis, monoclonal anti-CLDN18.2 antibodies can be incubated with, for example, CLDN18.2-positive tumor cells, such as SNU-16, DAN-G, KATO-III, or CLDN18.2-transfected tumor cells, at 37°C for approximately 20 hours. Cells can be harvested, washed in Annexin-V binding buffer (BD Biosciences), and incubated with Annexin-V conjugated with FITC or APC (BD Biosciences) for 15 minutes in the dark. All cells from each sample can be added to a PI solution (10 μg / ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected using commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, catalog number AD0200) is a nonisotopic immunoassay based on measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) during DNA synthesis in proliferating cells in microplates. Incorporated BrdU is detected using a europium-labeled monoclonal antibody. To enable antibody detection, cells are fixed with Fix solution and DNA is denatured. Unbound antibody is washed away, and DELFIA inducer is added to release europium ions from the labeled antibody into solution, where they form a highly fluorescent chelate with a component of the DELFIA inducer. Detection utilizes time-resolved fluorometry; measured fluorescence is proportional to DNA synthesis in cells in each well.

[0250] Preclinical trials Monoclonal antibodies that bind to CLDN18.2 can also be tested in in vivo models (e.g., in immunodeficient mice bearing xenograft tumors inoculated with CLDN18.2-expressing cell lines, such as DAN-G, SNU-16, or KATO-III, or with cell lines that express CLDN18.2 after transfection, such as HEK293) to measure their effectiveness in controlling the growth of tumor cells that express CLDN18.2.

[0251] In vivo testing can be performed using the antibodies described herein after xenografting CLDN18.2-expressing tumor cells into immunocompromised mice or other animals. Antibodies can be administered to tumor-free mice, followed by injection of tumor cells, to measure the antibody's ability to prevent tumor formation or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice, and the therapeutic effect of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms can be measured. Antibody administration can be combined with the administration of other substances, such as cytostatics, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies, to measure synergistic effects and potential toxicity of the combination. To analyze antibody-mediated toxic side effects, animals can be inoculated with the antibody or a control agent and thoroughly examined for symptoms potentially associated with CLDN18.2 antibody treatment. Potential side effects of in vivo administration of CLDN18.2 antibodies include toxicity, particularly 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 potential side effects mediated by the application of monoclonal CLDN18.2 antibodies in humans.

[0252] Mapping of epitopes recognized by antibodies can be performed as detailed in "Epitope Mapping Protocols (Methods in Molecular Biology)" by Glenn E. Morris ISBN-089603-375-9 and "Epitope Mapping: A Practical Approach" by Olwyn MRWestwood and Frank C. Hay, Practical Approach Series, 248.

[0253] The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

[0254] The pharmaceutical compositions will usually be presented in unit dosage form and may be prepared in a manner known per se The pharmaceutical composition may, for example, be in the form of a solution or suspension.

[0255] Pharmaceutical compositions may contain salts, buffering substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of substances that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0256] Pharmaceutically unacceptable salts can be used to prepare pharmaceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0257] Suitable buffering substances for use in the pharmaceutical compositions include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

[0258] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0259] Injectable formulations may contain pharmaceutically acceptable excipients such as lactated Ringer's solution.

[0260] The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate, enhance or enable application. According to the present invention, the term "carrier" also encompasses one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.

[0261] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

[0262] The term "excipient" as used herein is intended to refer to any substance that may be present in a pharmaceutical composition and that is not an active ingredient, such as a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent, or coloring agent.

[0263] The agents and compositions described herein can be administered by any conventional route, for example, parenteral administration, including injection or infusion. Administration is preferably parenteral, for example, intravenous, intraarterial, subcutaneous, intradermal, or intramuscular routes.

[0264] Compositions suitable for parenteral administration usually comprise sterile aqueous or non-aqueous preparations of the active compound, preferably isotonic with the recipient's blood. Examples of suitable carriers and solvents include Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are usually used as a solution or suspension medium.

[0265] The agents and compositions described herein are administered in an effective amount. "Effective amount" refers to an amount that achieves a desired response or a desired effect, either alone or together with further administrations. In the case of treating a specific disease or a specific condition, the desired response preferably relates to preventing the progression of the disease. This includes slowing the progression of the disease, particularly preventing or reversing the progression of the disease. The desired response in the treatment of a disease or condition can also be delaying or preventing the onset of the disease or the condition.

[0266] The effective amount of the agent or composition described herein depends on the condition to be treated, the severity of the disease, the patient's individual parameters, including the patient's age, physiological condition, size and weight, the duration of treatment, the type of concomitant treatment (if any), specific administration route and similar factors.Therefore, the dose of the agent described herein to be administered can depend on such various parameters.If the patient's response is insufficient at the initial dose, a higher dose (or an effectively higher dose achieved by a different, more limited administration route) can be used.

[0267] The agents and compositions described herein can be administered to patients, for example, in vivo, to treat or prevent various disorders such as those described herein. Preferred patients include human patients with disorders that can be corrected or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by an altered expression pattern of CLDN18.2.

[0268] For example, in one embodiment, the antibodies described herein can be used to treat a patient having a cancer disease, such as a cancer disease described herein characterized by the presence of cancer cells that express CLDN18.2.

[0269] The aforementioned pharmaceutical compositions and methods of treatment according to the present invention may also be used for immunization or vaccination to prevent the diseases described herein.

[0270] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0271] Example 1 A First-in-Human, Single-Dose, Multicenter, Phase I, Open-Label, Intravenous Infusion, Dose-Escalation Study Evaluating the Safety and Tolerability of IMAB362 in Hospitalized Patients with Advanced Gastroesophageal Cancer A first-in-human, single-dose, multicenter, Phase 1, open-label, iv infusion, dose-escalation study of IMAB362 was conducted to determine the maximum tolerated dose or maximum applicable single dose (MTD) of IMAB362, examine the safety, tolerability, and adverse event profile of IMAB362, determine the pharmacokinetic profile of single ascending doses of IMAB362, determine the immunogenicity of single-dose application of IMAB362, and determine the potential antitumor activity of IMAB362 in patients with advanced gastroesophageal (GE) cancer.

[0272] The study was designed as a first-in-human, Phase 1, multicenter, non-randomized, interpatient single-ascending dose, open-label clinical trial of a single intravenous infusion of IMAB362 and a 4-week treatment-free follow-up period.

[0273] To be included in the study, patients had to meet all of the following inclusion criteria: Histologically proven metastatic, refractory, or recurrent advanced gastroesophageal cancer CLDN18.2 expression confirmed by immunohistochemistry or availability of tumor tissue samples suitable for determining CLDN18.2 expression Prior standard chemotherapy including fluoropyrimidines, platinum compounds, and / or epirubicin, and - if clinically appropriate - docetaxel At least one measurable area of ​​disease according to RECIST criteria (computed tomography (CT) scan or magnetic resonance imaging (MRT) within 6 weeks prior to study enrollment) Age 18 or older Written informed consent after notification of the study ECOG performance status (PS) 0-1 or Karnofsky index 70-100% Life expectancy > 3 months ·Platelet count ≧100,000 / mm 3 Hemoglobin ≥ 10g / dl INR<1.5 Normal bilirubin AST and ALT < 2.5x upper limit of normal (ULN) (5x ULN if liver metastases are present) Creatinine <1.5×ULN For women of childbearing potential (last menstrual period less than 2 years prior to enrollment): Negative pregnancy test (β-HCG) at baseline and use of two highly effective methods of contraception for 8 weeks after study drug injection Male patients must use an accepted method of contraception for 8 weeks after the study drug injection.

[0274] Patients who presented with one or more of the following criteria were not included in the study: Pregnancy or breastfeeding · Prior allergic reaction or intolerance to monoclonal antibodies, including humanized and chimeric antibodies Pre-registration for this exam Less than 3 weeks after prior anti-tumor chemotherapy or radiation therapy Other investigational drugs or devices used concurrently with or within 4 weeks prior to this study Other concomitant anti-cancer drugs or therapies · History of positive test for human immunodeficiency virus (HIV) antibodies Known hepatitis ·Uncontrolled or serious illness, including but not limited to any of the following: - Ongoing or active infection requiring parenteral antibiotics Symptomatic congestive heart failure -Unstable angina - Uncontrolled hypertension - Clinically significant arrhythmias Myocardial infarction within the past 6 months - Gastric bleeding within the past 4 weeks -Symptomatic peptic ulcer - Clinical symptoms or confirmed brain metastases - Psychiatric illness or social conditions that preclude compliance with the study Coadministration of vitamin K antagonists (e.g., Coumadin) and anticoagulants · Concomitant administration of therapeutic doses of heparin (prophylactic doses are permitted).

[0275] From a total of 29 patients, 15 patients received the study drug and were assigned to dose cohorts (33, 100, 300, 600, or 1000 mg IMAB362 / m 2 ) were assigned to one of the dose groups. These patients formed the safety population (SP). Because potential dose-limiting toxicities did not occur in any of the dose groups, there was no need to test additional patients to identify potential dose-limiting toxicities. Therefore, only three patients in each dose cohort, or 15 patients overall, received study drug.

[0276] The patient assignments to the various IMAB362 dose cohorts are shown in Table 1 below.

[0277] [Table 1]

[0278] No patients prematurely terminated the study; all patients completed the study according to protocol.

[0279] A. Safety Assessment IMAB362 was found to be safe and well tolerated.

[0280] Only 25 adverse events (AEs) that occurred in eight patients were assessed as treatment-related. Treatment-related AEs were similar between dose groups. More than half of these AEs were gastrointestinal (primarily nausea and vomiting). Only one of these related AEs was assessed as severe (vomiting); all others were mild or moderate. All related AEs resolved, except for one case of dysgeusia (CTC grade 1 (mild)) with unknown outcome and one case of elevated GGT (CTC grade 2 (moderate)) that did not resolve.

[0281] No dose-limiting toxicities (DLTs), defined as treatment-related AEs that occurred during the study drug infusion or within 4 weeks after infusion and were either grade 3 toxicity (excluding nausea, vomiting, and alopecia) or grade 4 or 5 toxicity (per CTC version 3.0), were observed in any dose group. Therefore, the maximum tolerated dose or maximum applicable single dose (MTD) of IMAB362 determined in this study was 1000 mg / m 2 is.

[0282] No related SAEs or suspected unexpected serious adverse reactions (SUSARs) occurred in this study.

[0283] Only seven patients had at least one laboratory value outside the reference range rated as grade 3 (severe). No dose-response relationship or clear association with study drug was observed. No CTC grade 4 (life-threatening) or 5 (fatal) laboratory values ​​were reported.

[0284] In conclusion, no relevant differences in AE profile and other safety parameters were observed between dose groups. Generally, IMAB362 given as a single dose was found to be safe and well tolerated, with nausea and vomiting being the most common associated adverse events.

[0285] B. Pharmacokinetic and Immunogenicity Assessment For measurement of drug concentrations, IMAB362 serum levels were measured in all patients immediately before infusion of study drug, at the end of infusion, 3, 8, 12, and 24 hours after the end of infusion, and on days 3 (V3), 5 (V4), 8 (V5), 15 (V6), and 29 (V7).

