Therapies containing antibodies against claudin 18.2 for the treatment of cancer

Abstract

This invention provides therapeutic methods for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, particularly cancerous diseases such as gastroesophageal cancer. [Solution] Administration of anti-CLDN18.2 antibody to human patients with gastroesophageal cancer at a dose of at least 1000 mg / m² 2 Data is presented demonstrating that the antibody is safe and well-tolerated up to a certain dose. Furthermore, data is presented demonstrating that the antibody is fully functional to exert an antitumor cell effect in these patients and that evidence of its antitumor activity has been obtained. In one embodiment, a method is provided for treating or preventing a cancerous disease, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered to provide a serum level of at least 40 μg / ml.

Description

[Technical Field]

[0001] Cancers of the stomach and esophagus (gastroesophageal cancer: GE) are among the most malignant diseases with the greatest unmet medical needs. Gastric cancer is the second leading cause of cancer death worldwide. The incidence of esophageal cancer has increased over the past few decades, coinciding with changes in histological types and primary tumor sites. Esophageal adenocarcinoma is now more common than squamous cell carcinoma in the United States and Western Europe, with the majority of tumors located in the distal esophagus. The overall 5-year survival rate for GE cancer is 20–25%, despite the aggressive implementation of established standard treatments that are linked to 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 skeleton combined with a third compound (e.g., taxane or anthracycline). Even then, the best-case scenario is a progression-free survival of 5–7 months and an overall survival of 9–11 months.

[0003] The lack of significant benefit from various next-generation combination chemotherapy regimens for these cancers has driven research towards the use of targeted therapies. Recently, trastuzumab was approved for Her2 / neu-positive gastroesophageal cancer. However, since only about 20% of patients express the target and are not eligible for this treatment, the medical need remains high. [Background technology]

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

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

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

[0007] The chimeric IgG1 antibody against CLDN18.2, IMAB362, 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 (CLDN18.1) of claudin 18. IMAB362 exhibits precise tumor cell specificity and integrates four independent, highly potent mechanisms of action. After target binding, IMAB362 mediates cell death and direct inhibition of proliferation by inducing apoptosis induced by cross-linking of the target at ADCC, CDC, and the tumor cell surface. Therefore, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, both in vitro and in vivo. When treated with IMAB362, mice carrying CLDN18.2-positive cancer cell lines benefit from survival, with up to 40% of mice showing tumor regression.

[0008] The toxicity and PK / TK profile of IMAB362 have been thoroughly studied in mice and cynomolgus monkeys, including dose-finding studies, a 28-day repeated-dose toxicity study in cynomolgus monkeys, and a 3-month repeated-dose toxicity study in mice. Repeated administration of IMAB362 iv is well tolerated in both mice (maximum treatment period of 3 months with weekly administration, maximum dose level of 400 mg / kg) and cynomolgus monkeys (maximum of 5 weeks with weekly administration, up to 100 mg / kg). No signs of systemic or local toxicity are induced. In particular, gastric toxicity was not observed in any toxicity studies. 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 tissues lacking a target. The in vivo distribution in mice suggests that the absence of gastric toxicity is likely due to compartmentalization of tight junctions in the luminal region of healthy gastric epithelium, which significantly reduces the accessibility of the IMAB362 epitope. This compartmentalization is lost during malignant transformation, making the epitope druggable by IMAB362. [Overview of the project] [Problems that the invention aims to solve]

[0009] In this specification, the inventors describe the administration of an anti-CLDN18.2 antibody such as IMAB362 to human patients with gastroesophageal cancer at a dose of at least 1000 mg / m². 2 We present data demonstrating that the antibody is safe and well-tolerated up to that dose. Furthermore, the data presented herein demonstrate that the antibody is fully functional to exert antitumor cell activity in these patients and that evidence of its antitumor activity has been obtained. [Means for solving the problem]

[0010] The present invention generally provides therapeutic methods for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancerous diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, as well as their metastases, particularly gastric cancer metastases such as Krukenberg tumor, peritoneal metastases, and lymph node metastases. Particularly preferred cancerous diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary.

[0011] In a first embodiment, the present invention provides a method for treating or preventing cancer, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered to provide a serum level of at least 40 μg / ml. In a different embodiment, 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 a different embodiment, 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” means 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 at least 300 mg / m³ 2 For example, at least 600 mg / m² 2 Preferably 1500 mg / m² 2 Up to 1200 mg / m² 2 Up to 1000 mg / m² 2 This includes administering a dose of antibodies up to [a certain level].

[0012] In a second aspect, the present invention provides a method of treating or preventing a cancer disease, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered at a dose of at least 300 mg / m 2 , for example at least 600 mg / m 2 , preferably 1500 mg / m 2 up to, 1200 mg / m 2 up to or 1000 mg / m 2 up to.

[0013] In a third aspect, the present invention provides a method of treating or preventing a cancer disease, comprising administering to a patient an antibody having the ability to bind 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 with respect to surface expression of CLDN18.2. In this aspect, the present invention also provides a method of treating or preventing a cancer disease, comprising a. identifying a patient showing at least 50%, preferably 60%, 70%, 80% or 90% CLDN18.2 positive cancer cells and / or at least 40%, preferably 50% or 60% cancer cells positive with respect to surface expression of CLDN18.2, and b. administering to the patient an antibody having the ability to bind 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 with respect to surface expression of CLDN18.2.

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

[0015] In a fourth embodiment, the present invention provides a method for achieving a stable disease in a cancer patient, comprising administering to the patient an antibody having the ability to bind to CLDN18.2. In one embodiment, the stable disease is achieved for at least two months, at least three months, or at least six months.

[0016] In one embodiment of any of the methods 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 cancer, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered in multiple doses.

[0018] According to the present invention, when administering antibodies in multiple doses, the antibodies are preferably administered in at least 3, 4, 5, 6, 7, 8, 9, or 10 doses, preferably 30, 25, 20, 15, or 10 doses. The antibody administration is preferably carried out at time intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days. The antibody administration is preferably carried out at time intervals of 7 to 30 days, 10 to 20 days, or preferably about 14 days.

[0019] In one embodiment of the third, fourth, or fifth aspect of the method, the antibody is administered to provide a serum level of at least 40 μg / ml. In a different embodiment, 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 a different embodiment, 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 at least 300 mg / m² 2 For example, at least 600 mg / m² 2 Preferably 1500 mg / m² 2 Up to 1200 mg / m² 2 Up to 1000 mg / m² 2 This includes administering a dose of antibodies up to [a certain level].

[0020] In one embodiment of any of the above-described methods, the method further comprises administering one or more substances selected from the group consisting of antiemetics, antispasmodics, parasympathetic blockers, and gastric mucosal protective agents.

[0021] In a sixth aspect, the present invention provides a method for treating or preventing cancer, comprising administering to a patient one or more antibodies having the ability to bind to CLDN18.2, and one or more selected from the group consisting of antiemetics, antispasmodics, parasympathetic blockers, and gastric mucosal protective agents.

[0022] If the method of the present invention involves administering one or more substances selected from the group consisting of antiemetics, antispasmodics, parasympathetic blockers, and gastric mucosal protective agents, the method in various embodiments includes administering (i) an antiemetic and an antispasmodic, (ii) an antispasmodic and a gastric mucosal protective agent, (iii) an antiemetic and a gastric mucosal protective agent, or (iv) an antiemetic, an antispasmodic, and a gastric mucosal protective agent.

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

[0024] In one embodiment, butylscopolamine (Buscopan) is used as the antispasmodic.

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

[0026] In one embodiment, the method of the present invention involves administering to a 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 two or more combinations thereof, an antispasmodic such as butylscopolamine (Buscopan), and a proton pump inhibitor such as pantoprazole (e.g., Pantozol).

[0027] In one embodiment of any of the methods described above, the antibody is administered by IV infusion. In one embodiment, the IV infusion lasts for 1 to 4 hours, preferably about 2 hours.

[0028] In a sixth aspect, the present invention provides a method for determining the responsiveness of a cancer patient to the treatment or prevention of 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, the one or more markers being 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, biological samples such as blood can be taken from the patient before and after administration of the antibody capable of binding to CLDN18.2, for example, after administration of a single dose of the antibody, to establish the levels of one or more markers. Multiple samples can be taken from the same tissue, and the mean levels can be determined to account for possible variations in those levels. The levels of one or more markers after antibody administration are compared to the levels measured before administration. Thus, the effect of the antibody on the patient can be identified by the desired change in marker levels after administration of the antibody capable of binding to CLDN18.2. If a patient shows a desirable change in marker levels after administration of an antibody capable of binding to CLDN18.2, treatment with an antibody capable of binding to CLDN18.2 may be initiated.

[0029] In one embodiment, the level is measured in blood, plasma, or serum.

[0030] In one embodiment, one or more markers are selected from the group consisting of CA125, CA15-3, CA19-9, CEA, IL-2, IL-15, IFNγ, and TNFα, and a decrease in the level of at least one marker after antibody administration indicates that the patient is responsive to the treatment or prevention of cancer.

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

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

[0033] In this embodiment, before administering an antibody capable of binding to CLDN18.2, a biological sample, such as a tumor sample (e.g., tumor biopsy), may be taken from the patient to establish the level of CLDN18.2-positive cancer cells. Multiple samples may be taken, and the average level may be determined to account for possible variations in those levels. If the patient has a desirable level of CLDN18.2-positive cancer cells, an 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% of CLDN18.2-positive cancer cells indicates that the patient is fit for treatment or prevention of cancer. 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 CLDN18.2 surface expression indicates that the patient is fit for treatment or prevention of cancer.

[0035] Antibodies capable of binding to CLDN18.2 can bind to the native 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 the following: complement-dependent cell-mediated lysis (CDC), antibody-dependent cell-mediated lysis (ADCC), 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 a fragment of an antibody. In one embodiment, an antibody capable of binding to CLDN18.2 is an antibody selected from the group consisting of (i) an antibody produced by and / or available from a clone deposited under accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810, (ii) an antibody that is a chimeric or humanized form of the antibody contained in (i), (iii) an antibody having the specificity of the antibody contained in (i), and (iv) an antibody containing an antigen-binding portion or antigen-binding site, particularly a variable region, of the antibody contained in (i), and preferably having the specificity of the antibody contained in (i). In one embodiment, the antibody is conjugated to a therapeutic agent, such as a toxin, radioisotope, drug, or cytotoxic agent.

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

[0037] In one embodiment, the cancerous 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 cancerous disease may be Krukenberg tumor, peritoneal metastases, and / or lymph node metastases. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer. In a particularly preferred embodiment, the cancer is gastroesophageal cancer, e.g., metastatic, antitherapy-treated, or recurrent advanced gastroesophageal cancer. The patient may be a HER2 / neu-negative patient or a patient with a HER2 / neu-positive condition who is not eligible 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 antitumor drug treatment (e.g., calcium folinic acid and / or folinic acid). In one embodiment, the patient has an ECOG performance status between 0 and 1 and / or a Karnovski index 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 Sequence ID No. 1.

[0039] The present invention also provides antibodies having the ability to bind to the active substance described herein, such as CLDN18.2, for use in the methods described herein.