[0286] The IMAB362 serum levels over the course of the study for each patient are outlined in Table 2. 2 For one patient in the dose group (number 1201), low IMAB362 serum levels (12.633 μg / ml) were measured already before infusion of the study drug (V2, day 0) for unknown reasons.

[0287] [Table 2]

[0288] The observed mean peak concentrations (C max ) are shown in Table 3. C max The increasing mean values ​​for correspond to increasing infusion doses of IMAB362.

[0289] [Table 3]

[0290] A graphical representation of the mean blood concentrations of IMAB362 during the study period is shown in Figure 1.

[0291] The highest IMAB362 levels were measured immediately after the end of the infusion and up to 8 hours after the end of the infusion. At 3 hours after the end of the infusion, the mean IMAB362 concentration was 33 mg / m 2 14.1 μg / mL and 100 mg / m 2 In the 100 mg / mL group, 50.7 μg / mL and 300 mg / m 2 In the 164.2 μg / mL and 600 mg / m 2 307.8 μg / mL in the IV group, and 1000 mg / m 2 In the group, the value was 502.6 μg / mL.

[0292] The pharmacokinetics of IMAB362 are dose-dependent. The highest dose levels were observed within the first 8 hours after a 2-hour infusion. The mean half-life of IMAB362 was 8.5 days overall, ranging from approximately 5 to approximately 12 days in the various dose cohorts.

[0293] The present inventors have demonstrated from in vitro mechanism of action studies that IMAB362 at a concentration of 50 μg / ml is expected to exert robust antitumor cell effects through the inhibition of ADCC, CDC, and proliferation, and that the EC of ADCC and CDC, which are thought to be the main mechanism of action, are expected to be suppressed. 50 Based on this knowledge, it was determined that the value was covered by even half of this concentration level. 2 and 600 mg / m 2 A dose level of 300 mg / m was identified for more thorough evaluation in a multiple-dose study of IMAB362. 2 and 600 mg / m 2 Patients receiving IMAB362 significantly exceeded these levels on day 8 (V5) and were close to 50 μg / ml on day 15 (V6).

[0294] There was no evidence of anti-drug antibodies in patients after this single dose of IMAB362.

[0295] C. Evaluation of Antitumor Activity The primary endpoint for evaluation of potential antitumor activity was tumor status according to RECIST (version 1.0) classification at weeks 2–5 (V6 / V7) after IMAB362 infusion. Because all patients completed the study according to protocol, evaluation was exclusively performed at V7, i.e., weeks 4–5 after drug infusion. All patients were evaluated by CT.

[0296] Three patients did not have measurable disease (patients 1101 and 1201 did not have target lesions, and individual data were not available for patient 0302), but these patients were included in the population for analysis of antitumor activity because this was not a formal response assessment.

[0297] Overall, a complete or partial response could not be assessed for any patient. Stable disease was observed at 600 mg / m 2 This was observed in 1 of 15 patients in the 300 mg / m dose group. The percentage of tumor cells staining positive for CLDN18.2 in treated patients ranged from 1% to 80% (up to 50% of tumor cells had membrane staining), and in this patient, 90% or more of the tumor cells stained positive for CLDN18.2, with a large proportion of tumor cells showing membrane staining. 2 Two patients in the group also did not progress. Because these patients did not have target lesions, objective tumor response was not evaluable and they were classified as non-CR and non-PD. The duration of SD was approximately 2 months. The durations of non-CR and non-PD were approximately 2 months and 6 weeks, respectively.

[0298] A summary of the overall response by patient is shown in Table 4.

[0299] [Table 4]

[0300] The various parameters that contribute to the assessment of tumor status (overall response) are described below.

[0301] Table 5 summarizes the results of the evaluations after IMAB362 treatment (assessed at V7) with regard to the change in the total longest diameter (target lesions), the status of non-target lesions after IMAB362 treatment, and the occurrence of new lesions.

[0302] [Table 5]

[0303] The percentage change in the sum of the longest diameters of target lesions from V1 to V7 showed no obvious differences for different treatment doses.

[0304] Regarding non-target lesions, definite progression (V1 to V7) was reported more frequently in patients at lower dose levels, but not at 600 mg / m 2 and 1000 mg / m 2 Dose levels were not reported.

[0305] 300 mg / m 2 In one patient in the group (0403), clear progression was observed in non-target lesions and one target-lesion lymph node showed a decrease in its longest diameter.

[0306] No preference for one of the dose groups was observed for new lesions.

[0307] Patient 0302 (600mg / m 2 dose group) and 0205 (1000 mg / m 2 In the case of the 2000-mg dose group, the occurrence of new lesions was the reason for rating the overall response as progressive disease.

[0308] For assessment of non-target lesion status according to RECIST, levels of serum tumor antigens CA 125, CA 15-3, CA 19-9 and CEA were measured in a central laboratory at V2 (day 1, pre-infusion), V6 and V7.

[0309] A summary of serum tumor markers for the three patients with an overall response of at least stable disease is shown in Table 6.

[0310] [Table 6]

[0311] Of the three patients with stable disease or non-CR / non-PD by imaging, two patients had stable tumor markers during the observation period. One patient (0204) showed a significant decrease in all four tumor markers after treatment. In contrast, the majority of patients with progressive disease experienced an increase in tumor marker levels.

[0312] Tumor status (according to RECIST classification) 4-5 weeks after IMAB362 infusion (V6 / V7) was compared with baseline. Overall, a complete or partial response could not be assessed for any patient. One of 15 patients (600 mg / m 2 The 300 mg / m group had stable disease at the end of the study. 2 Two patients in the 100-dose group showed non-CR / non-PD. Consistent with this, tumor marker levels in these three patients remained stable (2 patients) or even declined significantly (1 patient). The majority of patients with progressive disease showed increasing tumor marker levels over time.

[0313] Regarding the parameters contributing to the assessment of tumor status (overall response), a reduction of one lesion was observed at 300 mg / m 2 At screening (V1), 13 of 15 patients had a total of 32 non-target lesions. After IMAB362 treatment (assessed at V7), definite progression of non-target lesions was reported in a total of 5 patients, with progression occurring in 33 mg / m 2 3 patients in the dose group, 100 mg / m 2 1 patient in the 300 mg / m 2 For none of these five patients, the overall response was assessed as progressive disease due solely to progression of their non-target lesions. A total of 17 new lesions were observed during the course of the study, evenly distributed across the dose groups. Two patients (600 mg / m 2 and 1000 mg / m 2 In the case of the 2000-mg dose group, the occurrence of new lesions was the reason for rating the overall response as progressive disease.

[0314] Additionally, ancillary data were collected in selected patients, which demonstrated that patient serum components and patient PBMCs were fully functional and potent in mediating CDC and ADCC, respectively, the primary mechanisms of action of IMAB362.

[0315] In conclusion, the indication of antitumor activity (stable disease, reduction of tumor markers) was observed at 300 mg / m 2 and 600 mg / m 2 The small sample sizes of the dose groups make it difficult to draw conclusions regarding the trend in effect.

[0316] C. Overall conclusion The trial was designed as a first-in-human, Phase 1, multicenter, non-randomized, interpatient, single-ascending dose, open-label clinical trial of a single intravenous infusion of IMAB362 and a 4-week treatment-free follow-up period.

[0317] A total of 15 patients received the study drug and were assigned to dose cohorts (33, 100, 300, 600, or 1000 mg IMAB362 / m 2 ) The dose groups can be considered comparable. No relevant imbalances in demographics and baseline characteristics were observed.

[0318] Regarding the primary objective of the study, no dose-limiting toxicities (DLTs) were observed in any dose group. Therefore, the applicable single dose of IMAB362 in this study was 1000 mg / m 2 IMAB362 was safe and well tolerated, with nausea and vomiting being the most common associated adverse events.

[0319] The AE profile and incidence rates were found to be similar in the various dose groups. No clear differences were observed between dose groups in the number of individual patients with clinically significant worsening of any hematology, biochemistry, or coagulation parameter.

[0320] Regarding the potential antitumor activity of IMAB362 according to RECIST criteria, no complete or partial response was observed in any patient. One of 15 patients (600 mg / m 2 The 300 mg / m group had stable disease at the end of the study. 2Two patients in the dose group showed non-CR / non-PD. Of these three patients with stable disease by imaging, two had stable tumor marker levels throughout the observation period. One patient showed a significant decrease in all four tumor markers after treatment.

[0321] This study and the target serum level of IMAB362 of 600 mg / m 2 Pharmacokinetic studies demonstrating that this can be achieved at this dose level support further evaluation of this dose.

[0322] Furthermore, supporting data confirm that the patient's immune effectors are fully functional and potent in mediating CDC and ADCC, respectively, the primary mechanisms of action of IMAB362.

[0323] Example 2 Drug efficacy The objectives of the in vitro analyses performed for this Phase I clinical trial included analyzing (i) whether effector cells present in the patient's blood are capable of inducing IMAB362-dependent ADCC, (ii) whether the patient's complement system is capable of inducing IMAB362-dependent CDC, and (iii) whether the ability of IMAB362 to induce ADCC and CDC is altered following administration in patients.

[0324] Various types of assays were performed to investigate the cytolytic activity induced by IMAB362 after administration in patients. Assays were performed using either patient serum or patient PBMCs isolated from blood samples (Table 7). For comparison and to verify the functionality of the CDC and serum ADCC assays, a human serum pool (generated from healthy human subjects) serially diluted with fresh IMAB362 was included in each assay in parallel. To test the functionality of the ADCC assay for PBMCs, blood cells isolated from a healthy donor were used as a positive control in the same assay for each patient.

[0325] A. Test Materials and Methods For various in vitro assays, patient serum samples were collected before IMAB362 infusion and 1, 7, 14, and 28–32 days after IMAB362 antibody administration (Table 7). These were used as a source of IMAB362 antibody and complement in CDC or as a source of antibody in serum ADCC assays. Patient pre-infusion serum was used as a "no IMAB362" negative control and for diluting patient serum samples to adjust the IMAB362 concentration to 0.5 μg / ml. Fresh blood samples were collected 14 days after infusion (7 days for patient 0203) and used as a source of effector cells for ADCC assays.

[0326] [Table 7]

[0327] Blood samples were collected from patients (Table 7), serum was collected, and serum aliquots were prepared and immediately stored at −80° C. Analysis of all these samples was performed in a single experiment after collection of all 24 serum samples.

[0328] For ADCC, fresh blood samples (15 ml Na2EDTA) were used to isolate PBMCs and the ADCC assay was performed the next day.

[0329] The ability of patient PBMCs to induce ADCC in conjunction with IMAB362 was tested ex vivo using fresh, 15 ml Na2EDTA-anticoagulated blood samples obtained from patients 14 days after IMAB362 administration (7 days for patient 0203). PBMCs from blood samples were isolated upon arrival using Ficoll density gradient centrifugation. PBMCs were cultured for 24 hours, and the following day, an ADCC assay was performed using luciferase-transfected CLDN18.2-positive NUGC4 human gastric cancer cells as targets, along with various concentrations of exogenously added IMAB362. PBMCs were added at an E:T ratio of 20:1, and the assay was incubated at 37°C and 5% CO2 for 24 hours. PBMCs from a healthy donor were tested in parallel in the same setting to analyze assay validity (positive assay control). This PBMC stock was stored in liquid N2 and for each ADCC assay using patient PBMCs, an aliquot from this PBMC stock was thawed and analyzed in parallel.