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

[0041] [Figure 1] Average blood concentration of IMAB362 during the trial. [Figure 2A] (Figure 2) ADCC activity of patient PBMCs. (Figure 2A) PBMCs were purified from blood samples of 6 patients on day 7 (white square) or day 14 (black square) after IMAB362 administration. Specific lysis rate of NUGC-4 gastric cancer target cells expressing CLDN18.2, obtained after 24 hours of addition of IMAB362 31.63 μg / ml and PBMCs from healthy donors or patients (E:T=20:1). [Figure 2B] NUGC-4 cells obtained 24 hours after the addition of PBMCs from various patients showed IMAB362 concentration-dependent specific lysis (graphs show mean ± standard deviation; p-values ​​were calculated using an independent t-test). [Figure 2C] ADCC response curves of healthy control PBMCs after the addition of gradually increasing concentrations of IMAB362. The assay was performed in parallel with individual ADCC analyses of patient PBMCs. [Figure 2D] ADCC response curves of patient PBMCs after the addition of gradually increasing concentrations of IMAB362 (sufficient PBMCs were obtained for patient 0202 to create a curve). [Figure 2E] The semi-maximal mortality rate for all patients and healthy donors was calculated using the built-in nonlinear regression analysis tool in GraphPad Prism software. [Figure 3]The 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 spiked with 0.3–10 μg / ml IMAB362 (positive control). Hi: Thermo-inactivated human serum pool spiked with 10 μg / ml IMAB362 (negative control). Number of patients is shown. Error bars: ± standard deviation. [Figure 4A] (Figure 4) The ability of patient complement components to interact with IMAB362 administered intravenously over time. A standardized CDC assay was performed by adjusting the IMAB362 concentration in each sample to 0.5 μg / ml using pre-infusion serum from each patient (dilution factor 10 to 680). (Figure 4A) The CDC assay was performed as shown in Figure 3. [Figure 4B] The CDC assay was performed as shown in Figure 3. [Figure 4C] Each dot represents the measurement result for one patient. White square: 0.5 μg / ml human serum IMAB362. The p-value was obtained using a paired t-test. Error bars: ± standard deviation. [Figure 5A] Dynamics of cytotoxicity induced by IV-administered circulating IMAB362. NUGC-4 target cells, healthy donor PBMCs (E:T=40:1), and patient serum samples (25% v / v) were used as antibody and complement sources in a total cytotoxicity assay to measure overall cytotoxic activity. Serum samples were collected from each patient 1, 7, 14, and 28–32 days after IMAB362 administration. Patients were treated with escalating doses of IMAB362 (33–1000 mg / m2). Antibody concentrations present in the assay are shown below each bar. HSC: Human serum pooled control (EC80–100) spiked with 200.0 μg / ml fresh IMAB362. PSC: Pre-infusion serum control from patients spiked with 200.0 μg / ml fresh IMAB362. na: Not available. [Figure 5B]Dynamics of cytotoxicity induced by IV-administered circulating IMAB362. NUGC-4 target cells, healthy donor PBMCs (E:T=40:1), and patient serum samples (25% v / v) were used as antibody and complement sources in a total cytotoxicity assay to measure overall cytotoxic activity. Serum samples were collected from each patient 1, 7, 14, and 28–32 days after IMAB362 administration. Patients were treated with escalating doses of IMAB362 (33–1000 mg / m2). Antibody concentrations present in the assay are shown below each bar. HSC: Human serum pooled control (EC80–100) spiked with 200.0 μg / ml fresh IMAB362. PSC: Pre-infusion serum control from patients spiked with 200.0 μg / ml fresh IMAB362. na: Not available. [Figure 6A] Dynamics of ADCC activity of IMAB362 in thermoactivated patient serum. The assay was performed as described in the previous figure, except that patient complement was thermoactivated (56°C, 30 minutes), ADCC activity was detected (black and gray bar portions), and the additional effects of serum components (white bar portions) were calculated. [Figure 6B] Dynamics of ADCC activity of IMAB362 in thermoactivated patient serum. The assay was performed as described in the previous figure, except that patient complement was thermoactivated (56°C, 30 minutes), ADCC activity was detected (black and gray bar portions), and the additional effects of serum components (white bar portions) were 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 33–1000 mg / m2. The antibody concentrations present in each assay are shown below each bar. HSC: Pooled healthy human serum control spiked with the indicated tapering concentrations of IMAB362. PC: Positive control (pre-infusion patient 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 33–1000 mg / m2. The antibody concentrations present in each assay are shown below each bar. HSC: Pooled healthy human serum control spiked with the indicated tapering concentrations of IMAB362. PC: Positive control (pre-infusion patient serum spiked with 10 μg / ml IMAB362). [Figure 8] Pharmacokinetic results of repeated infusions of IMAB362 in patients. Mean ± sd serum concentrations of IMAB362 (μg / ml) in 4 patients treated with repeated infusions of 300 mg / m2 (Cohort 1, left figure) and up to 30 patients treated with repeated infusions of 600 mg / m2 (30 patients for the first infusion, 12 patients for the fifth infusion) (Cohorts 2 and 3 combined, right figure). Arrows indicate IMAB362 infusions. The first infusion was performed on day 0. [Figure 9] Patient progression-free survival in the complete analysis set (FAS). [Figure 10] Patient progression-free survival time (n=20) in each protocol (PP) set. [Modes for carrying out the invention]

[0042] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, and that the scope of the present invention is limited only by the accompanying 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. These elements are listed together with specific embodiments, but it should be understood that they may be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the expressly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the expressly described embodiments with many of the disclosed elements and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by this description unless specifically indicated in the context.

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

[0045] The implementation of this invention shall, unless otherwise specified, be based on the literature in the art (e.g., Molecular Cloning: A Laboratory Manual, 2 nd Conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology are used, as described in Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0046] Throughout this specification and the following claims, unless specifically required by context, the word “includes” and variations such as “including” are understood to mean the inclusion of the member, integer or process or group of members, integers or processes described, but not the exclusion of any other member, integer or process or group of members, integers or processes; however, in some embodiments, such other members, integers or processes or groups of members, integers or processes may be excluded, i.e., the subject matter lies in the inclusion of the member, integer or process or group of members, integers or processes described. The terms “one” and “it” and similar references used in connection with the description of the invention (particularly in connection with the claims) should be interpreted as including both singular and plural unless specifically indicated herein or unless it is clearly inconsistent with the context. Enumerations of ranges of values ​​in this specification are intended simply as a simplified way of referring individually to each separate value belonging to that range. Unless specifically indicated herein, each individual value is incorporated herein as if it were individually enumerated. All methods described herein may be carried out in any suitable order, unless otherwise specifically indicated herein or if it is clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., "etc.") is intended solely to better illustrate the invention and not to limit the scope of the invention or the claims. No language herein should be construed as indicating that any non-claimed element is essential for carrying out the invention.

[0047] Throughout this specification, several sources are referenced. Each of the sources referenced herein (including all patents, patent applications, academic publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosures for the sake of prior art.

[0048] The term "CLDN18" refers to claudin 18 and includes 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 refers to human CLDN18.2, and more particularly to proteins comprising an amino acid sequence according to sequence number 1 of the sequence listing or a variant of said amino acid sequence.

[0050] The term "CLDN18.1" preferably refers to human CLDN18.1, and more particularly to proteins comprising an amino acid sequence according to sequence number 2 of the sequence listing or a variant of said amino acid sequence.

[0051] The term “mutant” in this invention refers, in particular, to mutants, splice mutants, conformational mutants, isoforms, allelic mutants, species mutants, and species homologs, especially those occurring in nature. Allelic mutants concern changes in the normal sequence of a gene, but their significance is often unclear. Complete gene sequencing often identifies numerous allelic mutants for a given gene. A species homolog is a nucleic acid sequence or amino acid sequence that originates from a different species than that of a given nucleic acid sequence or amino acid sequence. The term “mutant” encompasses any post-translational modification mutant and conformational mutant.

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

[0053] The term "cell surface" is used according to its usual meaning in this field, and therefore includes the area outside the cell that is accessible by binding of proteins and other molecules.

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

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

[0056] According to the present invention, CLDN18.2 is expressed in cells if its expression level is at least twice, preferably 10 times, 100 times, 1000 times, or 10000 times, higher than its expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in cells if its expression level is above the detection limit and / or high enough to allow binding by a CLDN18.2-specific antibody added to the cells. Preferably, the 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, and in particular the forms of cancer described herein. References herein to cancer or specific forms of cancer also include its metastases. In preferred embodiments, the disease to be treated according to this application includes cells expressing CLDN18.2.

[0058] The phrase "disease associated with cells expressing CLDN18.2" or similar expressions means, according to the present invention, that CLDN18.2 is expressed in cells of diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of diseased tissue or organ is increased compared to its state in healthy tissue or organ. Increase means an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more. In one embodiment, the expression is observed only in diseased tissue, and the expression in healthy tissue is suppressed. According to the present invention, cancer is an example of a disease associated with cells expressing CLDN18.2. Furthermore, according to the present invention, cancer is preferably characterized by cancer cells expressing CLDN18.2.

[0059] As used herein, “cancer disease” or “cancer” encompasses diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. The three malignant characteristics of cancer—uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body via lymph nodes or the blood)—distinguish cancer from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, but some, like leukemia, do not. “Cancer cells” means abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow after the stimulus that initiated new growth has ceased. Preferably, “cancer disease” is characterized by cells expressing CLDN18.2, and cancer cells express CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, preferably cancer cells of the cancers described herein.

[0060] According to the present invention, the terms “tumor” or “tumor disease” preferably refer to the abnormal proliferation of cells (called neoplastic cells, tumor-forming cells, or tumor cells) that form swelling or lesions. “Tumor cells” means abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow even after the stimulus that initiated new growth has ceased. Tumors exhibit a partial or complete absence of structural mechanisms and functional coordination with normal tissue, and usually form a distinct tissue mass, which may be benign, premalignant, or malignant.

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

[0062] Adenocarcinoma is a type of cancer that originates from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissues include skin, glands, and various other tissues that line the body's cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they possess 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 ones may not. By staining cells from a biopsy, pathologists determine whether a tumor is adenocarcinoma or some other type of cancer. Adenocarcinoma can occur in many tissues of the body due to the ubiquity of glands. Not all glands secrete the same substance, but as long as there is exocrine function to the cells, it is considered a gland, and therefore its malignant form is named adenocarcinoma. Malignant adenocarcinoma invades other tissues and often metastasizes if there is enough time for it to do so. Ovarian adenocarcinoma is the most common type of ovarian cancer. Ovarian adenocarcinomas include serous and mucinous adenocarcinomas, clear cell adenocarcinomas, and endometrioid adenocarcinomas.

[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 very complex process and depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then, after being carried by the blood, invasion of the target organ. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, because tumor cells or components may remain and exhibit metastatic potential. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis" relating to metastasis far from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention refers to lymph node metastasis. One particular form of metastasis treatable with the therapies of the present invention is metastasis arising from gastric cancer as the primary site. In preferred embodiments, such gastric cancer metastases are Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0064] Krukenberg tumor is a rare metastatic tumor of the ovary, accounting for 1%–2% of all ovarian tumors. The prognosis for Krukenberg tumor is still very poor, and there is no established treatment for it. 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 (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from cancers 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 an average age of 45, making them relatively young for patients with metastatic cancer. This young age distribution can be partly linked to the high incidence of signet-ring cell adenocarcinoma of the stomach in young women. The symptoms generally present are usually associated with ovarian dysfunction, most commonly abdominal pain and distension (primarily due to a large ovarian mass, usually bilateral). The remaining patients have nonspecific gastrointestinal symptoms or are asymptomatic. In addition, Krukenberg tumors are reported to be linked to masculinization resulting from hormone production by the ovarian bronchus. 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 swollen and have a raised contour. The surface of sections is yellow or white; they are usually solid but sometimes cystic. Importantly, the capsule surface of ovaries with Krukenberg tumors is typically smooth and free of adhesions or peritoneal deposits. It should be noted that other metastatic tumors to the ovaries tend to be associated with surface implants. This may explain why the macroscopic morphology of a Krukenberg tumor can sometimes superficially resemble that of a primary ovarian tumor. However, the bilaterality of Krukenberg tumors is consistent with their metastatic nature.

[0067] Patients with Krukenberg tumor have a significantly higher overall mortality rate. The majority of patients die within two years (mean survival time, 14 months). Several studies have shown that the prognosis is poor if the primary tumor is identified after ovarian metastases are discovered, and even worse if the primary tumor remains unidentified.

[0068] "To treat" means administering a compound or composition or combination of compounds or compositions to a subject in order to prevent or eliminate a disease, including by reducing the size or number of tumors in the subject; to stop or slow the progression of a disease in the subject; to prevent or delay the onset of a new disease in the subject; to reduce the frequency or severity of symptoms and / or recurrences in a subject who currently has or has previously had the disease; and / or to extend, i.e., increase, the lifespan of the subject.

[0069] In particular, the term “treatment of disease” includes curing, shortening the duration of, improving, preventing, slowing or halting the progression or worsening of a disease or its symptoms, or preventing or delaying its onset.

[0070] The term "patient" according to the present invention means a subject for treatment, particularly a disease subject, including humans, non-human primates, or other animals, especially mammals, such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents, such as mice and rats. In a particularly preferred embodiment, the patient is human.

[0071] According to the present invention, an antibody having the ability to bind to CLDN18.2 can be administered in combination with an active agent that stabilizes or increases the expression of CLDN18.2, i.e., simultaneously with the active agent, before the active agent and / or after the active agent.

[0072] The term "active agent that stabilizes or increases the expression of CLDN18.2" refers to an active agent or combination of active agents that, when provided to cells, causes an increase in the RNA and / or protein levels of CLDN18.2, preferably an increase in the CLDN18.2 protein level on the cell surface, compared to a situation in which the cells are not provided with the active agent or combination of active agents. Preferably, the cells are cancer cells, in particular cancer cells that express CLDN18.2, for example, the cancerous cells described herein. The term "active agent that stabilizes or increases the expression of CLDN18.2" refers, in particular, to an active agent or combination of active agents that, when provided to cells, causes a higher density of CLDN18.2 on the cell surface compared to a situation in which the cells are not provided with the active agent or combination of active agents. "Stabilizing the expression of CLDN18.2" includes situations in which an active agent or combination of active agents prevents or reduces a decrease in the expression of CLDN18.2, for example, if the active agent or combination of active agents is not provided, the expression of CLDN18.2 is thought to decrease, and the provision of the active agent or combination of active agents prevents or reduces the decrease in the expression of CLDN18.2. "Increasing the expression of CLDN18.2" includes situations in which an active agent or combination of active agents increases the expression of CLDN18.2, for example, if the active agent or combination of active agents is not provided, the expression of CLDN18.2 is thought to decrease, remain essentially constant, or increase, and the provision of the active agent or combination of active agents increases the expression of CLDN18.2 compared to a situation in which the active agent or combination of active agents is not provided, and the resulting expression is higher compared to a situation in which the expression of CLDN18.2 would decrease, remain essentially constant, or increase if the active agent or combination of active agents were not provided.

[0073] According to the present invention, the term "substance that stabilizes or increases the expression of CLDN18.2" includes chemotherapeutic agents or combinations of chemotherapeutic agents, such as cell proliferation inhibitors. A chemotherapeutic agent may affect a cell in one of the following ways: (1) damaging the cell's DNA so that the cell can no longer replicate; (2) inhibiting the synthesis of new DNA strands so that cell replication is impossible; or (3) halting the mitotic process of the cell so that the cell cannot divide into two cells.

[0074] According to the present invention, the term “active agent for stabilizing or increasing the expression of CLDN18.2” preferably relates to an active agent or a combination of active agents, such as a cell growth inhibitory compound or a combination of cell growth inhibitory compounds, provided to cells, particularly cancer cells, to cause the cells to arrest or accumulate in one or more phases of the cell cycle, preferably other than the G1 and G0 phases, preferably one or more phases of the cell cycle other than the G1 phase, preferably the G2 or S phase of the cell cycle, for example, the G1 / G2 phase, S / G2 phase, G2 phase or S phase, one or more 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 the aforementioned one or more phases of the cell cycle increases. Each cell goes through a cycle that consists of four phases in order to replicate itself. The first phase, called G1, is the stage in which the cell prepares to replicate its chromosomes. The second phase is called S, and in this phase DNA synthesis occurs and DNA is replicated. The next phase is the G2 phase, where RNA and proteins are replicated. The final stage is the M phase, which is the actual stage of cell division. In this final stage, the replicated DNA and RNA separate and move to separate ends of the cell, causing the cell to actually divide into two identical functional cells. Chemotherapy agents, which are DNA damaging agents, typically cause an accumulation of cells in the G1 and / or G2 phases. Chemotherapy agents that block cell proliferation by interfering with DNA synthesis, such as antimetabolites, typically cause an accumulation of cells in the S phase. Examples of these agents are 6-mercaptopurine and 5-fluorouracil.

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

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

[0077] Under certain circumstances, cancer cells can enter a lethal stress pathway that leads to the release of spatiotemporalally defined signaling combinations 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 are sensed by innate immune effectors such as dendritic cells, initiating the release of signals that trigger cognitive immune responses, including CD8+ T cell and IFN-γ signaling, so that tumor cell death can induce an effective anti-cancer immune response. These signals include preapoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, preapoptotic secretion of ATP, and postapoptotic release of the nuclear protein HMGB1. Considered together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatins, and gamma irradiation can induce all the signals that define ICD, but cisplatin, for example, lacks the ability to induce CRT transposition from the ER to the surface of dead cells, a process that requires ER stress, and requires supplementation with thapsigargin, an ER stress inducer.

[0078] According to the present invention, the term “immunogenic cell death-inducing agent” 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 produces a tumor-specific immune response. In particular, an immunogenic cell death-inducing agent when provided to cells induces the cells to release a spatiotemporally defined combination of signals, including, in particular, preapoptotic exposure to calreticulin (CRT), an endoplasmic reticulum (ER) chaperone on the cell surface, preapoptotic secretion of ATP, and postapoptotic release of the nucleoprotein HMGB1.

[0079] According to the present invention, the term "substance that induces immunogenic cell death" includes anthracyclines and oxaliplatins.

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

[0081] Anthracyclines preferably produce one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by intercalating between base pairs of DNA / RNA strands, thus hindering the replication of rapidly proliferating cancer cells. 2. Inhibit topoisomerase II enzyme, preventing the relaxation of supercoiled DNA, 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" preferably relates to a substance that induces apoptosis by inhibiting the recombination of DNA by topoisomerase II, and preferably to an anticancer agent.

[0083] Preferably, according to the present invention, the term "anthracycline" generally refers to the following ring structure: [ka] This refers to a class of compounds that possess and include analogues and derivatives thereof, pharmaceutically acceptable salts, hydrates, esters, conjugates, and prodrugs.

[0084] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pirarubicin, barurubicin, N-trifluoro-acetyldoxorubicin-14-valerate, acrasinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and acrasinomycin A (acrarubicin). Mitoxantrone is a member of the anthracendione class of compounds, which are anthracycline analogs that lack the sugar moiety of anthracycline 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] In the formula, R1 is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and OMe, R3 is selected from the group consisting of H and OH, and R4 is selected from the group consisting of H and OH.

[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 yet another embodiment, R1 is OH, R2 is OMe, R3 is OH, and R4 is H. In yet another embodiment, R1 is H, R2 is H, R3 is H, and R4 is OH.

[0087] In connection with the present invention, epirubicin is particularly intended as an anthracycline. Epirubicin is given by the following formula: [ka] It is an anthracycline drug that has the following characteristics and is marketed under the trade names Ellence in the United States and Pharmorubicin or Epirubicin 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 considered to cause fewer side effects and is therefore preferred over doxorubicin, the most common anthracycline, in some chemotherapy regimens.

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

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

[0090] The term "carboplatin" is derived from the following formula: [ka] This refers to the compound cis-diammine(1,1-cyclobutanedicarboxylate)platinum(II).

[0091] The term "oxaliplatin" is derived from the following formula: [ka] This refers to a compound that is a platinum compound complexed with a diaminocyclohexane support ligand.

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

[0093] The term "nucleoside analog" refers to structural analogs of nucleosides, encompassing both purine analogs 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) (marketed under the trademark names Adrucil, Carac, Efudix, Efudex, and Fluoroplex) is defined by the following formula: [ka] It is a compound that is a pyrimidine analog of [the compound in question].

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

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

[0097] In particular, this term refers to the compound pentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidine-4-yl]carbamate.

[0098] Taxanes are a class of diterpene compounds initially derived from natural sources such as yew plants, but some are now artificially synthesized. The primary mechanism of action of taxane-class drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Examples of taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0099] According to the present invention, the term "docetaxel" is defined by the following formula: [ka] This refers to compounds that possess [a certain characteristic].

[0100] According to the present invention, the term "paclitaxel" is defined by the following formula: [ka] This refers to compounds that possess [a certain characteristic].

[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]indolidino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog" refers to the following structure: [ka] This refers to compounds that contain [the specified element].

[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 is represented by the following formula: [ka] It is a semi-synthetic analog of the natural alkaloid camptothecin, which possesses [specific properties].

[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]-indolidino[1,2-b]quinoline-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0105] Topotecan is, 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]indolidino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0107] According to the present invention, the active agent that stabilizes or increases the expression of CLDN18.2 may be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, or a combination of agents, for example, a combination of agents established for use in cancer treatment. Such a combination of agents may be a combination of agents used in chemotherapy, and may be a combination of agents 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 combination of drugs used in EOX chemotherapy includes epirubicin, oxaliplatin, and capecitabine. The combination of drugs used in ECF chemotherapy includes epirubicin, cisplatin, and 5-fluorouracil. The combination of drugs used in ECX chemotherapy includes epirubicin, cisplatin, and capecitabine. The combination of drugs used in EOF chemotherapy includes epirubicin, oxaliplatin, and 5-fluorouracil.

[0109] Epirubicin is usually administered at 50 mg / m². 2 Cisplatin is 60 mg / m² 2 Oxaliplatin is 130 mg / m² 2 It is administered at a dose of 200 mg / m², with 5-fluorouracil being 200 mg / m². 2 Capecitabine is administered by continuous intravenous infusion / day and orally at a dose of 625 mg / m². 2 It is administered twice a day in a 3-week cycle for a total of 8 doses.

[0110] The combination of drugs used in FLO chemotherapy is 5-fluorouracil, folinic acid, and oxaliplatin (usually 2,600 mg / m² of 5-fluorouracil). 2 Inject this for 24 hours, folinic acid 200 mg / m² 2 and oxaliplatin 85 mg / m² 2 (including every two weeks)

[0111] FOLFOX is a chemotherapy regimen consisting of folinic acid (leucovorin), 5-fluorouracil, and oxaliplatin. The recommended dose schedule, administered every two weeks, is as follows: Day 1: Oxaliplatin 85 mg / m² 2 IV infusion and leucovorin 200 mg / m² 2 IV infusion of [unclear], followed by 5-FU 400 mg / m². 2 IV bolus, followed by 5-FU 600 mg / m² 2 IV infusion as a 22-hour continuous infusion; Day 2: Leucovorin 200 mg / m² 2 IV infusion over 120 minutes, followed by 5-FU 400 mg / m² 2An IV bolus is administered over 2-4 minutes, followed by 5-FU 600 mg / m². 2 IV infusion as a 22-hour continuous infusion.

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

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

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

[0115] The terms "folic acid" or "leucovorin" refer to compounds useful in synergistic combinations with the chemotherapeutic agent 5-fluorouracil. Folic acid is given by the following formula: [ka] It holds.

[0116] In particular, this term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridine-6-yl)methylamino]benzoyl]aminopentanedioic acid.

[0117] According to the present invention, an antibody having the ability to bind to CLDN18.2 can be administered in combination with a γδ T cell stimulating agent, i.e., simultaneously with the stimulating agent, before and / or after the stimulating agent.

[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. Most T cells have a TCR consisting of two glycoprotein chains called the α-TCR chain and the β-TCR chain. In contrast, γδ T cells have a TCR composed of one γ chain and one δ chain. This group of T cells is usually much rarer than αβ T cells. Human γδ T cells play a crucial role in stress surveillance responses such as infection and autoimmunity. Transformation-inducible changes in tumors have also been suggested to produce γδ T cell-mediated stress surveillance responses, enhancing anti-tumor immunity. Importantly, after antigen engagement, activated γδ T cells at the lesion site provide chemokines that mediate the recruitment of cytokines (e.g., INFγ, TNFα) and / or other effector cells, exhibiting immediate effector functions such as cytotoxicity (via cell 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 they increase dramatically in many acute infections, such as tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria, where they can outnumber all other lymphocytes within days. Therefore, they are presumed to play an early and essential role in sensing the "danger" of invading pathogens.