[0330] The characterized materials used were as follows: CLDN18.2-positive target cells: NUGC4-10cH11E10 gastric cancer cells transiently transfected with luciferase Positive control effector cells: PBMCs obtained from a healthy donor (frozen N2 stock lot ID: 276-SMS-09-00706, 4e7c / vial, MNZ, 08.07.07.SJA) Functional control antibody: IMAB362 in serial dilutions (0.4ng / ml to 126.5µg / ml) Assay negative control antibody: isotype control (rituximab, 126.5 μg / ml).

[0331] The ability of patient serum components to induce complement-dependent cytotoxicity (CDC) in conjunction with IMAB362 was analyzed ex vivo over time. Serum samples were collected and stored at -80°C, and all patient samples were assayed in parallel in the same experiment.

[0332] A fixed amount of 0.5 μg / ml IMAB362 (in vitro EC 50In addition to pre-infusion serum spiked with exogenously added IMAB362 (corresponding to the IMAB362 concentration), samples collected 1, 7, 14, and 28-32 days after IMAB362 administration were also tested. Circulating IMAB362 in these samples had to be adjusted to 0.5 μg / ml (normalized CDC). The final serum concentration in each assay was adjusted to 20%. Luciferase-transfected CHO-K1 cells stably transfected with CLDN18.2 were used as targets. A serum pool from a healthy human donor spiked with IMAB362 was tested for comparison.

[0333] The characterized materials used were as follows: CLDN18.2-positive target cells: stably transfected CHO-K1 p740 luci #2A5 cells. Assay positive control: IMAB362 serial dilutions (1:3.16) prepared in a pool of human serum from healthy donors to final concentrations ranging from 31.6 ng / ml to 10.0 μg / ml. Functional control antibody: IMAB362 adjusted to a final assay concentration of 0.5 μg / ml in each patient's pre-infusion serum sample. Assay negative control antibody: Isotype control antibody (rituximab) diluted in human serum pool.

[0334] The kinetics of overall cytotoxicity mediated by IMAB362 in the human circulation, integrating its ability to induce ADCC and CDC, was analyzed in a "one-tube" assay.

[0335] Serum samples were collected 7, 14, and 28–32 days after intravenous administration of IMAB362. These samples contained the patient's complement factors plus circulating IMAB362. Serum was applied to a final serum concentration of 25% (v / v) in each assay. PBMCs from healthy controls were added as effector cells, and NUGC-4 cells were used as target cells, with an E:T ratio of 40:1.

[0336] In an additional setting, serum was heat-inactivated to destroy complement activity, and this second assay therefore reflects exclusively the ADCC activity induced by IMAB362 present in patient serum.

[0337] During the Phase I study, serum samples were collected and stored at −80° C. All patient samples were assayed in parallel in the same experiment.

[0338] The characterized materials used were as follows: CLDN18.2-positive target cells: NUGC-4 10CH11 luci eGFP#2 gastric cancer cells stably transfected with luciferase. Effector cells: PBMCs (fresh buffy coat) from healthy donors. Functional control antibody: IMAB362 serial dilutions (0.26ng / ml to 200.0µg / ml) spiked in a human serum pool. Sample positive control: IMAB362 (200.0 μg / ml) (EC 200.0 μg / ml for IMAB362 in this setting) 80~100 The patient's pre-infusion serum sample was spiked with 100 mg of ribonucleotides (RI). Assay negative control antibody: Isotype control antibody (rituximab) in human serum pool.

[0339] The ability of IMAB362 to interact with and activate complement present in patient serum after prolonged circulation in the patient's blood, inducing complement-dependent cytotoxicity (CDC), was analyzed ex vivo at 1, 7, 14, and 28-32 days after IMAB362 administration. The assay was performed by using patient serum samples directly in the assay (non-normalized CDC). As a positive control, a fixed amount of 10 μg / ml IMAB362 (in vitro EC 90~100 Pre-infusion serum was exogenously spiked with 1000 mg of CLDN18.2 (corresponding to a 1000 mg / mL concentration). The final serum concentration in each assay was adjusted to 20%. Luciferase-transfected CHO-K1 cells stably transfected with CLDN18.2 were used as targets. A serum pool from healthy human donors spiked with IMAB362 was tested for comparison.

[0340] During the Phase I study, serum samples were collected and stored at −80° C. All patient samples were assayed in parallel in the same experiment.

[0341] The characterized materials used were as follows: CLDN18.2-positive target cells: stably transfected CHO-K1 p740 luci #2A5 cells. Functional control antibody: IMAB362 serial dilutions (1:3.16) prepared in a pool of human serum from healthy donors to final concentrations ranging from 31.6 ng / ml to 10.0 μg / ml. Sample positive control: IMAB362 (10.0 μg / ml) (in vitro CDC-EC 90~100 Pre-infusion patient serum samples spiked with 1000 mg of ribonucleotides (1000 mg / mL) at 2000 mg / mL of ribonucleotides (1000 mg / mL). Assay negative control antibody: Isotype control antibody diluted in human serum pool.

[0342] B. Results Ability of patient PBMCs to mediate ADCC To analyze the ability of patient immune cells to lyse CLDN18.2-expressing tumor cells, NUGC-4 gastric cancer cells, which endogenously express CLDN18.2, were incubated with increasing concentrations of IMAB362 and with patient PBMCs. Assays with PBMCs from healthy donors were included as a functional control.

[0343] Patient PBMCs showed IMAB362 dose-dependent lysis rates, with a maximum of 27-77% at concentrations of approximately 30 μg / ml. This was not significantly different from the maximum lysis rates of 14-56% obtained with healthy control PBMCs tested in the same assay (unpaired t-test) (Figure 2). ADCC activity was greatest for patient 0204.

[0344] These data indicate that PBMCs from gastric cancer patients are not inferior to PBMCs from healthy donors in inducing ADCC of human CLDN18.2-positive gastric cancer cells in conjunction with IMAB362.

[0345] The ability of the patient's complement system to induce CDC The ability of patient complement to interact with IMAB362 present in serum and induce CDC was tested. Pre-infusion serum samples were spiked with fresh 0.5 μg / ml IMAB362, and CDC activity was compared with the same antibody concentration spiked in a human serum pool. Serum / antibody samples were incubated with CHO-K1 p740 luci #2A5 cells, and lysis was determined after 80 minutes by measuring luciferase activity.

[0346] All patients were able to induce significant CDC within 80 minutes (Figure 3). Five of the six patients showed maximum lysis rates ranging from 50 to 71%. This is comparable to the data we obtained in a parallel study using pooled serum from healthy controls (64.5%). Notably, patient 0204 showed the highest CDC activity with fresh IMAB362 (93.9%).

[0347] Ability of soluble effectors in patient serum to induce cell death by intravenously circulating IMAB362 Next, the ability of patient sera to interact with intravenously administered IMAB362 throughout its circulation in patients was examined by testing serum samples collected at various time points after IMAB362 administration on CLDN18.2-positive CHO-K1 target cells in a CDC assay. The serum samples served as a source of patient-specific soluble effectors, including complement, as well as IMAB362. IMAB362 concentrations in serum samples were measured by ELISA (vivoScience) (Table 8) and adjusted to a final IMAB362 concentration of 0.5 μg / ml (the mean EC50 of IMAB362) using each patient's corresponding pre-infusion serum as a diluent. Because IMAB362 concentrations varied depending on the therapeutic dose and the time of blood collection, the dilution factors for the samples varied widely between patients, ranging from 4.6-fold to 688-fold. A serum pool from healthy donors (HSCs) was used as a control (Figure 4).

[0348] Although killing activity is maintained within the first 24 hours compared to the positive control (pre-infusion serum from each patient plus fresh IMAB362), serum samples collected after 1 week show a decline in cytolytic activity, which progresses further over the following weeks (Figure 4). Even so, significant cytotoxicity was still achieved by patient serum even after 2 weeks of IMAB362 administration. The loss of CDC activity after 28-32 days was significant, most pronounced in patients treated with the low dose of IMAB362 (Figure 4). The high dose (0.204; 600 mg / m 2 and 0205;1000 mg / m 2 CDC activity appeared to be better preserved throughout the period studied in patients treated with CDC. Based on currently available data, the mechanism underlying this decline is not yet understood.

[0349] [Table 8]

[0350] Effect of serum components on IMAB362-induced cytotoxicity The ADCC activity of mAbs can be impaired in the presence of human serum. The effect of patient serum on ADCC activity was examined. For this study, serum samples from each patient were collected 7, 14, and 28–32 days after IMAB362 administration, corresponding to the patient's complement factors plus circulating IV-administered IMAB362. All patient serum samples were diluted to a final serum concentration of 25% (v / v), and the residual IMAB362 concentration in each patient's assay sample was calculated (Table 8). In this ADCC assay, PBMCs from one healthy donor were used as effectors, and NUGC-4 cells were used as target cells (E:T ratio = 40:1). To ensure comparability, all assays for all patients were performed in parallel in a single experiment using the same conditions, target cells, and donor PBMCs. As a functional assay control, a pool of healthy human serum was spiked with IMAB362 (200.0 μg / ml). As an additional positive control, individual patient pre-infusion serum samples were diluted with 200.0 μg / ml IMAB362 (in vitro EC for IMAB362 in this system).80~100 The sample was spiked with 1000 kJ / ml (equivalent to 1000 kJ / ml).

[0351] In all assays, we observed that the IMAB362 antibody present in patient serum after administration was highly active and induced cytotoxicity (Figure 5). IMAB362 bioactivity was maintained for 28-32 days after administration, with specific killing still exceeding 48% in all dose groups. The overall differences between dose groups were surprisingly small, suggesting a saturation effect. Lower doses (33-300 mg / m 2 In patients treated with 600 or 1000 mg / m, a moderate decline in specific killing from 77.7–87.4% to 48.3–66.8% was observed over time, correlating with the decline in serum antibody concentrations (Figure 5, top panel). The highest activity, which was stably maintained over time, was observed at 600 or 1000 mg / m 2 observed in patients treated with IMAB362 (lower panel of Figure 5).

[0352] This assay was repeated using serum samples in which the complement factors were inactivated by incubation at 56°C for 30 minutes. Cytotoxicity with heat-inactivated patient serum samples was lower in all cases compared to that obtained with untreated serum. A similar reduction was observed with the heat-inactivated pool from healthy donors (HSCs, Figure 6).

[0353] In summary, these data indicate that patient serum does not inhibit the ADCC capacity of soluble serum components but rather enhances the total cytolytic activity induced by IMAB362.

[0354] Kinetics of IMAB362-mediated CDC in patient sera To determine the kinetics of CDC ability of IMAB362 in serum from patients in the different dose groups, serum samples were collected at 1, 7, 14 and 28 days after IMAB362 administration.

[0355] Again, this serum served as a source of complement as well as IMAB362. Final serum concentrations were adjusted to 20% (v / v) of the final volume. The final IMAB362 concentrations in each CDC assay sample are listed in Table 7. As a positive control, a patient's pre-infusion sample was spiked with fresh IMAB362 antibody to a concentration of 10 μg / ml (the in vitro EC value of IMAB362 in this CDC assay system). 95 ) to a final concentration of 0.032 μg / ml. Additionally, for functional control of the CDC assay, serial dilutions of IMAB362 (0.032–10 μg / ml) were prepared in a human serum pool. Standardized assays on CHO-K1 cells stably transfected with CLDN18.2 and luciferase were used as target cells. All serum samples were thawed and tested in parallel in the same experiment.

[0356] CDC activity correlates well with the antibody concentration in each serum sample (Figure 7). Most importantly, the data suggest that CDC-mediated cytotoxic activity is maintained over 4 weeks. Notably, patients in the high-dose group do not show a decline in CDC activity over this period.

[0357] Summary and Conclusion Patients with GEC appear to be unimpaired in their ability to induce both ADCC and CDC of CLDN18.2-expressing target cells in conjunction with IMAB362. Of note, the maximum specific lysis seen in ADCC and CDC and the EC measured for ADCC were significantly lower. 50 was highest for patient 0204, who had the most pronounced clinical and serum tumor antigen response.

[0358] Ex vivo analysis of CDC on circulating IMAB362 at various time points after administration of IMAB362 showed that even two weeks after administration, there was still enough active IMAB362 present in the circulation of patients to induce potent ADCC and CDC.

[0359] CDC activity in patients with circulating IMAB362 decreases over time for reasons that are currently unknown.

[0360] Example 3 cytokines Serum levels of cytokines can serve as indicators of a patient's immune status. In this clinical trial, the purpose of analyzing cytokines was primarily to support safety monitoring. We investigated cytokines within this ancillary analysis with a view to defining potential biomarker candidates.

[0361] Cytokine levels were measured 1 day before IMAB362 infusion and on days 3 and 5 of the treatment cycle. Cytokines examined included pro-inflammatory (IL-1, IL-6, IL-12, IFNγ, TNFα) and anti-inflammatory (IL-4, IL-10) cytokines, as well as cytokines required for T cell proliferation and function (IL-2) and NK cell proliferation (IL-2, IL-15).

[0362] Cytokines were analyzed by ELISA and flow cytometry (Interlab) according to Interlab's SOP-MU-IMM.M.0144.05 "Flow Cytomix Cytokin-Check IL4, IL6, IL13, TNF-alpha, IFN-gamma, MCP-1, IL10, IL2, IL1-beta, IL12p70, IL8, IL17A, IL23" and SOP-MU-IMM.M.0151.02 "Human Interleukin 15."

[0363] Cytokine serum levels of IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, IFNγ, and TNFα were analyzed for 14 of the 15 patients (Table 9). Patient 0403 (300 mg / m 2 Cytokine levels were not measured for the 24-hour period. Only serum cytokine level values ​​above the reference range were analyzed for temporal changes. Reference range values ​​were defined by Interlab (see CSR GM-IMAB-001).

[0364] Table 9: Cytokine serum levels on days 1, 3 and 5 Cytokine serum levels in all patients were measured on days 1, 3, and 5. Reference ranges for each cytokine are shown. Values ​​below or above the detection limit were set to the respective detection limit for calculations. [Table 9]

[0365] Proinflammatory cytokine levels (IL-1, IL-6, IL-12, IFNγ, and TNFα) were above their respective reference ranges in 9 of 14 patients (0104, 0105, 0201, 0203, 0204, 0112, 1202, 0112, and 0205). IFNγ levels were elevated in 2 patients (0201 and 1202), and TNFα levels were elevated in 1 of these 2 patients (0201). In both patients, IFNγ and TNFα levels were elevated before IMAB362 administration and decreased on subsequent days. IL-6 levels were elevated in 8 patients (0104, 0105, 0203, 1101, 0204, 0112, 1202, and 0205). No clear pattern of change in IL-6 levels emerged with respect to IMAB362 administration and a dose-response relationship. Patient 0204 (600mg / m 2 IL-6 levels in IMAB362 were not elevated before administration but rose significantly 2 days after infusion, a pattern not seen in any of the other patients. IL-1 and IL-12 levels remained within their respective reference ranges for all patients.

[0366] Anti-inflammatory cytokine levels (IL-4, IL-10) were above their respective reference ranges in 6 of 14 patients (0103, 0104, 0201, 0202, 0203, 1101). IL-10 levels were elevated in 6 patients (0103, 0104, 0201, 0202, 0203, 1101), and IL-4 levels were elevated in 2 of these patients (0201, 0202). Variations in anti-inflammatory cytokine levels showed no clear pattern with respect to IMAB362 administration and dose-response relationship.

[0367] Levels of IL-2 and IL-15, cytokines responsible for T-cell and NK-cell function and proliferation, were above their respective reference ranges in 9 of 14 patients (0104, 0201, 0202, 0203, 1101, 1201, 0204, 1202, and 0205). IL-2 levels were above the reference range in 6 patients (0201, 0202, 0203, 1101, 1202, and 0205), and IL-15 levels were above the reference range in 5 patients (0104, 0202, 1201, 0204, and 1202). Seven of the nine patients (0104, 0201, 0202, 0203, 1201, 1202, 0205) with high pre-dose IL-2 / IL-15 levels showed a decrease in cytokine levels on subsequent days: IL-2 / IL-15 levels were above their respective reference ranges before IMAB362 administration and decreased on days 2 and 4 after IMAB362 administration. The most significant decrease in this group was observed for IL-2 serum concentrations. All five patients (0201, 0202, 0203, 1202, 0205) with high pre-infusion levels of IL-2 experienced a decrease to less than 50% of their pre-infusion levels on day 4 after administration. This decrease was observed at 33 mg / m 2 This could also be observed in one patient (0201) who had a significantly higher IL-2 level (354 pg / mL) before administration of IMAB362.

[0368] A different IL-2 concentration profile was observed in patient 1101 (300 mg / m 2IMAB362), which had IL-2 levels within the reference range before and 2 days after infusion, but had high IL-2 concentrations on day 4 after infusion.

[0369] IL-15 levels decreased by day 4 after administration in all four patients (0104, 0202, 1201, 1202) who had high pre-infusion IL-15 levels. This concentration profile is very similar to the observed IL-2 concentration profile, although the relative decrease in levels is less pronounced.

[0370] No dose-effect relationship was observed for any of the cytokines analyzed.

[0371] To summarize the above, analysis of patient pretreatment levels showed that IL-6, IL-10, IL-2, and IL-15 were elevated in a substantial proportion of late-stage patients with gastroesophageal disease, whereas none or only one patient had elevated levels of IL-1, IL-12, IL-4, IFNγ, and TNFα.

[0372] Analysis of changes in cytokine levels within the first 5 days after IMAB362 treatment yielded the following observations: A significant decrease in these levels was observed in all 5 patients with high IL-2 levels, with 4 of the 5 patients reaching normal baseline values. Similarly, a moderate decrease was observed in all 4 patients with high IL-15 levels after IMAB362 administration. A significant decrease in levels after treatment was also observed in one patient with high IFNγ and one patient with high TNFα levels. IL-6, in contrast, increased after IMAB362 administration, with 4 patients showing IL-6 levels above baseline before treatment and 7 of 14 patients showing IL-6 levels above baseline 5 days after treatment.

[0373] Example 4 An international, multicenter, open-label, phase IIa, multiple-dose study evaluating the efficacy and safety of multiple doses of IMAB362 in patients with advanced adenocarcinoma of the stomach or lower esophagus We conducted an international, multicenter, open-label, phase IIa, multiple-dose study to evaluate the efficacy and safety of multiple-dose IMAB362 in patients with advanced adenocarcinoma of the stomach or lower esophagus. The primary objective of this study was to evaluate response rates (CR, PR) by RECIST. Secondary objectives included the frequency and severity of adverse events and tolerability of multiple-dose IMAB362 by CTCAE v3.0, progression-free survival (PFS) (time from the start of the first infusion to the date of first observed disease progression or death from any cause, whichever occurred first), immunogenicity by analysis of the human anti-chimeric antibody, quality of life, clinical benefit (CR, PR, and SD by RECIST), and pharmacokinetics of IMAB362 by serum levels.

[0374] Patients were screened for the presence of the IMAB362 target, CLDN18.2, in their tumors. CLDN18.2 status was determined by immunohistochemistry using an anti-claudin-18 antibody, performed according to a standardized protocol. Patients with tumors in which at least 50% of cells stained with at least 2+ (double-strength) staining intensity were enrolled in the trial. Inclusion and exclusion criteria were verified during the screening visit (V1). Patients were recruited from a university hospital specializing in the treatment of gastroesophageal cancer.

[0375] Patients had to meet all of the following inclusion criteria: Histologically proven metastatic, refractory, or recurrent advanced adenocarcinoma of the stomach or lower esophagus CLDN18.2 expression confirmed by immunohistochemistry in paraffin-embedded tumor tissue samples with a staining intensity of at least 2+ (scale of 0 to 3+) in at least 50% of tumor cells At least one measurable area of ​​disease according to RECIST criteria (CT scan or MRI within 2 weeks prior to the second visit) Age ≥ 18 years old Written informed consent ECOG performance status (PS) 0-1 or Karnofsky index 70-100% Life expectancy > 3 months ·Platelet count ≧100,000 / mm 3 Hemoglobin ≥ 10g / dl Normal bilirubin AST and ALT < 2.5x upper limit of normal (ULN) (5x ULN if liver metastases are present) Creatinine <1.5×ULN For women of childbearing potential (last menstrual period less than 2 years prior to enrollment): Negative pregnancy test (β-HCG) at baseline and use of two highly effective methods of contraception during treatment and for 8 weeks after the last infusion of study drug. Male patients whose sexual partners are females of childbearing potential must use generally accepted methods of contraception during treatment and for 8 weeks after the last infusion of study drug.

[0376] Patients who met any one or more of the following exclusion criteria were not eligible for study enrollment: Pregnancy or breastfeeding · Prior severe allergic reaction or intolerance to monoclonal antibodies, including humanized or chimeric antibodies Less than 3 weeks after prior anti-tumor chemotherapy or radiation therapy Other investigational drugs or devices used concurrently with or within 4 weeks prior to this study Other concomitant anticancer therapies (not for the indication under investigation) Known HIV infection or known active hepatitis (A, B, C) Concomitant use of vitamin K antagonists (e.g., Coumadin, Malkumar) and anticoagulants Heparin in therapeutic doses (prophylactic doses are acceptable) ·Uncontrolled illnesses, including but not limited to any of the following: - Ongoing or active infection requiring parenteral antibiotics Symptomatic congestive heart failure -Unstable angina - Uncontrolled hypertension - Clinically significant arrhythmias Myocardial infarction within the past 6 months - Gastric bleeding within the past 4 weeks -Symptomatic peptic ulcer - Clinical manifestations of brain metastases · Mental illness or social circumstances that make compliance with the study impossible.