[0120] γδ T cells respond to small non-peptide phosphorylated antigens (phosphoantigens), such as pyrophosphates 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 that inhibit farnesyl pyrophosphate synthase (FPPS), an enzyme in the mevalonate pathway. Among many, zoledronic acid (ZA, zoledronate, Zometa®, Novartis) is a representative such aminobisphosphonate and is already clinically administered to patients for the treatment of osteoporosis and metastatic bone disease. After treatment of PBMCs in vitro, ZA is taken up particularly by monocytes. IPP accumulates in monocytes and differentiates into antigen-presenting cells that stimulate γδ T cell expression. In this situation, the addition of interleukin-2 (IL-2) as a proliferation and survival factor for activated γδ T cells is preferred. Finally, certain alkylated amines have been described as activating Vγ9Vδ2 T cells in vitro, but only at millimolar concentrations.

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

[0122] One specific group of compounds that stimulate γδ T cells are bisphosphonates, particularly 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 their analogues and derivatives, pharmaceutically acceptable salts, hydrates, esters, conjugates, and prodrugs: [1-Hydroxy-2-(1H-imidazole-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, e.g., zoledronate; (Dichlorophosphonomethyl)phosphonic acid, e.g., clodronate; {1-Hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronate, e.g., ibandronate; (3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic acid, e.g., pamidronate; (1-hydroxy-1-phosphono-2-pyridine-3-ylethyl)phosphonic acid, risedronate, e.g., risedronate; (1-hydroxy-2-imidazo[1,2-a]pyridine-3-yl-1-phosphonoethyl)phosphonic acid, minodronate; [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid; [4-amino-1-hydroxy-1-(hydroxy-oxide-phosphoryl)-butyl]phosphonic acid, alendronate, 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), chyrdronic acid.

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

[0125] In one particularly preferred embodiment, the γδ T cell stimulating agent according to the present 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 protein that attracts lymphocytes and is part of the body's natural response to microbial infections and to distinguish between foreign (non-self) and self. IL-2 mediates its action by binding to IL-2 receptors expressed by lymphocytes.

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

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

[0129] 5-HT3 receptor antagonists block serotonin receptors in the central nervous system and gastrointestinal tract. Examples include, but are not limited to, ondansetron (Zofran), which can be administered as an oral tablet, orally dissolvable tablet, or by injection; drasetron (Anzemet), which can be administered as a tablet or by injection; granisetron (Kytril, Sancuso), which can be administered as a tablet (Kytril), oral solution (Kytril), injection (Kytril), or as a single transdermal patch to the upper arm (Sancuso); tropisetron (Navoban), which can be administered as an oral capsule or by injection; and palonosetron (Aloxi) and mirtazapine (Remeron), which can be administered as an injection or oral capsule.

[0130] Aprepitant (Emend) is an example of an NK1 receptor antagonist, but it is not the only one.

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

[0132] Further antiemetics that can be used according to the present invention, particularly in combination with 5-HT3 receptor antagonists and / or NK1 receptor antagonists, include, but are not limited to, metoclopramide (Reglan), lorazepam, atropine, arizaprid (Litican, Plitican, Superan, Vergentan), and dimenhydrinates (Dramamine, Driminate, Gravol, Gravamin, Vomex, Vertirosan), which act on the GI duct as motility enhancers.

[0133] According to the present invention, an antispasmodic (synonym: anticonvulsant) can be administered. According to the present invention, the term "antispasmodic" refers to a compound, composition, or drug that suppresses muscle spasms. Preferably, the antispasmodic is useful for smooth muscle contraction. According to the present invention, an antispasmodic that is effective in treating spastic effects in the digestive system is preferred. Therefore, a preferred antispasmodic is effective in reducing gastrointestinal spasms.

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

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

[0136] According to the present invention, the term "proton pump inhibitor" relates to a compound, composition, or agent whose primary effect is a significant and long-term reduction of gastric acid production.

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

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

[0139] For example, other compounds, compositions, or drugs can be administered to prevent the common adverse effects of NSAIDs on gastric ulcer formation, and in particular to prevent NSAID-induced gastric ulcers. In one embodiment, misoprostol, a synthetic prostaglandin E1 (PGE1) analog used for the prevention of NSAID-induced gastric ulcers, can be administered. Misoprostol acts on parietal cells to inhibit gastric acid secretion by G protein-coupled receptor-mediated inhibition of adenylyl cyclase, which results in a decrease in intracellular cyclic AMP levels and a decrease in proton pump activity on the apical surface of parietal cells.

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

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

[0142] One of the main adverse drug reactions (ADRs) associated with NSAIDs relates to their gastrointestinal (GI) effects. These effects are severe enough to pose a risk of ulcer perforation and upper gastrointestinal bleeding in many cases. NSAID patients experience dyspepsia, NSAID-associated upper gastrointestinal 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 reduced nutritional value to the epithelial mucosa. Therefore, NSAIDs are preferably not administered according to the present invention. Placetamol or "acetaminophen," which are not classified as NSAIDs because they exert only a weak anti-inflammatory effect, can be administered as analgesics according to the present invention, but may not be efficient for pain management, and therefore, administration of an NSAID may be necessary, especially to avoid the administration of opioids.

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

[0144] The term “antigen” refers to an active substance, such as a protein or peptide, that contains an epitope to which an immune response is directed and / or should be directed. In preferred embodiments, the antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a component of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus, particularly as an intracellular or surface antigen on cancer cells, and is preferably produced in large quantities.

[0145] In relation to the present invention, the term “tumor-associated antigen” preferably refers to a protein that is specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage under normal conditions, and is expressed or abnormally expressed in one or more tumor or cancerous tissues. In relation to the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and preferably are not expressed at all or are rarely expressed in normal tissues.

[0146] The term "epitope" refers to an antigenic determinant within a molecule, i.e., a portion of a molecule recognized by the immune system, such as by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes typically consist of a group of chemically active surface groups on a molecule, such as an amino acid or sugar side chain, and usually possess specific three-dimensional structural and charge properties. Conformational epitopes and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. The epitopes of proteins such as CLDN18.2 preferably comprise continuous or discontinuous portions of the protein and have an amino acid length of 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25. For example, the epitopes may have an amino acid length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0147] The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any molecule containing its antigen-binding moiety. The term “antibody” includes, but is not limited to, monoclonal antibodies and antibody fragments or derivatives, including human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, e.g., scFv, and antigen-binding antibody fragments such as Fab and Fab' fragments, and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, non-glycosylated 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 more conserved, hypervariable regions called complementarity-determining regions (CDRs) with a region called a framework region (FR) interposed between them. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins 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. Human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation).

[0149] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, where the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may include a complete variable domain fused to a constant domain, or it may include only a complementation-determining region (CDR) transplanted into an appropriate framework region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble a human immunoglobulin. 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 altered CDRs 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 derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another antibody. Typically, the variable regions of both the light and heavy chains mimic the variable region of an antibody derived from one species of mammal, while the constant region is homologous to the sequence of an antibody derived from another species. One obvious advantage of such a chimeric morphology is that the variable region can be conveniently induced from currently known sources using B cells or hybridomas from readily available non-human host organisms, for example, by combining it with a constant region derived from human cell preparations. The variable region has the advantage of ease of preparation and its specificity is not affected by the source, although the human constant region is less likely to elicit an immune response from human subjects than a constant region from a non-human source when the antibody is injected. However, the definition is not limited to this particular example.

[0151] The terms “antigen-binding portion” (or simply “binding portion”) or “antigen-binding fragment” (or simply “binding fragment”) of an antibody, or similar terms, refer to one or more fragments of an antibody that possess 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) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) Fd fragments, which consist of VH and CH domains; (iv) Fv fragments, which consist of the VL and VH domains of one arm of the antibody; (v) dAb fragments, which consist of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination by synthetic linkers that enable the production of single-chain proteins (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair up to form a monovalent molecule. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of the antibody. Further examples include binding-domain immunoglobulin fusion proteins, which include (i) a binding-domain polypeptide fused to an immunoglobulin hinge-domain 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 may be a heavy chain variable region or a light chain variable region.The binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Applications 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0152] The term “bispecific molecule” is intended to encompass any active substance having two distinct binding specificities, such as a protein, peptide, or protein or peptide complex. For example, the molecule may bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The terms “multispecific molecule” or “heterospecific molecule” are intended to encompass any active substance having three or more distinct binding specificities, such as a protein, peptide, or protein or peptide complex. For example, the molecule may 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. Therefore, the present invention encompasses, but is not limited to, bispecific, triplicate, quadruplespecific and other multispecific molecules for CLDN18.2 and other targets, such as Fc receptors on effector cells. The term “bispecific antibody” also encompasses diabodies. Diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby pairing those domains with complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0153] Antibodies can bind to therapeutic components or active substances such as cytotoxicities, drugs (e.g., immunosuppressants), or radioisotopes. Cytotoxic or cytotoxic drugs include any active substance that is harmful to cells, particularly those that kill them. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Suitable therapeutic agents for antibody conjugate formation include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxylamine) Examples of therapeutic agents include, but are not limited to, doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or lysine A.

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

[0155] The antibody conjugates of the present invention can be used to modulate a given biological response, and the drug component should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug component may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, enzyme-activated toxins or their active fragments, such as abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors.

[0156] Techniques for binding such therapeutic components to antibodies are well known, for example, 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 Prospective Of The Therapeutic Use Of Radiolabeled Antibody" 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 specific germline sequence if the antibody is obtained by immunizing an animal or by screening an immunoglobulin gene library, and the antibody selected in the screening 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 specific germline sequence exhibits 10 or fewer amino acid differences, more preferably 5, or even more preferably 4, 3, 2, or 1 or fewer amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0158] As used herein, the term “heteroantibody” refers to two or more antibodies, derivatives thereof, or antigen-binding regions linked together, of which at least two have different specificities. These different specificities include binding specificity to Fc receptors on effector cells and binding specificity to antigens or epitopes on target cells, such as tumor cells.

[0159] The antibodies described herein may be monoclonal antibodies. As used herein, the term “monoclonal antibody” refers to a preparation of an antibody molecule with a single molecular composition. Monoclonal antibodies exhibit single-binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma containing B cells obtained from a non-human animal, such as a mouse, fused to immortalized cells.

[0160] The antibodies described herein may be recombinant antibodies. The term “recombinant antibody” as used herein includes all antibodies produced, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) or hybridomas produced therefrom that are transgenic or transchromosomal with respect to immunoglobulin genes, (b) antibodies isolated from host cells transformed to express antibodies, such as transfectomas, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies produced, expressed, created or isolated by any other means, including splicing immunoglobulin gene sequences to other DNA sequences.

[0161] The antibodies described herein may originate from a variety of species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0162] The antibodies described herein encompass polyclonal and monoclonal antibodies, and include IgA antibodies such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more specifically 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] As used herein, the term "transfectoma" encompasses recombinant eukaryotic host cells that express antibodies, such as CHO cells, NS / O cells, HEK293 cells, HEK293T cells, plant cells, or fungal cells, including yeast cells.

[0164] As used herein, “heterogeneous antibody” is defined in relation to transgenic organisms that produce such antibodies. The term refers to antibodies that are not composed of transgenic organisms and have an amino acid sequence or coding nucleic acid sequence that corresponds to one found in organisms generally derived from non-transgenic species.

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

[0166] For the purposes of this invention, the term “antibody” encompasses all antibodies and antibody derivatives described herein. The term “antibody derivative” refers to any modified form of an antibody, such as a conjugate of an antibody with another active substance or antibody, or an antibody fragment.

[0167] The antibodies described herein are preferably isolated. As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to CLDN18.2 substantially does not contain 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, exhibit cross-reactivity to other related antigens, e.g., 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, a combination of “isolated” monoclonal antibodies relates to antibodies having different specificities and combined in a well-defined composition or mixture.

[0168] The term "binding" in this invention preferably refers to specific binding.

[0169] According to the present invention, if an antibody has a significant affinity for a predetermined target in a standard assay and binds to the predetermined target, then the antibody can bind to the predetermined target. "Affinity" or "binding affinity" is often expressed using the equilibrium dissociation constant (K). D ) is measured by. Preferably, the term “significant affinity” is 10 -5 M or smaller, 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, or 10 -12 M or a dissociation constant less than or equal to (K D This refers to binding to a predetermined target.

[0170] If an antibody does not have significant affinity to a target in a standard assay and does not significantly bind to the target, particularly not detectably, then the antibody cannot (substantially) bind to the target. Preferably, if the antibody is present at concentrations up to 2 μg / ml, preferably up to 10 μg / ml, more preferably up to 20 μg / ml, particularly up to 50 μg / ml or 100 μg / ml or higher, then the antibody is not detectably bound to the target. Preferably, the antibody has a K for binding to a predetermined target to which the antibody can bind. D At least 10 times, 100 times, 10 3 double, 10 4 double, 10 5 double or 10 6 K is twice as high D When the antibody binds to the target, it does not have significant affinity for the target. For example, K regarding binding to a target to which the antibody can bind. D 10 -7 If M, then K regarding binding to a target for which the antibody does not have significant affinity. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3M, 10 -2 M or 10 -1 It is M.

[0171] An antibody is specific to a predetermined target if it can bind to a predetermined target but cannot bind to other targets, i.e., it does not have significant affinity for other targets in a standard assay and does not significantly bind to other targets. According to the present invention, an antibody is specific to CLDN18.2 if it can bind to CLDN18.2 but cannot (substantially) bind to other targets. Preferably, an antibody is specific to 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 some other specific polypeptide. Preferably, an antibody has a K for binding to targets that it is not specific to. D At least 10 times, 100 times, 10 3 double, 10 4 double, 10 5 double or 10 6 K is twice as low D When the antibody binds to a predetermined target, the antibody is specific to that predetermined target. For example, regarding the binding of the antibody to a target to which it is specific, K D 10 -7 If M, then K regarding the binding of the antibody to a non-specific target. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It is M.

[0172] The binding of antibodies to targets 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, e.g., by equilibrium dialysis; by using the BIAcore 2000 instrument, using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigens; or by other methods known to those skilled in the art. Affinity data can be analyzed by, for example, Scatchard et al., Ann NYAcad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may differ when measured under different conditions, e.g., different salt concentrations, pH. Therefore, affinity and other antigen-binding parameters, e.g., K D ,I C 50 The measurement is preferably performed using standard solutions and standard buffers of the antibody and antigen.

[0173] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[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 other Ig classes.

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

[0176] As used herein, the term “reorganized” essentially refers to the configuration of a heavy-chain or light-chain immunoglobulin locus in which the V segment is directly adjacent to the DJ or J segment in the conformation encoding the complete VH or VL domain, respectively. Reorganized immunoglobulin (antibody) loci can be identified by comparison with germline DNA, and a reorganized locus has at least one recombinant heptameric / nocaper homology element.

[0177] As used herein with respect to the V segment, the terms “unreorganized” or “germ cell configuration” refer to a configuration in which the V segment has not been rearranged to be directly 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 within the extracellular domain of CLDN18.2, particularly within the first extracellular domain, and preferably within amino acid positions 29-78 of CLDN18.2. In certain embodiments, an antibody capable of binding to CLDN18.2 is an antibody that can bind to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NO: 3, 4, and 5; (ii) an epitope localized on CLDN18.2-loop 1, preferably SEQ ID NO: 8; (iii) an epitope localized on CLDN18.2-loop 2, preferably SEQ ID NO: 10; (iv) an epitope localized on CLDN18.2-loop D3, preferably SEQ ID NO: 11; (v) an epitope encompassing CLDN18.2-loop 1 and CLDN18.2-loop D3; or (vi) a non-glycosylated epitope localized 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 preferably an antibody capable of binding to CLDN18.2 expressed on the cell surface. In certain preferred embodiments, 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 protein, peptide or its immunogenic fragment or derivative. Antibodies capable of binding to CLDN18.2 can be obtained by a method comprising the step of immunizing an animal with a protein or peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50, or with nucleic acids or host cells expressing the said protein or peptide. Preferably, the antibody binds to cancer cells, particularly the aforementioned cancerous cells, and preferably does not substantially bind to non-cancerous cells.

[0180] Preferably, binding of an antibody capable of binding to CLDN18.2 to cells expressing CLDN18.2 induces or mediates the death of the CLDN18.2-expressing cells. The cells expressing CLDN18.2 are preferably cancer cells, particularly selected from the group consisting of neoplastic 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 cell-mediated lysis (CDC), antibody-dependent cell-mediated lysis (ADCC), apoptosis, and inhibition of proliferation of CLDN18.2-expressing cells. Preferably, ADCC-mediated lysis of cells occurs in the presence of effector cells, which in certain embodiments are selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs. Inhibition of cell proliferation can be measured in vitro by 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 in replicating cells (during the S phase of the cell cycle) and replace thymidine during DNA replication. For example, by detecting the incorporated chemical using an antibody specific to BrdU, cells that were actively replicating their DNA can be identified.

[0181] In preferred embodiments, the antibodies described herein have the following characteristics: a) Specificity for CLDN18.2; b) Binding affinity to CLDN18.2 at about 100 nM or less, preferably about 5 to 10 nM or less, more preferably about 1 to 3 nM or less; c) Ability to induce or mediate CDC on CLDN18.2-positive cells; d) Ability to induce or mediate ADCC on CLDN18.2-positive cells; e) Ability to inhibit the proliferation of CLDN18.2-positive cells; f) Ability to induce apoptosis in CLDN18.2-positive cells It may be characterized by one or more of the following.

[0182] In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 is produced by a hybridoma having the following name and accession number, deposited in DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany): 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, Deposit No. DSM ACC2809, deposited on October 26, 2006 n.182 - D1106 - 362, Deposit No. DSM ACC2810, deposited on October 26, 2006.

[0183] Preferred antibodies according to the present invention are those produced by and obtained from the above - described hybridomas, i.e., 37G11 in the case of 182 - D1106 - 055, 37H8 in the case of 182 - D1106 - 056, 38G5 in the case of 182 - D1106 - 057, 38H3 in the case of 182 - D1106 - 058, 39F11 in the case of 182 - D1106 - 059, 43A11 in the case of 182 - D1106 - 062, 61C2 in the case of 182 - D1106 - 067, 26B5 in the case of 182 - D758 - 035, 26D12 in the case of 182 - D758 - 036, 28D10 in the case of 182 - D758 - 040, 42E12 in the case of 182 - D1106 - 061, 125E1 in the case of 182 - D1106 - 279, 163E12 in the case of 182 - D1106 - 294, and 175D10 in the case of 182 - D1106 - 362; and their chimeric and humanized forms.

[0184] Preferred chimeric antibodies and their sequences are shown in the following table.

Table 1 - 1

[0185] In a preferred embodiment, an antibody, particularly a chimeric form of antibody according to the present invention, includes an antibody having 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 a further preferred embodiment, an antibody, particularly a chimeric form of antibody according to the present invention, includes an antibody having 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 a specific preferred embodiment, an antibody, particularly a chimeric form of antibody according to the present invention, includes an antibody 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, an antibody having the ability to bind to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising a mouse κ variable light chain, a human κ light chain constant region allotype Km(3), a mouse heavy chain variable region, and a human IgG1 constant region, allotype G1m(3).

[0187] In a specific preferred embodiment, a chimeric form of antibody includes an antibody 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 a specific preferred embodiment, a chimeric form of antibody includes an antibody comprising a combination of a heavy chain and a light chain selected from the following possibilities (i)-(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 or fragment thereof represented by SEQ ID NO: 15, and the light chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 20. (iii) The heavy chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 16, and the light chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 22. (iv) The heavy chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 18, and the light chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 25. (v) The heavy chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 17, and the light chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 24. (vi) The heavy chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 19, and the light chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 23. (vii) The heavy chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 19, and the light chain comprises the amino acid sequence or fragment represented by SEQ ID NO: 26. (viii) The heavy chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 19, and the light chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 27, and (ix) The heavy chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 19, and the light chain comprises the amino acid sequence or fragment thereof represented by SEQ ID NO: 28.

[0189] Antibodies following (v) are particularly preferred.

[0190] The terms “fragment” or “amino acid sequence fragment” used above relate to a portion of an antibody sequence, i.e., an antibody sequence shortened at the N-terminus and / or C-terminus, which, when replaced in the antibody, maintains the binding of the antibody to CLDN18.2 and, preferably, the function of the antibody as described herein, such as CDC-mediated lysis or ADCC-mediated lysis. Preferably, the amino acid sequence fragment contains at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues from the amino acid sequence. The amino acid sequence fragment selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28, preferably relates to the sequence from which the 17th, 18th, 19th, 20th, 21st, 22nd, or 23rd amino acid at the N-terminus has been removed.

[0191] In a preferred embodiment, the antibody capable of binding to CLDN18.2 includes 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 its fragments.

[0192] In a preferred embodiment, the antibody capable of binding to CLDN18.2 includes 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 its fragments.

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

[0194] Antibodies following (v) are particularly preferred.

[0195] In a preferred embodiment, the antibody having the ability to bind 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 45-52 of SEQ ID NO: 14, CDR2: Positions 70-77 of SEQ ID NO: 14, CDR3: Positions 116-125 of SEQ ID NO: 14, (ii) CDR1: Positions 45-52 of sequence number 15, CDR2: Positions 70-77 of sequence number 15, CDR3: Positions 116-126 of sequence number 15, (iii) CDR1: Positions 45-52 of sequence number 16, CDR2: Positions 70-77 of sequence number 16, CDR3: Positions 116-124 of sequence number 16, (iv) CDR1: Positions 45-52 of sequence number 17, CDR2: Positions 70-77 of sequence number 17, CDR3: Positions 116-126 of sequence number 17, (v) CDR1: Positions 44-51 of SEQ ID NO: 18, CDR2: Positions 69-76 of SEQ ID NO: 18, CDR3: Positions 115-125 of SEQ ID NO: 18, (vi) CDR1: Positions 45-53 of sequence number 19, CDR2: Positions 71-78 of sequence number 19, CDR3: Positions 117-128 of sequence number 19.

[0196] In a preferred embodiment, the antibody having the ability to bind 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 47-58 of sequence number 20, CDR2: Positions 76-78 of sequence number 20, CDR3: Positions 115-123 of sequence number 20, (ii) CDR1: Positions 49-53 of SEQ ID NO: 21, CDR2: Positions 71-73 of SEQ ID NO: 21, CDR3: Positions 110-118 of SEQ ID NO: 21, (iii) CDR1: Positions 47-52 of sequence number 22, CDR2: Positions 70-72 of sequence number 22, CDR3: Positions 109-117 of sequence number 22, (iv) CDR1: Positions 47-58 of sequence number 23, CDR2: Positions 76-78 of sequence number 23, CDR3: Positions 115-123 of sequence number 23, (v) CDR1: Positions 47-58 of SEQ ID NO: 24, CDR2: Positions 76-78 of SEQ ID NO: 24, CDR3: Positions 115-123 of SEQ ID NO: 24, (vi) CDR1: positions 47 - 58 of SEQ ID NO: 25, CDR2: positions 76 - 78 of SEQ ID NO: 25, CDR3: positions 115 - 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 - 58 of SEQ ID NO: 26, CDR2: positions 76 - 78 of SEQ ID NO: 26, CDR3: positions 115 - 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 - 58 of SEQ ID NO: 27, CDR2: positions 76 - 78 of SEQ ID NO: 27, CDR3: positions 115 - 123 of SEQ ID NO: 27, and (ix) CDR1: positions 47 - 52 of SEQ ID NO: 28, CDR2: positions 70 - 72 of SEQ ID NO: 28, CDR3: positions 109 - 117 of SEQ ID NO: 28.

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

[0198] In a further preferred embodiment, the antibody having the ability to bind to CLDN18.2 is preferably a monoclonal antibody against CLDN18.2, preferably comprising one or more complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL) of the monoclonal antibody against CLDN18.2 as described herein, preferably at least the CDR3 variable region, and preferably comprising one or more complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL) as described herein, preferably at least the CDR3 variable region. In one embodiment, the one or more of the complementarity-determining regions (CDRs) are selected from the set of complementarity-determining regions CDR1, CDR2 and CDR3 as described herein. In particularly preferred embodiments, the antibody capable of binding to CLDN18.2 preferably comprises a monoclonal antibody against CLDN18.2, preferably the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of the monoclonal antibody against CLDN18.2 as described herein, and more preferably the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) as described herein.