[0377] All patients in all cohorts received repeat doses of IMAB362 every 2 weeks at visits 2, 5, 6, 7, and 8 (5 applications). The dose escalation procedure encompassed the following cohorts at two different doses (antibody / body surface area) of IMAB362: Cohort 1: 300 mg / m 2 Cohort 2: 600 mg / m 2 Cohort 3: 600 mg / m 2

[0378] The antibody solution was administered as a 2-hour intravenous infusion every 2 weeks. It was important that the infusion time be at least 2 hours. The infusion had to use an infusion system (e.g., Infusomat® fmS) to control the infusion time. An infusion set delivered the study drug, inspected for compatibility by the manufacturer, had to be used for drug administration. The infusion of the study drug had to be in the morning. A qualified physician had to be available during the infusion and for 24 hours afterwards.

[0379] Thirty-seven patients received at least one treatment. Unfortunately, three of these patients were not fully documented in the database, so 34 patients were included in the All Patient Treatment Set (APT set) and used for safety analysis. 4, 6, and 24 patients received 300 mg / m 2 Cohort 1 of IMAB362, 600 mg / m 2 Cohort 2 of IMAB362 and 600 mg / m 2 They were assigned to cohort 3 of IMAB362.

[0380] During the treatment period, one patient in Cohort 1, three patients in Cohort 2, and 12 patients in Cohort 3 discontinued the study before receiving five infusions of IMAB362 and completed the 9th visit (including the second tumor imaging) 2 weeks after the 5th infusion. These patients were replaced.

[0381] Two patients in Cohort 2 had no measurable disease at baseline and were excluded from efficacy analyses. Minor protocol deviations, such as baseline tumor assessment >14 days before the second visit (n = 3; 8.8%), hemoglobin <10 g / dL (n = 5; 14.7%), abnormal bilirubin (n = 3; 8.8%), ALT or AST >2.5 ULN (>5 ULN in cases of liver metastases) (n = 2; 5.9%), creatinine >1.5 ULN (n = 1; 2.9%), and a long time window (>15 days) between the screening period and the start of treatment (n = 2; 5.9%), occurred but did not result in exclusion from analyses. One patient had a myocardial infarction within the past 6 months. Waivers were accepted.

[0382] In cohorts 2 and 3, patients received 600 mg / m 2 Since all patients received the same dose of 300 mg / m², it was decided to analyze these patients as one group. All patients in the APT set (n=34) were Caucasian. The mean age was 18.5 years. 2 In the dose group, 62 years (range 45–65 years) and 600 mg / m 2 In the dose group, the age was 61 years (range 42-77 years).

[0383] The results of cancer localization and histopathological grading are summarized in Table 10. The mean time from initial diagnosis to the screening visit for this study was 16 months (min 2.7 months / max 56 months). HER2 / neu expression status was 600 mg / m 2 The disease status of most patients was unknown, except for five patients treated with HER2 / neu. One of these five patients was HER2 / neu positive.

[0384] TNM classification was determined for gastric (n = 16) and esophageal or gastroesophageal junction (n = 19) cancers. In the APT set, 25% of patients presented with gastric primary tumors classified as T1 or 2, 31% with T3, 25% with T4, and 19% with unknown. At the time of diagnosis, 69% of patients in the APT set had at least one or two involved lymph nodes, as indicated by N1 classification, and 56% of patients had peritoneal metastases (M1). 69% of patients with esophageal or gastroesophageal junction cancer were diagnosed as ≥ T3. At least one or two involved lymph nodes (N1) were reported for 84% of patients. In addition, 84% of patients presented with peritoneal metastases.

[0385] Table 10: Summary of tumor location and type at time of initial diagnosis (One patient had esophageal and gastric cancer; several patients had gastric cancer affecting different parts of the stomach.) [Table 10]

[0386] Based on MedDRA SOC, the most frequent clinically relevant pre-existing conditions were surgery in 25 patients (73.5%), chemotherapy in 30 patients (88.2%), and radiation in 7 patients (79.4%). In the majority of cases, surgery consisted of surgical resection of an organ, including gastrectomy (72%), esophagectomy (16%), lymphadenectomy (32%), and cholecystectomy (20%).

[0387] All but four patients had received at least one prior treatment for their study disease. Based on the WHO DD ATC, the most frequently used medications were pyrimidine analogues (fluorouracil and / or capecitabine), platinum compounds (cisplatin and / or oxaliplatin), and antidotes for antineoplastic treatment (calcium folinate and / or folinic acid). Other prior drug treatments (ended no later than the day of infusion) were also recorded.

[0388] A total of 30 of the 34 patients (88.2%) had at least one comorbid condition, i.e., an ongoing illness on the day of study drug infusion. Based on the MedDRA System of Clinical Organization Classification (SOC), the most common diagnoses were "gastrointestinal disorders" in 19 patients (56%), "general and systemic disorders" in 12 patients (35%), "metabolic and nutritional disorders" in 10 patients (29%), and "musculoskeletal and connective tissue disorders" in 8 patients (23.5%). Concomitant medications were primarily medications for acid-related disorders (17 patients; 50%), analgesics (12 patients; 35.3%), and medications for GI disorders (10 patients; 29.4%).

[0389] A. Safety Assessment Overall compliance with the study protocol was ensured because study drug infusions were administered by investigators at the study site and patients had to remain at the hospital for observation for at least 24 hours and up to 72 hours. Allocation of eligible subjects to dose cohorts was carried out exactly as specified by the study protocol (overseen by the DSMB). Study duration, defined as the time from the first screening visit to the last study date, ranged from a minimum of 18 days to a maximum of 355 days. The mean study duration was 106 days. Sixteen patients terminated the study early, before the ninth target visit.

[0390] Patients in all dose groups received an average of 4.5 to 5 infusions of IMAB362. The mean duration of one infusion of IMAB362 in the APT set was 125 minutes. One patient had a duration of less than the protocol-specified 120 minutes. This patient discontinued the infusion early due to vomiting.

[0391] 300 mg / m 2 (n=4) or 600 mg / m 2A safety analysis was performed on the APT set, including all 34 patients who received at least one dose of rituximab (n=30). 241 physician-described adverse events were coded according to the MedDRA dictionary and converted to preferred terms. A preferred term adverse event was counted only once for each patient (even if the same adverse event occurred more than once for that patient during the study period). The highest NCI-CTC grade occurring in each patient was recorded. 32 (94%) patients had at least one adverse event (regardless of relationship) during the study period. Two patients had no adverse events recorded. Overall, 6 patients (18%) did not experience any adverse events potentially related to the drug. 104 drug-related adverse events by preferred term were reported for 28 patients. 8 serious adverse events potentially related to the drug were reported for 4 patients. The lowest dose group (300 mg / m 2 The small number of patients in the 300 mg / m 2 Cohort and 600 mg / m 2 The incidence of patients with related adverse events in the groups (cohorts 2 and 3) is 75% and 83%, respectively.

[0392] Overall, the most frequently reported AEs from the SOC were "gastrointestinal disorders" (27 / 34 patients, 79.4%) and "general disorders and administration site conditions" (26 / 34 patients, 76.5%). Based on MedDRA PTs, the most frequently recorded AEs were "nausea" (57 events in 18 patients), "vomiting" (52 events in 16 patients), and "fatigue" (20 events in 14 patients). Overall, only 192 of the recorded AEs were assessed by the investigator as related to the study drug. These treatment-related AEs were classified into 104 different preferred terms and were observed in 28 / 34 patients.

[0393] Most related adverse events were mild to moderate. There were 8 (23.5%) patients with moderate drug-related treatment-emergent events and 12 (35.3%) patients with serious drug-related treatment-emergent events.

[0394] Severe drug-related AEs were vomiting at 300 mg / m 2 Two patients in the 600 mg / m dose group were reported, one of whom experienced nausea. 2 Ten patients in the dose group experienced serious drug-related adverse events; of the six patients who vomited, three additionally experienced nausea, one patient experienced hypersensitivity (allergic reaction), one patient experienced hypersalivation, one patient experienced dehydration, and one patient experienced hypoalbuminemia. The final two patients also reported vomiting and nausea. Two patients suffered related hypersensitivity (allergic reaction) during the study drug infusion, one of which was classified as moderate and one as severe. Both patients recovered after the infusion was discontinued.

[0395] Of all treatment-emergent events reported, 12 / 34 patients required study drug intervention for the AE. In 7 (21%) cases, the AE led to permanent study discontinuation. The underlying adverse event was drug-related in 3 patients (hypersensitivity (allergic reaction) (n=2), vomiting, and abdominal pain) and non-drug-related in the other 4 patients (worsening general health (n=3), pneumonia). One patient had a dose reduction, and one patient had a 4-day drug postponement due to severe vomiting with nausea. Three patients had their infusion interrupted / extended. 27 patients (79%) received concomitant therapy for the AE. Eleven patients were hospitalized.

[0396] Thirteen patients had 31 recorded SAEs. One patient died during the second screening period of the study. Twelve patients had other serious adverse events, and the adverse events were study drug-related in four patients. Related adverse events such as vomiting, nausea, and GI bleeding and dehydration were determined by the investigator to be study drug-related. There were four SARs and two SUSARs (vomiting and vomiting with GI bleeding) in this study. The final outcome was death in seven cases. None of the deaths were classified by the investigator as study drug-related.

[0397] One patient was a 45-year-old Caucasian man with a lean diet (BMI 19.3) and good general health (ECOG performance status grade 1, Karnofsky index 80%).

[0398] Patients received 300 mg / m every 2 weeks on November 4, 2011, November 22, and a third dose on December 6. 2 The patient received an infusion of IMAB362. Prior to the study, the patient had already experienced grade 1 nausea and vomiting. Grade 3 vomiting was diagnosed on November 7, 2010. This was assessed as severe, requiring hospitalization. The vomiting progressed to grade 1 on November 17, 2010, and finally stopped completely, allowing the patient to be discharged the same day. Before the second and third infusions of IMAB362, the patient was treated with potent premedication (alizapride, aprepitant, metoclopramide, and dimehydrinate) to prevent nausea and vomiting, and thus did not experience further episodes of nausea or vomiting. The investigator assessed the vomiting as related to the study drug. The report was accepted by the sponsor on January 19, 2011. The SAE was determined to be unexpected but related to the study drug and was therefore reported as a SUSAR.

[0399] One patient was a 77-year-old Caucasian man. He had a normal diet (BMI 24) and was in excellent general condition (ECOG performance status: Grade 0, Karnofsky index: 100%) at screening. Prior to the study, the patient was already suffering from nausea and was therefore treated with metoclopramide as needed. The patient was discontinued on November 9, 2011, at 600 mg / m because the study had to be discontinued early due to death. 2The patient received only a single application of IMAB362. A pleural effusion in the left lung was diagnosed by X-ray before the infusion and reported as an SAE. Hematemesis occurred the following morning. After administration of pantoprazole and ondansetron 8 mg intravenously, vomiting decreased and hematemesis resolved the same day. Vomiting decreased from grade 3 to grade 2 and finally stopped on November 12, 2011, allowing the patient to be discharged on November 13, 2011. The investigator assessed the event as study drug-related. The report was accepted by the sponsor on November 10, 2011, and the event was determined to be unexpectedly related to the study drug and therefore reported as a SUSAR. The patient's general condition deteriorated, he developed renal failure, and unfortunately, he died on December 6, 2011.