[0199] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises the CDRs along with their intervening framework regions. Preferably, the portion comprises at least about 50% of either or both of the first and fourth framework regions, wherein the 50% is 50% of the C-terminus of the first framework region and 50% of the N-terminus of the fourth framework region. The construction of the antibody by recombinant DNA technology may result in the introduction of residues on the N-terminal or C-terminal side of the variable region encoded by a linker introduced to facilitate cloning or other manipulative steps, including the introduction of a linker to link the variable region of the present invention to an immunoglobulin heavy chain, other variable domains (e.g., in the construction of a diabody), or a further protein sequence including a protein label.

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

[0201] References herein to antibodies containing a specific chain, region, or sequence in relation to the heavy chain of the antibody preferably refer to a situation in which all of the heavy chains of the antibody contain the specific chain, region, or sequence. This also applies to the light chain of the antibody.

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

[0203] According to the present invention, the term "expression" is used in its most common sense and includes the production of RNA or the production of RNA and protein / peptide. This term also includes the partial expression of nucleic acids. Furthermore, expression can be carried out transiently or stably.

[0204] Any teachings given herein with respect to a particular amino acid sequence, for example, those shown in the sequence listing, should be interpreted to also apply to sequences that are functionally equivalent to the particular sequence, for example, variants of the particular sequence that produce amino acid sequences exhibiting properties identical or similar to those of the particular amino acid sequence. One important property is to maintain the antibody's binding to its target or to maintain the antibody's effector function. Preferably, a sequence that is a variant with respect to a particular sequence, when it replaces the particular sequence in the antibody, maintains the antibody's binding to CLDN18.2 and, preferably, the antibody's function as described herein, such as CDC-mediated lysis or ADCC-mediated lysis.

[0205] Those skilled in the art will recognize that the sequences of the CDR, hypervariable region, and variable region can be modified without losing their ability to bind to CLDN18.2. For example, the CDR region is identical or highly homologous to the region of the antibody specified herein. "Highly homologous" means that 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2 substitutions may be made within the CDR. In addition, the hypervariable region and variable region can be modified to exhibit substantial homology to the region of the antibody specified and disclosed herein.

[0206] For the purposes of this invention, the term "mutant" of an amino acid sequence includes amino acid insertion mutants, amino acid addition mutants, amino acid deletion mutants, and / or amino acid substitution mutants. Amino acid deletion mutants, which involve deletions at the N-terminus and / or C-terminus of a protein, are also called N-terminal and / or C-terminal cleavage mutants.

[0207] Amino acid insertion mutants involve the insertion of one, two, or more amino acids within a specific amino acid sequence. In amino acid sequence mutants with insertions, one or more amino acid residues are inserted at specific locations within the amino acid sequence, although random insertions are also possible, provided that the resulting products are properly screened.

[0208] Amino acid addition mutants include amino-terminal and / or carboxyl-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

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

[0210] Amino acid substitution mutants are characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. It is preferable that the modification is located at a non-conserved position in the amino acid sequence among homologous proteins or peptides and / or that an amino acid is substituted with another amino acid having similar properties. Preferably, the amino acid change in a protein mutant is a conserved amino acid change, i.e., a substitution of a similar charged or uncharged amino acid. A conserved amino acid change encompasses the substitution of one of the families of amino acids related to the side chain. 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 (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 the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given with respect to 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 total 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 with respect to 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 with respect to the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with a standard setting, 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 have conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between those sequences.

[0213] The term "identity percentage" is intended to represent the percentage of amino acid residues that are identical between two sequences being compared, obtained after best alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are conventionally performed by comparing them after optimal alignment, and such comparisons are performed segment by segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be achieved manually, by local homology algorithms (Smith and Waterman, 1981, Ads App.Math.2, 482; Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; 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 Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

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

[0215] The term “transgenic animal” refers to an animal having a genome that includes one or more transgenes, preferably heavy chain and / or light chain transgenes, or a transchromosome (which may or may not be integrated into the animal’s natural genomic DNA), and which is preferably capable of expressing the transgenes. For example, a transgenic mouse may 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 cells expressing the CLDN18.2 antigen and / or CLDN18.2. The human heavy chain transgene may be integrated into the mouse’s chromosomal DNA, as in transgenic mice such as HCo7 or HCol2 mice, e.g., HuMAb mice, or the human heavy chain transgene may be maintained extrachromosomally, as in transchromosomal (e.g., KM) mice described in International Publication No. 02 / 43478. Such transgenic and transchromosomal mice can produce numerous isotypes (e.g., 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” means the ability to cause an overall reduction or overall decrease of a level, for example, a level of cell expression or proliferation, 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” preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more.

[0218] Mechanism of action of mAbs The following provides a discussion of the mechanism underlying the therapeutic effect of the antibody of the present invention, but this should not be considered a limitation to the present invention in any sense.

[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 directly kill tumor cells by targeting a payload (e.g., a radioisotope, drug, or toxin), or to attack tumors through complementary mechanisms of action, which may include an antitumor immune response that may be impaired due to the cytotoxic side effects of chemotherapeutic agents on T lymphocytes, in synergistic action with traditional chemotherapeutic agents. However, the antibodies described herein can also exert their effects simply by binding to CLDN18.2 on the cell surface, and thus, for example, by blocking cell proliferation.

[0220] Antibody-dependent cell-mediated cytotoxicity ADCC represents the ability to kill effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells be marked with an antibody.

[0221] ADCC preferably occurs when an antibody binds to an antigen on tumor cells and the antibody's 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 certain cell populations characteristically express defined Fc receptors. ADCC can be considered a mechanism that directly induces varying degrees of immediate tumor destruction, resulting in antigen presentation and induction of a T-cell response 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 cell injury CDC is another method of cell death that can be directed by antibodies. IgM is the most effective isotype for complement activation. 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 involves the C of an antibody molecule, such as an IgG molecule. H This results in the exposure of numerous very close C1q binding sites on the 2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interactions into high-avidity interactions, which initiate a cascade of events involving a series of other complement proteins, leading to the proteolytic release of effector cytochemotactic / activating substances C3a and C5a. Preferably, the complement cascade terminates with the formation of membrane-damaging complexes, which create 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 described in Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, can also be used.

[0224] The mouse is the preferred animal system for producing hybridomas that secrete monoclonal antibodies. Hybridoma production in mice is a very widely established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are well known in this field. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also well known.

[0225] Other preferred animal strains for producing hybridomas that secrete monoclonal antibodies are rat and rabbit strains (see, for example, Spieker-Polet et al., Proc.Natl.Acad.Sci.USA92:9348 (1995); also see Rossi et al., Am.J.Clin.Pathol.124:295 (2005)).

[0226] In yet another preferred embodiment, human monoclonal antibodies can be produced using transgenic or transchromosomal mice that carry a portion of the human immune system rather than a 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.” The production of human antibodies in such transgenic mice can be carried out as detailed with respect to CD20 in International Publication No. 2004 / 035607.

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

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

[0229] During the course of the immunization protocol, the immune response can be observed for plasma and serum samples obtained by tail vein or retroorbital blood collection. Mice with sufficient immunoglobulin titers can be used for fusion. To increase the proportion of hybridomas that secrete specific antibodies, mice can be additionally immunized intraperitoneally or intravenously with antigen-expressing cells three days before sacrificial death and splenectomy.

[0230] To produce hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells can be isolated from immunized mice and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can then be screened for antigen-specific antibody production. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies specific to the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. The antibody-secreting hybridomas can be re-plated and screened again. If still positive for monoclonal antibodies, they can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro, and antibodies can be produced in tissue culture medium for characterization.

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

[0232] For example, in one embodiment, a gene of interest, such as an antibody gene, can be ligated to an expression vector, such as a eukaryotic expression plasmid, as used in the GS gene expression system disclosed in International Publication No. 87 / 04462, WO89 / 01036, and European Patent No. 338841, or other expression systems well known in the art. The purified plasmid containing the cloned antibody gene can be introduced into eukaryotic host cells such as CHO cells, NS / O cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells such as plant-derived cells, fungal or yeast cells. The method used to introduce these genes may be one of the methods 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, and they can then be amplified in terms of expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0233] Alternatively, the cloned antibody gene can be expressed in other expression systems, including microorganisms, such as prokaryotic cells like Escherichia coli. Furthermore, antibodies can be produced in transgenic non-human animals, such as milk from sheep and rabbits or eggs from hens, or in transgenic plants; see, for example, Verma, R., et al. (1998) J.Immunol.Meth.216:165-181; Pollock, et al. (1999) J.Immunol.Meth.231:147-157; and Fischer, R., et al. (1999) Biol.Chem.380:825-839.

[0234] Chimera transformation Mouse monoclonal antibodies, when labeled with toxins or radioisotopes, can be used as therapeutic antibodies in humans. Unlabeled mouse antibodies are highly immunogenic in humans when repeatedly applied, leading to reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimerizing or humanizing the respective antibodies. A chimeric antibody has different parts derived from antibodies of different animal species, for example, a variable region derived from a mouse antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by ligating the variable regions of the heavy and light chains of a mouse antibody with the constant regions of the heavy and light chains of a human antibody (as described, e.g., in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by ligating the human κ light chain constant region with the mouse light chain variable region. In another preferred embodiment, chimeric antibodies can be produced by ligating a human λ light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for the production of chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for the production of chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0235] Humanization Antibodies primarily interact with target antigens via amino acid residues located within six heavy-chain and light-chain complementarity-determining regions (CDRs). Therefore, the amino acid sequences within CDRs are more diverse among individual antibodies than the sequences outside the CDRs. Since CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors containing CDR sequences from specific naturally occurring antibodies, transplanted onto framework sequences from different antibodies 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 constructed variable genes formed by V(D)J ligation during B cell maturation. Germline gene sequences also differ uniformly and individually across the entire variable region from the sequences of high-affinity secondary repertoire antibodies.

[0236] The ability of an antibody to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blotting, immunofluorescence, and flow cytometry).

[0237] To purify the antibody, the selected hybridoma can be grown in a 2-liter spinner flask for monoclonal antibody purification. Alternatively, the antibody can be produced in a dialysis-based bioreactor. The supernatant can be filtered and concentrated as needed, then 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 replaced with 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] Site-specific or multi-site mutagenesis can be used to determine whether the selected monoclonal antibody binds to a unique epitope.

[0239] To determine the antibody isotype, isotyping ELISA can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics). Wells on a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with a monoclonal antibody or purified isotyping control at ambient temperature for 2 hours. The wells can then be reacted with either mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or a mouse IgM-specific peroxidase-binding probe. 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 no. 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 living cells expressing antigens. Cell lines that express antigens naturally or after transfection, and 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 at 4°C for 30 minutes. After washing, APC-labeled or Alexa647-labeled anti-IgG antibodies can be bound to antigen-binding monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument that utilizes the side-scattering properties to gate single living cells. To distinguish antigen-specific monoclonal antibodies from non-specific conjugates in a single measurement, a simultaneous transfection method can be used. Cells transiently transfected with plasmids encoding antigens and fluorescent markers can be stained as described above. Transfected cells can be detected using different fluorescence channels than antibody-stained cells. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies selectively bind to cells expressing the fluorescent marker, while non-specific antibodies bind to non-transfected cells at a similar rate. A selective assay using fluorescence microscopy can be used in addition to or instead of the flow cytometry assay. Cells can be stained precisely as described above and examined by fluorescence microscopy.

[0241] Immunofluorescence microscopy can be used to determine the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing the antigen. For example, cell lines expressing the antigen spontaneously or after transfection, along with negative controls lacking antigen expression, are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen at 25°C for 30 minutes. After washing, the cells can be reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0242] Cell extracts from antigen-expressing cells 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 a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed on an ECL substrate.

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

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

[0245] Antibody-dependent cell-mediated cytotoxicity (ADCC) In short, 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. The washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum to express CLDN18.2. 51 Cr-labeled target cells can be mixed with effector cells in various ratios to target cells. Alternatively, target cells may be labeled with a fluorescence-enhancing ligand (BATDA). Highly fluorescent chelates of europium containing the enhancing ligand released from dead cells can be measured by fluoroscopy. Another selective technique can utilize the transfection of target cells with luciferase. The added Lucifer Yellow can then be oxidized only by living cells. Purified anti-CLDN18.2 IgG can then be added at various concentrations. Unrelated human IgG can be used as a negative control. The assay can be performed at 37°C for 4–20 hours, depending on the effector cell type used. 51 Samples can be assessed for cell lysis by measuring Cr release or the presence of EuTDA chelates. Alternatively, luminescence resulting from the oxidation of Lucifer Yellow can be a measure of viability.

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

[0247] Complement-dependent cell injury (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 by 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 increased membrane permeability can be evaluated using a propidium iodide (PI) exclusion assay. Briefly, target cells are washed and 5 × 10⁻⁶ 5 The mAbs / ml can be incubated with various concentrations of mAbs at room temperature or 37°C for 10-30 minutes. Then, serum or plasma can be added to a final concentration of 20% (v / v), and the cells can be incubated at 37°C for 20-30 minutes. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be immediately analyzed by flow cytometry using a FACS array.

[0248] In a selective assay, the induction of CDC can be measured in adherent cells. In one embodiment of this assay, cells are placed in a tissue culture flat-bottom microtiter plate in a 3 × 10⁶ layer 24 hours before the assay. 4 Inoculate at a density of / well. The following day, remove the growth medium and incubate the cells in triplicates with the antibody. Incubate the control cells with growth medium or growth medium containing 0.2% saponin for background lysis and maximum lysis measurements. After incubation at room temperature for 20 minutes, take the supernatant and add 20% (v / v) human plasma or serum (preheated to 37°C) in DMEM to the cells, and incubate for a further 20 minutes at 37°C. Add all cells from each sample to propidium iodide solution (10 μg / ml). Next, replace the supernatant with PBS containing 2.5 μg / ml ethidium bromide and measure the fluorescence emission at 600 nm with 520 nm excitation using a Tecan Safire. Calculate the percentage of specific lysis 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 the ability to initiate apoptosis, monoclonal anti-CLDN18.2 antibodies can be incubated with CLDN18.2-positive tumor cells, such as SNU-16, DAN-G, KATO-III, or CLDN18.2-transfected tumor cells, at 37°C for approximately 20 hours. Cells can be collected, washed in Annexin-V binding buffer (BD biosciences), and incubated with Annexin-V conjugated to FITC or APC (BD biosciences) in the dark for 15 minutes. All cells from each sample can be added to PI solution (10 μg / ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, catalog number AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) integration during DNA synthesis in cells proliferating in a microplate. The integrated BrdU is detected using a europium-labeled monoclonal antibody. To enable antibody detection, cells are fixed and DNA denatured using Fix solution. The unbound antibody is washed away, and a DELFIA inducer is added to dissociate the europium ion from the labeled antibody into solution. In solution, this ion forms a highly fluorescent chelate with the components of the DELFIA inducer. Time-resolved fluorescence spectroscopy is used for detection, and the measured fluorescence is proportional to DNA synthesis in the cells of each well.

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

[0251] In vivo studies can be performed using the antibodies described herein after xenotransplantation of tumor cells expressing CLDN18.2 into immunocompromised mice or other animals. The antibodies can be administered to tumor-free mice, followed by injection of tumor cells to measure the antibody's effect in preventing tumor formation or tumor-related symptoms. The antibodies can be administered to tumor-bearing mice to measure the therapeutic effect of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibody application can be combined with the application of other substances, such as cell proliferation inhibitors, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies, to measure synergistic effects and potential toxicity. To analyze antibody-mediated toxic side effects, animals can be inoculated with the antibody or a control reagent and thoroughly examined for symptoms that may be associated with CLDN18.2 antibody treatment. Possible side effects of in vivo application of CLDN18.2 antibodies include toxicity in CLDN18.2-expressing tissues, particularly 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] The mapping of antibody-recognized epitopes 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 active ingredients described herein may be administered in the form of any suitable pharmaceutical composition.

[0254] Pharmaceutical compositions are usually provided in unit dose forms and can be prepared by methods known to the public. Pharmaceutical compositions may be in the form of solutions or suspensions, for example.

[0255] The pharmaceutical composition may contain salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" means that the substance is non-toxic and does not interact with the action of the active ingredient of the pharmaceutical composition.

[0256] Medicinally unacceptable salts can be used to prepare medicinally acceptable salts and are included in the present invention. Such medicinally acceptable salts 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. Medicinally 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 pharmaceutical compositions include acetic acid in salts, citric acid in salts, boric acid in salts, and phosphoric acid in salts.

[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 Ringer's lactate solution.

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

[0261] Suitable carriers for parenteral administration include, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0262] As used herein, the term “excipient” is intended to refer to all substances that may be present in a pharmaceutical composition and are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings, or colorants.

[0263] The active substances and compositions described herein may be administered by any conventional route, for example, by parenteral administration including injection or infusion. Administration is preferably parenteral, for example, by intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular routes.

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

[0265] The active substances and compositions described herein are administered in effective amounts. “Effective amount” means an amount that, alone or in combination with further doses, achieves the desired response or effect. In the case of treating a particular disease or condition, the desired response preferably relates to halting the progression of the disease. This includes slowing the progression of the disease, in particular preventing or reversing its progression. The desired response in the treatment of a disease or condition may also be a delay in the onset of the disease or condition or a prevention of its onset.

[0266] The effective amount of the active substance or composition described herein depends on the condition being treated, the severity of the disease, the patient's age, physiological state, size and weight, and other individual patient parameters, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the active substance described herein may depend on these various parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0267] The active substances and compositions described herein can be administered to patients, for example, in vivo, to treat or prevent a variety of disorders as described herein. Preferred patients include human patients with disorders that can be corrected or improved by administration of the active substances and compositions described herein. This includes cellular disorders characterized by altered expression patterns of CLDN18.2.

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

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

[0270] The present invention will be further illustrated by the following embodiments, but these should not be construed as limiting the scope of the invention. [Examples]

[0271] (Example 1) A clinical first-in-human, single-dose, multicenter, open-label phase I IV dose-escalation study evaluating the safety and tolerability of IMAB362 in hospitalized patients with advanced gastroesophageal cancer. To determine the maximum tolerated dose or maximum applicable single dose (MTD) of IMAB362, to examine the safety, tolerability, and adverse event profile of IMAB362, to determine the pharmacokinetic profile of IMAB362 at single escalating doses, to determine the immunogenicity of IMAB362 at single dose applications, and to determine the potential antitumor activity of IMAB362 in patients with advanced gastroesophageal (GE) cancer, a human clinical first-in-human single-dose multicenter phase I open-label IV infusion dose-escalation study was conducted.

[0272] This trial was designed as a first-in-human, multicenter, non-randomized, patient-to-patient single-dose escalation, open-label clinical trial comparing a single intravenous infusion of IMAB362 with a 4-week treatment-free follow-up period.

[0273] To be included in the trial, patients had to meet all of the following selection criteria: • Metastatic, anti-treatment, or recurrent advanced gastroesophageal cancer as confirmed by histological examination. • Availability of CLDN18.2 expression confirmed by immunohistochemistry or tumor tissue samples suitable for determining CLDN18.2 expression. Prior standard chemotherapy including fluoropyrimidines, platinum compounds and / or epirubicin, and—where clinically appropriate—docetaxel. • At least one measurable site of the disease as determined by RECIST criteria (computed tomography (CT) scan or magnetic resonance imaging (MRT) within 6 weeks prior to clinical trial enrollment) • Age 18 or older • Written informed consent after notification of the exam • ECOG Performance Status (PS) 0-1 or Karnovsky Index 70-100% • Average life expectancy > 3 months ·Platelet count ≧100,000 / mm 3 • Hemoglobin ≥ 10 g / dl INR < 1.5 • Bilirubin levels are normal. • AST and ALT levels < 2.5 times the upper limit of normal (ULN) (5 times ULN if liver metastases are present) • Creatinine <1.5 × ULN • For women with potential pregnancy (last menstrual period less than 2 years prior to registration): Use of two highly effective methods of contraception for 8 weeks after baseline pregnancy test (β-HCG) injection and 8 weeks after administration of the test strip. Male patients must use generally accepted methods of contraception for 8 weeks after the injection of the test drug.

[0274] Patients meeting 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 antitumor chemotherapy or radiation therapy • Other test drugs or devices used concurrently with or within 4 weeks prior to this test • Other combination anticancer drugs or therapies • History of positive testing for human immunodeficiency virus (HIV) antibodies Known hepatitis • Uncontrolled or serious illnesses, including but not limited to any of the following: - Progressive or active infection requiring parenteral antibiotics - Symptomatic congestive heart failure - Unstable angina - Uncontrolled hypertension - Clinically important arrhythmias - Myocardial infarction within the past 6 months - Gastric bleeding within the past 4 weeks -Symptomatic peptic ulcer - Clinical symptoms or confirmed metastases of the brain - A mental illness or social condition that makes compliance with the exam impossible. • Simultaneous administration of a vitamin K antagonist (e.g., coumadin) and an anticoagulant. • Concurrent administration of therapeutic doses of heparin (prophylactic doses are acceptable).

[0275] Out of a total of 29 patients, 15 patients were administered the study drug to determine the dose cohort (33, 100, 300, 600, or 1000 mg IMAB362 / m²). 2 Each patient was assigned to one of the dose groups. These patients formed the safety population (SP). Since no potential dose-limiting toxicities occurred in any of the dose groups, it was not necessary to test additional patients to confirm potential dose-limiting toxicities. Therefore, only three patients in each dose cohort, i.e., 15 patients in total, received the study drug.

[0276] Table 1 below shows the patient assignments to various IMAB362 dose cohorts.

[0277] [Table 1]

[0278] No patients terminated the trial early; in other words, all patients completed the trial according to the protocol.

[0279] A. Safety Evaluation IMAB362 has been found to be safe and well-tolerated.

[0280] Only 25 adverse events (AEs) occurring in 8 patients were evaluated as treatment-related. Treatment-related AEs were similar across dose groups. More than half of these AEs were gastrointestinal disorders (primarily nausea and vomiting). Only one of these AEs was evaluated as severe (vomiting), while all others were mild or moderate. All AEs resolved with the exception of one case of dysgeusia (CTC grade 1 (mild)) with an undetermined outcome and one case of elevated GGT (CTC grade 2 (moderate)) that did not recover.

[0281] No dose-limiting toxicities (DLTs), defined as treatment-related adverse events (AEs) occurring during the study drug infusion period or within 4 weeks after infusion and classified as either Grade 3 toxicity (excluding nausea, vomiting, and alopecia) or Grade 4 or 5 toxicity (according to 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 is 1000 mg / m². 2 That is the case.

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

[0283] Only seven patients had at least one out-of-range clinical laboratory value rated as Grade 3 (severe). No dose-response relationship or clear association with the study drug was observed. No clinical laboratory values ​​of Grade 4 (life-threatening) or 5 (death-causing) were reported.

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

[0285] B. Evaluation of pharmacokinetics and immunogenicity For the measurement of drug concentration, the IMAB362 serum levels of all patients were measured immediately before the injection of the test drug, at the end of the injection, 3, 8, 12 and 24 hours after the end of the injection, and on the 3rd day (V3), 5th day (V4), 8th day (V5), 15th day (V6) and 29th day (V7).

[0286] A summary of the IMAB362 serum levels during the test for each patient is shown in Table 2. For the 300 mg / m 2 dose group, for one patient (number 1201), an already low IMAB362 serum level (12.633 μg / ml) was measured before the injection of the test drug (V2, day 0) for unknown reasons.

[0287] [Table 2]

[0288] The observed average peak concentration (C max ) for each dose group is shown in Table 3. The incremental average value for C max corresponds to the incremental injection dose of IMAB362.

[0289] [Table 3]

[0290] A graphical representation of the average blood concentration of IMAB362 during the test period is shown in Figure 1.

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

[0292] The pharmacokinetics of IMAB362 are dose-dependent. The peak dose level was 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 12 days across various dose cohorts.