[0400] One patient was a 42-year-old Caucasian man with a well-nourished diet (BMI 26) and excellent general condition (ECOG performance status grade 0; Karnofsky index: 100%). 2The patient received two infusions of IMAB362. On March 20, 2012, the patient received his first dose of study medication. He developed nausea and severe vomiting, and the infusion rate had to be reduced 35 minutes after the infusion. Symptoms were treated with pantoprazole 40 mg and granisetron 3 mg, as well as two vials of butylscopolamine butylsulfate and aprepitant 80 mg intravenously. This serious adverse event resulted in prolonged hospitalization. The investigator assessed the event as related to the study medication. The SAE report was accepted by the sponsor on March 21, 2012, and the event was determined to be expected and related to the study medication. Several days later, on March 24, 2012, the patient had to be hospitalized again due to severe dehydration caused by nausea and vomiting. Additionally, the patient suffered from epigastric pain. The patient received metamizole 1 g intravenously, a buprenorphine patch, and an infusion for hydration. By March 30, 2012, the symptoms had subsided, and the patient recovered from dehydration. The investigator assessed the event as unrelated to the study drug. The SAE report was received by the sponsor on March 26, 2012, and the event was determined to be unexpected and unrelated to the study drug. On April 3, 2012, the patient received a second infusion, which again resulted in nausea and vomiting. The patient was treated with 30 drops of metoclopramide PO and 1 vial of dimenhydrinate IV. On April 5, 2012, the symptoms worsened and were assessed as severe. In addition, the patient suffered from dysphagia, which significantly reduced food intake. The symptoms resolved on April 15, 2012. The investigator assessed the event as related to the study drug. The SAE report was received by the sponsor on April 19, 2012, and the event was determined to be expected and related to the study drug.

[0401] One patient was a 73-year-old Caucasian man with a well-nourished diet (BMI 26) and good general health (ECOG performance status grade 1; Karnofsky index: 90%). From November 8, 2011, to January 3, 2012, the patient received 600 mg / m every 2 weeks. 2The patient received five scheduled study drug applications of IMAB362. The patient received his first application of IMAB362 on November 8, 2011. During and after this infusion, the patient developed nausea and vomiting. Symptoms became severe on November 9, 2011. After treatment with metoclopramide, symptoms resolved one day later. The investigator assessed the event as related to the study drug. The SAE report was received by the sponsor on November 10, 2011, and the event was determined to be expected and related to the study drug. The third infusion was administered on December 6, 2011. The patient experienced moderate and mild vomiting and was treated with clemastine, ranitidine, and ondansetron. The vomiting lasted for one day. The nausea persisted for seven days. The study was terminated on January 16, 2012, due to disease progression. No follow-up visits were conducted.

[0402] In conclusion, IMAB362 was found to be safe and well tolerated in a heavily pretreated patient population with advanced adenocarcinoma of the stomach, esophagus, or gastroesophageal junction. Overall, the most frequently reported AEs from SOC were "gastrointestinal disorders" (27 / 34 patients, 79.4%) and "general disorders and administration site conditions" (26 / 34 patients, 76.5%).

[0403] On a MedDRA PT basis, the most frequently recorded AEs were "nausea" (57 events in 18 patients), "vomiting" (52 events in 16 patients) and "fatigue" (20 events in 14 patients).

[0404] Overall, only 192 of the recorded AEs were assessed by the investigator as related to the study drug. These treatment-related AEs were observed in 28 of 34 patients. 83% of these related AEs were gastrointestinal disorders (68%, 130 AEs) recorded in 25 patients and general / systemic disorders (15%, 29 AEs) recorded in 16 patients.

[0405] On a MedDRA PT basis, most related adverse events were mild to moderate, with nausea (50%), vomiting (47%), fatigue (27%), abdominal pain (15%), peripheral edema (15%), decreased appetite (12%), and diarrhea (12%) occurring in more than 10% of patients.

[0406] Two patients developed related hypersensitivity (allergic) reactions during the study drug infusion, one classified as moderate and one classified as severe, both of which recovered after the infusion was stopped.

[0407] No abnormal study drug-related laboratory values ​​of CTC grade 4 (life-threatening) or 5 (fatal) were reported.

[0408] Twelve patients (35.3%) had serious related treatment-emergent events. Severe drug-related AEs included vomiting at 300 mg / m 2 Two patients in the 600 mg / m dose group were reported, one of whom experienced nausea. 2 Ten patients in the dose group experienced severe drug-related adverse events: of the six patients who had vomiting, three additionally experienced nausea, one patient experienced hypersensitivity (allergic reaction), one patient experienced hypersalivation, one patient experienced dehydration, and one patient experienced hypoalbuminemia. The final two patients also reported vomiting and nausea.

[0409] At the time of analysis, 13 patients had recovered from all drug-related adverse events, 2 patients were in ongoing recovery, 11 patients had not recovered from at least one AE, and the status was unknown for 2. Of the 11 patients who had not recovered from at least one drug-related adverse event, 9 had gastrointestinal disorders (nausea in 4, vomiting in 2).

[0410] Thirteen patients had 31 documented SAEs, including seven deaths. One patient died during the screening period, i.e., before the start of the study drug infusion, and was therefore classified as a screening event. Four patients had treatment-emergent gastrointestinal SAEs that were determined to be treatment-related, including vomiting (n=4), nausea (n=2), dehydration (n=1), and GI bleeding (n=1). One of these patients with vomiting received 300 mg / m 2 and the other three were treated with 600 mg / m 2 They were treated with IMAB 362. Three of these four patients recovered, except for one who died of unrelated renal failure.

[0411] The incidence of drug-related adverse events was 300 mg / m 2 Dose groups and 600 mg / m 2 The incidence was comparable between the dose groups, occurring in 75% and 83% of patients, respectively. The frequency and severity of nausea, vomiting, and fatigue were also comparable between the dose groups. There was no clear relationship between dose and frequency / severity of adverse events.

[0412] The adverse event profile for most AEs reported in the gastrointestinal tract is consistent with the underlying disease and is also consistent with the CLDN18.2 expression profile. CLDN18.2 is also expressed in gastric epithelial cells (in tight junctions), suggesting that nausea and vomiting are on-target effects.

[0413] Generally speaking, 300 mg / m 2 and 600 mg / m 2 IMAB362, given in multiple doses, was found to be safe and well tolerated, with vomiting and nausea being the most common associated adverse events.

[0414] B. Pharmacokinetic and Immunogenicity Assessment Preliminary drug concentration data for multiple dose applications of IMAB362 are 300 mg / m 2 and 600 mg / m 2Data is available for four patients in the first cohort and 34 patients in the second and third cohorts who received IMAB362.

[0415] [Table 11]

[0416] Blood samples were collected before each infusion. After the first infusion, additional samples were collected at the end of the infusion and at 1, 1.5, 2, 3, 4, 6, 12, 24 hours, 3, and 6 days after the end of the infusion. After the final infusion, samples were collected at the end of the infusion and at 1, 1.5, 2 hours, and 14 days and weeks 4 through 8 after the end of the final infusion. No analytes were detectable in predose samples from individual patients assigned to cohorts 1 through 3.

[0417] After the first IMAB362 injection, c max Values ​​ranged from 208.9 μg / mL to 349.6 μg / mL for the first cohort. For the second and third cohorts combined, c max Values ​​ranged from 269.1 μg / mL to 575.1 μg / mL after the first application.

[0418] A time-dependent decrease in IMAB362 concentrations was observed in serum samples collected at subsequent time points (V3 through V5) (Figure 8). At visit 5, before the second infusion, minimum serum levels of between 11.3 μg / mL and 36.8 μg / mL (mean 22.5 ± 10.5 μg / mL) were measured for Cohort 1, and between 17.0 μg / mL and 100.2 μg / mL (mean 54.5 ± 29.0 μg / mL) for Cohorts 2 and 3 combined.

[0419] At visit 8 (day 57), before the fifth infusion, minimum serum levels between 32.4 μg / mL and 67.1 μg / mL (mean value 46.1 ± 18.5 μg / mL) were measured for cohort 1, and between 28.3 μg / mL and 301.6 μg / mL (mean value 147.2 ± 93.1 μg / mL) for cohorts 2 and 3 (Table 12).

[0420] After injection at the 8th visit, c max Values ​​ranged from 259.1 μg / mL to 326.5 μg / mL for the first cohort and from 278.1 μg / mL to 642.6 μg / mL for cohorts 2 and 3 (Table 11).

[0421] For Cohort 1, the mean C max Values ​​were measured 90 minutes after the first IMAB362 infusion (270.6 ± 63.9 μg / mL) and 90 minutes after the fifth infusion (279.2 ± 27.7). For combined cohorts 2 and 3, mean C max Values ​​were measured at the end of the first IMAB362 infusion (340.8±80.2 μg / mL) and 60 minutes after the fifth infusion (443.3±97.7) (Table 12).

[0422] In summary, the measured serum levels of IMAB362 were 300 mg / m 2 showed that serum concentrations of IMAB362 fell below the desired level of 50-100 μg / ml during every 2-week cycles in patients treated with 600 mg / m 2 In contrast, in the majority of patients, serum levels of IMAB362 remained above 50 μg / ml even 2 weeks after the first application. Seven to 29 days (mean 15 days) after the fifth dose, dose levels remained above 50 μg / ml (mean 151.3 ± 90.1 μg / mL).

[0423] Table 12: 300 and 600 mg / m 2 Descriptive pharmacokinetic data for multiple doses of IMAB362 300 mg / m 2Four patients (Cohort 1) were treated with repeated doses of 600 mg / m 2 Mean ± sd concentration (μg / ml) of IMAB362 in serum from up to 30 patients (30 in the 1st infusion, 12 in the 5th infusion) treated with multiple doses of [Table 12]

[0424] Mild accumulation of IMAB362 was observed between cycles, with accumulation rates ranging from 1.03- to 3.52-fold before the second infusion based on the first pre-dose value (mean 2.04).

[0425] Table 13: Accumulation of IMAB362 after repeated injections To determine the accumulation rate, the ratio of IMAB362 concentrations before visits 6, 7, 8, and 9.x (responder treatment) to before the second infusion (visit 5) was calculated. [Table 13]

[0426] In conclusion, the pharmacokinetics of IMAB362 were observed to be dose-dependent.

[0427] After the first IMAB362 injection, c max Values ​​ranged from 208.9 μg / mL to 349.6 μg / mL for the first cohort. For the second and third cohorts combined, c max Values ​​ranged from 269.1 μg / mL to 575.1 μg / mL after the first application.

[0428] A time-dependent decrease in IMAB362 concentrations was observed in serum samples collected at subsequent time points (V3 through V5): at visit 5, before the second infusion, minimum serum levels between 11.3 μg / mL and 36.8 μg / mL (mean 22.5 ± 10.5 μg / mL) were measured for Cohort 1, and between 17.0 μg / mL and 100.2 μg / mL (mean 54.5 ± 29.0 μg / mL) for Cohorts 2 and 3 combined.

[0429] At the eighth visit (day 57), before the fifth infusion, minimum serum levels between 32.4 μg / mL and 67.1 μg / mL (mean value 46.1 ± 18.5 μg / mL) were measured for cohort 1, and between 28.3 μg / mL and 301.6 μg / mL (mean value 147.2 ± 93.1 μg / mL) for cohorts 2 and 3.

[0430] After the fifth injection at the eighth visit, c max Values ​​ranged from 259.1 μg / mL to 326.5 μg / mL for the first cohort and from 278.1 μg / mL to 642.6 μg / mL for cohorts 2 and 3.

[0431] For Cohort 1, the mean C max Values ​​were measured 90 minutes after the first IMAB362 infusion (270.6 ± 63.9 μg / mL) and 90 minutes after the fifth infusion (279.2 ± 27.7). For combined cohorts 2 and 3, mean C max Values ​​were measured at the end of the first IMAB362 infusion (340.8±80.2 μg / mL) and 60 minutes after the fifth infusion (443.3±97.7).

[0432] In summary, the measured serum levels of IMAB362 were 300 mg / m 2 showed that serum concentrations of IMAB362 fell below the desired level of 50-100 μg / ml during every 2-week cycles in patients treated with 600 mg / m 2In contrast, in the majority of patients, serum levels of IMAB362 remained above 50 μg / ml even 2 weeks after the first application. Seven to 29 days (mean 15 days) after the fifth dose, dose levels remained above 50 μg / ml (mean 151.3 ± 90.1 μg / mL).

[0433] C. Evaluation of Antitumor Activity Full Analysis Set (FAS): All subjects who received at least one dose of study medication and for whom post-treatment efficacy data were available were included.

[0434] At the time of analysis, 50 patients received 600 mg / m 2 Nine of these patients were recently included, and further data are not currently available due to their recent enrollment. Ten patients did not undergo second tumor imaging, and therefore these patients are not included in the FAS set. The FAS set includes 31 patients.

[0435] The mean age was 57 years, ranging from 35 to 77 years. Patients in the FAS set had a mean Karnofsky index of 90% (range 70-100%). The majority of patients (81%) had been previously treated with at least one chemotherapy regimen. Six patients had not received any prior chemotherapy regimen.

[0436] [Table 14]

[0437] The mean number of prior chemotherapy regimens was 2.0 (range 0-5). Chemotherapy regimens for gastroesophageal cancer consisted primarily of various combinations of 5-FU derivatives, platinum compounds, taxanes, epirubicin, irinotecan, trastuzumab for HER2 / neu-positive patients, and other investigational agents. In the FAS set, 81% of patients received at least one dose of 5-FU or capecitabine, and 74% had been treated with at least one platinum compound before enrollment. Six (19%) patients had been previously treated with trastuzumab or other investigational agents. Six (19%) patients also received radiation therapy before the start of the study.

[0438] Due to the late stage of disease, patients had an average of 2.0 metastatic sites (range 1.0-4.0), most notably lymph nodes (19 patients, 61%), liver (13 patients, 42%), ascites (8 patients, 26%), and peritoneum (7 patients, 23%).

[0439] The overall disease control rate was 39% (Table 15). Four patients had a confirmed partial response, and eight patients had disease stabilization. The first re-evaluation for these patients occurred 8 to 11 weeks after the first infusion, except for two patients, for whom the first tumor re-evaluation occurred 6 weeks later.

[0440] [Table 15]

[0441] Six of the 12 patients with clinical disease control had at least one tumor marker (CEA; CA19-9; CA125; CA15-3) that was elevated at baseline and decreased by 35-76% over the study period. All tumor markers were below the cutoff values ​​in three patients, and tumor marker results were unavailable for one patient.

[0442] Interestingly, the four patients with progressive disease as their best response also had tumor marker reductions of between 29 and 54% over the course of the study.

[0443] Partial responses were achieved after 2.3 months (2 patients), 6.5 months (1 patient), and 4.8 months (1 patient) of treatment, respectively. PR was confirmed for one patient and persisted for an additional 4.4 months, resulting in a PFS of 9.2 months for this patient. Confirmation for the other three patients occurred after 6 weeks (1 patient) and 12 weeks (2 patients), respectively. Further details can be found in Table 16.

[0444] Table 16: Detailed evaluation of each patient-based FAS set na - data not yet available; nd - not detectable. * - Tested because the event did not occur until November 2012 or the exact date is currently unknown. The last date of follow-up was used for each case. # - Tumor marker below cutoff value; therefore not counted in the text. [Table 16-1] [Table 16-2]

[0445] The mean progression-free survival for patients in the FAS set was 10 weeks (min 4 weeks; max 40 weeks). Due to limited event availability, the mean progression-free survival for patients with clinical benefit (PR+SD) shown in Figure 9 is of limited value. Patients without clinical benefit (PD) had a mean progression-free survival of 9 weeks (min 4 weeks; max 11 weeks) (Figure 9).

[0446] There were no differences between patients with clinical benefit (PR or SD as best response) or progressive disease (PD as best response) with regard to age (mean 56 vs. 59 years), no prior chemotherapy regimen (mean 1.9 vs. 2.1), or Karnofsky index (mean 89 vs. 88%). Only the number of metastatic sites was lower in the responder group, a mean of 1.9 compared with a mean of 2.3 in the non-responder group. The difference was not statistically significant.

[0447] The intensity of IHC staining (mean and maximum) was similar between patients with clinical benefit and those with progressive disease. The number of stained cells differed between the two groups. The maximum number of stained cells and the mean number of stained cells were higher in patients with clinical benefit: 77% vs. 67% and 71% vs. 60%, respectively.

[0448] Differences were also observed regarding the location of metastases: pleural effusion (25% vs. 5%), peritoneal carcinomatosis (42% vs. 11%), and ascites (42% vs. 16%) were more frequent in patients with clinical benefit compared with patients with progressive disease as best response. The presence of liver metastases was much less common in patients with clinical benefit (17% vs. 58%).

[0449] Per Protocol (PP) set: The PP population included all patients who completed the treatment period (up to visit 9) without any significant protocol deviations.

[0450] Of the 31 patients in the FAS set, 2 had significant protocol violations (no target lesions), and 9 patients did not complete the study protocol until the 9th visit and therefore received fewer than 5 required infusions of IMAB362. The PP set included 20 patients.

[0451] The mean age was 60 years, ranging from 35 to 77 years. Patients in the PP set had a mean Karnofsky index of 90% (range 70-100%). The majority of patients (80%) had been previously treated with at least one chemotherapy regimen. Four patients had not received any prior chemotherapy regimen.

[0452] [Table 17]

[0453] The mean number of prior chemotherapy regimens was 2.0 (range 0-5). Chemotherapy regimens for gastroesophageal cancer consisted primarily of various combinations of 5-FU derivatives, platinum compounds, taxanes, epirubicin, irinotecan, trastuzumab for HER2 / neu-positive patients, and other investigational agents. In the PP set, 80% of patients received at least one dose of 5-FU or capecitabine, and 75% were treated with at least one platinum compound before enrollment. Five (25%) patients had been previously treated with trastuzumab or other investigational agents. Further details can be found in Table 17. Five (25%) patients also received radiation therapy before the start of the study.

[0454] Due to the late stage of disease, patients had an average of 3.0 metastatic sites (range 1.0-4.0), most notably lymph nodes (13 patients, 65%), liver (9 patients, 45%), ascites (6 patients, 30%), and lungs (5 patients, 25%).

[0455] The overall disease control rate was 50%. Four patients had a confirmed partial response, and six patients had disease stabilization. The first re-evaluation for these patients occurred 8 to 11 weeks after the first infusion, except for one patient, for whom the first tumor re-evaluation occurred 6 weeks later (Table 18).

[0456] [Table 18]

[0457] Six of the 10 patients with clinical disease control had at least one tumor marker (CEA; CA19-9; CA125; CA15-3) that was elevated at baseline and decreased by 35-76% over the study period. All tumor markers were below the cutoff values ​​in two patients, and tumor marker results were unavailable for one patient.

[0458] Interestingly, the three patients with progressive disease as their best response also had tumor marker reductions of 29 to 54% over the course of the study.

[0459] Partial responses were achieved after 2.3 months (2 patients), 6.5 months (1 patient), and 4.8 months (1 patient) of treatment, respectively. PR was confirmed for one patient and persisted for an additional 4.4 months, resulting in a PFS of 9.2 months for this patient. Confirmation for the other three patients occurred after 6 weeks (1 patient) and 12 weeks (2 patients), respectively. Further details can be found in Table 19.

[0460] Table 19: Detailed evaluation of each patient-based PP set na - data not yet available; nd - not detectable. * - Event was censored because it did not occur until November 2012 or the exact date is currently unknown. The last date of follow-up was used for each case. # - Tumor marker below cutoff value; therefore not counted in the text. [Table 19-1] [Table 19-2]

[0461] The mean progression-free survival for patients in the PP set was 18 weeks (min 9 weeks; max 40 weeks). Due to limited event availability, the mean progression-free survival for patients with clinical benefit (PR+SD) shown in Figure 10 is of limited utility. Patients without clinical benefit (PD) had a mean progression-free survival of 10 weeks (min 9 weeks; max 10 weeks) (Figure 10).

[0462] There were no differences between patients with clinical benefit (PR or SD as best response) or patients with progressive disease (PD as best response) with regard to age (mean 57 vs. 62 years), no prior chemotherapy regimen (mean 2.1 vs. 2.0), or Karnofsky index (mean 88 vs. 88%). Only the number of metastatic sites was higher in patients with clinical benefit, with a mean of 3.0 compared with a mean of 2.2 in patients without clinical benefit. The difference was not statistically significant.

[0463] The intensity of IHC staining (mean and maximum) was similar between patients with clinical benefit and those with progressive disease. The number of stained cells differed between the two groups. The maximum number of stained cells and the mean number of stained cells were higher in patients with clinical benefit: 76% vs. 66% and 70% vs. 66%, respectively.

[0464] Differences were also observed regarding the location of metastases: pleural effusion (30% vs. 10%), peritoneal carcinomatosis (40% vs. 0%), and ascites (40% vs. 20%) were more frequent in patients with clinical benefit compared with patients with progressive disease as best response. The presence of liver metastases was much less common in patients with clinical benefit (20% vs. 70%).

[0465] In conclusion, tumor status (per RECIST) was compared with baseline at 2 weeks after the fifth IMAB362 infusion (V9). At least one post-baseline staging was available for 31 patients (FAS). Patients were enrolled at an advanced stage of disease, with a mean of 2.0 prior chemotherapy regimens and a mean of 2.0 metastatic sites.

[0466] Confirmed partial responses were assessed in four patients, resulting in an overall response rate of 13%. Three of these patients are currently undergoing treatment, and the duration could not be calculated. In addition, eight patients had disease stabilization, resulting in a disease control rate of 39%. At the time of analysis, the progression-free survival time for these 12 patients with clinical benefit ranged from 6 to 40 weeks. Seven of these patients had no events recorded to date, so a mean value could not be calculated. Nine of the patients with clinical benefit had at least one elevated tumor marker at baseline, and six of these patients had a concurrent decrease of -35 to -76%. Interestingly, the four patients with progressive disease as their best response also had a decrease of -29 to -54% in at least one elevated tumor marker over the course of the study. The mean overall progression-free survival time was 10 weeks, ranging from 4 to 40 weeks.

[0467] In patients with clinical benefit (4PR+8SD=39%), the incidence of peritoneal carcinomatosis, pleural effusion, and ascites was higher, and the incidence of liver metastases was lower than in patients without clinical benefit. On the other hand, one patient with a confirmed partial response had a high incidence of liver metastases.

[0468] For the current data set, it appears that patients with clinical benefit had higher numbers of cells with positive IHC staining.

[0469] Additionally, ancillary data were collected in selected patients, which demonstrated that patient serum components and patient PBMCs were fully functional and potent in mediating CDC and ADCC, respectively, the primary mechanisms of action of IMAB362.

[0470] In conclusion, antitumor activity (partial response, stable disease, reduction of tumor markers) was observed and IMAB362 merits further investigation.

[0471] D. Overall conclusion The trial was designed as a three-cohort, phase IIa, multicenter, non-randomized, inter-patient, multiple-dose escalation, open-label clinical trial. Eligible patients were required to be refractory to standard treatment or naive to accepted therapies.

[0472] For this interim report, 34 patients were evaluable for the safety analysis (APT set), of which 4 were in Cohort 1 (300 mg / m 2 ), and 6 patients in Cohort 2 (600 mg / m 2 ) and 20 patients in Cohort 3 (600 mg / m 2 ) was enrolled in the Phase IIa study. IMAB362, given in a multiple-dose schedule, was safe and well tolerated in many pretreated patients with gastroesophageal cancer, with nausea and vomiting being the most common associated adverse events. Most associated adverse events were mild to moderate. Only two patients experienced allergic reactions, one moderate and one severe. There were no grade 4 or grade 5 adverse events (including laboratory parameters) in this Phase IIa study or the previous Phase I study. Because the majority of registered monoclonal antibodies are associated with life-threatening grade 4 and 5 side effects, it is noteworthy that IMAB362 has not caused any grade 4-related adverse events to date. The indication for bevacizumab in metastatic breast cancer was revoked by the FDA in November 2011 after initial preapproval in 2008. Bevacizumab did not extend life expectancy and caused severe hypertension and bleeding, accompanied by intestinal and nasal septum perforation. Cetuximab causes acne-like rash and grade 3-4 infusion reactions, anaphylaxis, and cardiac arrest requiring pretreatment prophylaxis with the antihistamine diphenhydramine. Trastuzumab, still widely used, causes symptomatic cardiac dysfunction in 2-7% of patients, a condition that has been known for over a decade.

[0473] The primary measure for evaluation of potential antitumor activity was tumor status by RECIST. Thirty-one patients had at least one such assessment after baseline and were therefore included in the FAS. The four PRs and eight SDs among 31 heavily pretreated patients (RR 13%, DCR 39%) compare very favorably with response results in other phase II trials of targeted monotherapies approved for second-line or advanced-stage treatment.

[0474] The EGFR antagonist cetuximab achieved a 3% RR (plus 7% SD) in advanced EGFR-positive metastatic cancer (mCRC) measured after 8 weeks in a phase II study of 30 patients, most of whom had two or more metastatic sites and prior therapy. In a second study of 55 advanced-stage patients, cetuximab resulted in a 5% RR and an additional 11% SD measured after 8 weeks. Similar response rates were achieved in patients with EGFR-positive refractory mCRC, and cetuximab was later approved for this indication.

[0475] Sunitinib and erlotinib have been tested in patients with advanced GEC in various phase II trials involving a total of approximately 150 patients. DCR after 6 to 8 weeks varied between 16 and 39%, with response rates reported of 3 to 7%, respectively.

[0476] The phase II objective response rate for trastuzumab as second-line therapy in breast cancer was 11%, with a 9% ≥6-month SD. A 9% response rate was reported for erlotinib in pretreated lung cancer. Sorafenib achieved a 2%–18% RR in two phase II trials in renal cancer, and a 7% RR for temsirolimus was reported in a renal cancer trial. These targeted therapy compounds were subsequently further developed in combination with chemotherapy and registered in these indications.

[0477] IMAB362 is a safe and effective antibody. As expected from the excellent tissue specificity of the target surface molecule and the high-precision binding of the antibody, the investigational drug is well tolerated compared to other commercially available targeted therapies. Furthermore, evidence of clinical activity in several patients has been observed that is comparable to or better than the phase II results of other commercially available targeted therapies.

[0478] Example 5 IMAB362-induced nausea / vomiting IMAB362 has been shown to induce nausea / vomiting up to NCI-CTC grade 3. The symptomatology can be described as follows: (i) non-dose-dependent, (ii) acute onset, primarily within 5 minutes of infusion, may persist after the end of the infusion, (iii) begins with epigastric cramps and hypersalivation, (iv) vomiting can begin without warning, (v) is uncommon in patients who have undergone total gastrectomy, and (vi) the reaction occurs with the first infusion, while symptoms increase with each cycle.

[0479] The fact that these adverse reactions occur rarely in patients who have undergone total gastrectomy suggests that the underlying mechanism is an on-target effect. With IMAB362, vomiting is more frequent than nausea and often occurs without any preceding nausea. Onset can be both acute and delayed. We hypothesize that small amounts of IMAB362 bind to a tight junction epitope with limited access. This results in localized disruption of tight junctions and leakage of gastric acid into the submucosa. The resulting tissue reaction and spasm initiate the nausea / vomiting cascade.

[0480] Therefore, the recommended strategy is prophylaxis with effective antiemetics and protection of the gastric mucosa.

[0481] For example, patients should receive antiemetic prophylaxis before starting medication. For both preventive and therapeutic intervention, a combination of NK-1 receptor (e.g., aprepitant / Emend) and 5-HT3 receptor blockers (e.g., ondansetron / Zofran) is recommended, and can be expanded to additional compounds. Antiemetic administration is preferably carried out for at least the first 3 days of each cycle. Prophylactic administration of butylscopolamine / buscopan immediately before each IMAB362 infusion may be considered.

[0482] Any measures to protect the mucosa may also reduce gastric symptoms. In this regard, proton pump inhibitors and / or misoprostol may be used, for example, on days 1-2 or 3 of each cycle. Nonsteroidal anti-inflammatory drugs (NSAIDs) should not be used, but acetaminophen is acceptable. If acetaminophen is not effective for pain management, NSAIDs can be used if necessary to avoid opioid therapy. Patients taking NSAIDs are preferably treated with proton pump inhibitors and / or misoprostol.

[0483] Therefore, antiemetic prophylaxis and protection of the gastric mucosa may be initiated immediately prior to infusion of IMAB362. For example, the following combination may be administered, with intravenous application being preferred: NK-1 RA: e.g. aprepitant / Emend (150 mg IV) 5-HT3 RAs: e.g., palonosetron (0.25 mg IV), ondansetron / Zofran (8 mg IV), granisetron (3 mg IV) Butylscopolamine / Buscopan Proton pump inhibitor: Pantoprazole / Pantozol

[0484] Optionally, metoclopramide / MCP, lorazepam and / or atropine may also be administered.

[0485] IMAB362 is an antibody that relies heavily on an immunological mechanism of action, which can be impaired by immunosuppressive compounds. For this reason, steroids should be avoided in antiemetic prophylaxis and should only be used if other compounds have failed.

[0486] Furthermore, exposure to IMAB362 should be monitored carefully. For example, close monitoring is recommended for the first 15-30 minutes. If necessary, the infusion rate should be slowed (e.g., to 4 hours instead of 2 hours) and interruptions to the infusion should be included.

[0487] Antiemetic prophylaxis and gastric mucosal protection can be continued, for example, until day 3 of each cycle.

Claims

1. Use of an antibody capable of binding to claudin-18 splice variant 2 (CLDN18.2) for the preparation of a medicament for treating or preventing a cancer disease in a human patient, comprising: The cancer disease is characterized by cancer cells that express CLDN18.2; The treatment comprises administering the antibody at least 300 mg / m ２ in at least three doses spaced at least seven days apart, The antibody comprises an antibody heavy chain comprising a set of HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 17, and an antibody light chain comprising a set of LCDR1, LCDR2, and LCDR3 of the amino acid sequence represented by SEQ ID NO: 24, HCDR1 consists of amino acid positions 45-52 of SEQ ID NO: 17, HCDR2 consists of amino acid positions 70-77 of SEQ ID NO: 17, HCDR3 consists of amino acid positions 116-126 of SEQ ID NO: 17, LCDR1 consists of amino acid positions 47-58 of SEQ ID NO: 24, LCDR2 consists of amino acid positions 76-78 of SEQ ID NO: 24, and LCDR3 consists of amino acid positions 115-123 of SEQ ID NO:

24.

2. 2. The use of claim 1, wherein the serum level of the antibody in the patient is between 40 μg / ml and 700 μg / ml.

3. The use of claim 1 or 2, wherein at least 50% of the cancer cells of the human patient are CLDN18.2 positive and / or at least 40% of the cancer cells of the human patient are positive for surface expression of CLDN18.

2.

4. The use according to any one of claims 1 to 3, further comprising administering one or more selected from the group consisting of antiemetics, antispasmodics, parasympatholytics and agents that protect the gastric mucosa.

5. The use of any one of claims 1 to 4, wherein the treatment is a combination therapy with an agent that stabilizes or increases the expression of CLDN18.2, and the agent is selected from the group consisting of epirubicin, oxaliplatin, cisplatin, 5-fluorouracil, capecitabine, docetaxel, irinotecan, topotecan, and combinations of these agents.

6. The use according to any one of claims 1 to 5, wherein the antibody is administered by intravenous infusion.

7. 7. The use of claim 6, wherein the intravenous infusion lasts for 1 to 4 hours.

8. 8. The use according to any one of claims 1 to 7, wherein the antibody mediates cell death by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular lysis (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation.

9. 9. The use of any one of claims 1 to 8, wherein the antibody is an antibody selected from the group consisting of: (i) an antibody produced by and / or obtainable from the clone deposited under accession number DSM ACC2810; (ii) an antibody that is a chimerized or humanized form of the antibody included in (i); (iii) an antibody having the specificity of the antibody included in (i); and (iv) an antibody containing the variable region of the antibody included in (i) and having the specificity of the antibody included in (i).

10. 10. The use according to any one of claims 1 to 9, wherein the cancer is gastric cancer, esophageal cancer, gastroesophageal junction cancer, or gastroesophageal cancer.

11. 11. The use of any one of claims 1 to 10, wherein the human patient has received prior treatment with at least one agent selected from the group consisting of pyrimidine analogs, platinum compounds, epirubicin, docetaxel, and antidotes for antineoplastic drug treatment.

12. 12. The use according to any one of claims 1 to 11, wherein the human patient has an ECOG performance status between 0 and 1 and / or a Karnofsky index between 70 and 100%.

13. 13. The use according to any one of claims 1 to 12, wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO: 1.

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