[0293] Based on in vitro mechanism of action studies, the inventors believe that a robust antitumor cell effect can be expected at a concentration of 50 μg / ml of IMAB362 by inhibiting ADCC, CDC, and proliferation, and that the main mechanism of action is the EC of ADCC and CDC. 50 It was determined that the value would be covered even by half of this concentration level. Based on this knowledge, 300 mg / m² 2 and 600 mg / m² 2 The dose level was identified for more detailed evaluation in multi-dose studies of IMAB362. 300 mg / m² 2 and 600 mg / m² 2 Patients who were taking IMAB362 showed significantly higher levels on day 8 (V5), and were close to 50 μg / ml on day 15 (V6).

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

[0295] C. Evaluation of antitumor activity The primary measure for evaluating potential antitumor activity was tumor status according to the RECIST (version 1.0) classification at 2–5 weeks post-IMAB362 infusion (V6 / V7). Since all patients completed the trial according to the protocol, evaluation was performed exclusively at V7, i.e., 4–5 weeks post-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 was not available for patient 0302), but since this was not a formal efficacy evaluation, these patients were included in the population for the analysis of antitumor activity.

[0297] Overall, complete or partial responses could not be evaluated for any patient. Stable disease was observed in 1 of 15 patients in the 600 mg / m 2 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 having membrane staining), with 90% or more of the tumor cells in this patient staining positive for CLDN18.2 and a large percentage of tumor cells showing membrane staining. Two patients in the 300 mg / m 2 group also did not progress; since these patients had no target lesions, they were not evaluable for objective tumor response and were classified as non-CR, 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 responses by patient is shown in Table 4.

[0299]

Table 4

[0300] Various parameters contributing to the assessment of tumor status (overall response) are described below.

[0301] A summary of the evaluation results (evaluated at V7) after IMAB362 treatment is shown in Table 5 for the total change in the 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 total longest diameter of target lesions from V1 to V7 did not show a clear difference for different treatment doses.

[0304] Regarding non-target lesions, clear progression (from V1 to V7) was reported more frequently in patients at lower dose levels, but not at 600 mg / m². 2 and 1000 mg / m² 2 No dose-level findings were reported.

[0305] 300 mg / m² 2 In one patient in the group (0403), clear progression was observed in non-target lesions, and a decrease in the longest diameter was observed in one target lesion lymph node.

[0306] Regarding new lesions, no preference trend was observed for any particular dose group.

[0307] Patient 0302 (600mg / m 2 Dose group) and 0205 (1000 mg / m²) 2 In the dose group, the development of new lesions was the reason for evaluating the overall response as a progressive disease.

[0308] To assess the status of non-target lesions using RECIST, serum tumor antigen levels CA125, CA15-3, CA19-9, and CEA were measured in the central laboratory on V2 (Day 1, pre-infusion), V6, and V7.

[0309] Table 6 shows a summary of serum tumor markers for three patients with at least a stable overall disease response.

[0310] [Table 6]

[0311] Of the three patients with stable disease or non-CR / non-PD as detected by imaging, two 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) at 4-5 weeks (V6 / V7) after IMAB362 injection was compared to baseline. Overall, no complete or partial response could be evaluated in any patient. One out of 15 patients (600 mg / m²) 2 The dose group showed stable disease at the end of the study. The 300 mg / m² group with unmeasurable disease... 2 Two patients in the dose group showed non-complete response (CR) / non-progressive disease (PD). Consistent with this, tumor marker levels in these three patients remained stable (2 patients) or decreased significantly (1 patient). The majority of patients with progressive disease showed an increase in tumor marker levels over time.

[0313] Regarding parameters that contribute to the evaluation of tumor status (overall response), a reduction of 300 mg / m² in one lesion corresponds to a reduction of 300 mg / m². 2 Observed in the dose group. At screening (V1), 13 out of 15 patients had a total of 32 non-target lesions. After IMAB362 treatment (evaluated at V7), clear progression of non-target lesions was reported in a total of 5 patients at 33 mg / m². 2 Three patients in the dose group received 100 mg / m². 2 One person in the dose group and 300 mg / m² 2 One patient in the dose group was affected. In all five patients, the overall response was not evaluated as progressive disease solely due to the progression of non-target lesions. A total of 17 new lesions were observed during the course of the study and were evenly distributed across the dose groups. Two patients (600 mg / m²) 2 and 1000 mg / m² 2 In the dose group, the development of new lesions was the reason for evaluating the overall response as a progressive disease.

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

[0315] In conclusion, the suggestion of antitumor activity (stable disease, reduction of tumor markers) was observed at 300 mg / m². 2 and 600 mg / m² 2 This was observed in the dose group. Due to the small sample size of the dose group, it is difficult to draw conclusions regarding the trend of the effect.

[0316] C. Overall conclusion This trial was designed as a first-in-human, multicenter, non-randomized, patient-to-patient single-dose escalation, open-label clinical trial of IMAB362 as a single intravenous infusion followed by a 4-week no-treatment follow-up period.

[0317] A total of 15 patients were administered the study drug, and dose cohorts were established (33, 100, 300, 600, or 1000 mg IMAB362 / m²). 2 Each patient was assigned to one of the dose groups. The dose groups can be considered comparable. No relevant imbalances were observed in patient statistics or baseline characteristics.

[0318] Regarding the primary objective of the study, no dose-limiting toxicity (DLT) was observed in any dose group. Therefore, the applicable single dose of IMAB362 in this study is 1000 mg / m². 2 IMAB362 was safe and well-tolerated, with nausea and vomiting being the most common associated adverse events.

[0319] The AE profile and incidence of AEs were similar across the various dose groups. There were no significant differences between the dose groups in the number of individual patients with any clinically significant worsening of hematological, biochemical, or coagulation parameters.

[0320] Regarding the potential antitumor activity of IMAB362 according to RECIST criteria, no complete or partial response was observed in any patient. One out of 15 patients (600 mg / m²) showed no response. 2 The dose group showed stable disease at the end of the study. The 300 mg / m² group with unmeasurable disease... 2Two patients in the dose group showed non-complete response (CR) / non-perioded disease (PD). Of these three patients with stable disease on 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 is 600 mg / m². 2 Pharmacokinetic studies demonstrating that the desired effect is achieved at the dose level support the need for further evaluation of this dose.

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

[0323] (Example 2) Drug efficacy The objectives of the in vitro analysis conducted for this Phase I clinical trial included analyzing (i) whether effector cells present in the patient's blood could induce IMAB362-dependent ADCC, (ii) whether the patient's complement system could induce IMAB362-dependent CDC, and (iii) whether the ability of IMAB362 to induce ADCC and CDC would change after administration in the patient.

[0324] Various assays were performed to investigate in detail the cytolytic activity induced by IMAB362 after administration in patients. The assays were performed using either patient serum or patient PBMCs isolated from blood samples (Table 7). For comparison and to validate the functionality of the CDC and serum ADCC assays, serially diluted human serum pools (prepared from healthy human subjects) containing fresh IMAB362 were included in parallel with each assay. To test the functionality of the ADCC assay for PBMCs, blood cells isolated from healthy donors were used as positive controls 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 sources of IMAB362 antibodies and complement in CDC or as antibody sources in serum ADCC assays. Patient pre-infusion serum was used as a negative control for "no IMAB362" 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 extracted, serum aliquots were prepared, and immediately stored at -80°C. All of these samples were analyzed in a single experiment after the collection of all 24 serum samples.

[0328] For ADCC, PBMCs were isolated using a fresh blood sample (15 ml of Na2EDTA), and the ADCC assay was performed the following day.

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

[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 healthy donors (frozen N2 stock lot ID: 276-SMS-09-00706, 4e7c / vial, MNZ, 08.07.07.SJA) • Functional control antibody: IMAB362 in serial dilutions (0.4 ng / ml to 126.5 μg / ml) • Assay-negative control antibody: Isotype control (rituximab, 126.5 μg / ml).

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

[0332] Fixed dose of 0.5 μg / ml IMAB362 (in vitro EC) 50In addition to pre-infusion serum to which an equivalent concentration was exogenously added, samples collected 1, 7, 14, and 28–32 days after IMAB362 administration were also tested, and in these samples, circulating IMAB362 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 the target. For comparison, a serum pool from healthy human donors spiked with IMAB362 was tested.

[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 a final concentration 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: Isotyped control antibody (rituximab) diluted in a human serum pool.

[0334] The dynamics of IMAB362-mediated global cytotoxicity in human circulation, integrating its ability to induce ADCC and CDC, were analyzed in a "one-tube" assay.

[0335] Serum from each patient, collected 7, 14, and 28–32 days after IV administration of IMAB362, and therefore containing the patient's complement factors plus circulating IMAB362, was tested in this assay. In each assay, serum was applied up to a final serum concentration of 25% (v / v). Healthy control PBMCs were added as effector cells, and NUGC-4 cells were used as target cells, resulting in an E:T ratio of 40:1.

[0336] In an additional setting, serum was thermally inactivated, disrupting complement activity. This second assay therefore reflects ADCC activity induced solely by IMAB362 present in patient serum.

[0337] During the Phase I trial period, serum samples were collected and stored at -80°C. All patient samples were tested 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 that have been stably transfected with luciferase. • Effector cells: PBMCs (fresh pia mater) from healthy donors. • Functional control antibody: IMAB362 serial dilutions (0.26 ng / ml to 200.0 μg / ml) spiked in a human serum pool. • Sample positive control: IMAB362 (200.0 μg / ml) (EC for IMAB362 under this setting) 80~100 Pre-infusion serum sample from a patient who spiked (is). • Assay-negative control antibody: Isotype control antibody (rituximab) from a 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, thereby inducing complement-dependent cell-mediated cytotoxicity (CDC), was analyzed ex vivo at 1, 7, 14, and 28–32 days after IMAB362 administration. The assay was performed by directly using patient serum samples in the assay (unnormalized CDC). A fixed dose of 10 μg / ml IMAB362 (in vitro EC) was used as a positive control. 90~100 Pre-injection serum to which an exogenous dose (corresponding to the concentration) was added. The final serum concentration in each assay was adjusted to 20%. Luciferase-transfected CHO-K1 cells stably transfected with CLDN18.2 were used as the target. For comparison, a serum pool from healthy human donors spiked with IMAB362 was tested.

[0340] During the Phase I trial period, serum samples were collected and stored at -80°C. All patient samples were tested 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 human serum pool 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 at different concentrations. • Assay-negative control antibody: Isotyped control antibody diluted in a human serum pool.

[0342] B. Results Patient PBMC capacity mediated by ADCC To analyze the ability of patient immune cells to lyse tumor cells expressing CLDN18.2, NUGC-4 gastric cancer cells endogenously expressing CLDN18.2 were incubated with escalating concentrations of IMAB362 and with patient PBMCs. Assays on PBMCs from healthy donors were included as a functional control.

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

[0344] These data demonstrate that PBMCs from gastric cancer patients are no less effective than PBMCs obtained from healthy donors in inducing ADCC in human CLDN18.2-positive gastric cancer cells in conjunction with IMAB362.

[0345] The patient's complement system's ability 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 to 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 by measuring luciferase activity after 80 minutes.

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

[0347] Ability of soluble effectors in patient serum to induce cell death by intravenous IMAB362 Next, the ability of patient serum to interact with IV-administered IMAB362 throughout the patient's circulatory period was investigated by testing serum samples collected at various time points after IMAB362 administration in a CDC assay against CLDN18.2-positive CHO-K1 target cells. Serum samples were 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 the corresponding pre-infusion serum from each patient was used as a diluent to adjust the final IMAB362 concentration to 0.5 μg / ml (mean EC50 of IMAB362). Since IMAB362 concentrations vary depending on the therapeutic dose and time of blood collection, the dilution factors for samples varied considerably among patients, ranging from 4.6 to 688 times. A serum pool from healthy donors (HSCs) was used as a control (Figure 4).

[0348] Compared to positive controls (pre-infusion serum from each patient + fresh IMAB362), cytotoxic activity was maintained within the first 24 hours, but cytolytic activity in serum samples collected one week later was reduced, and this progressed further in subsequent weeks (Figure 4). Even so, considerable cytotoxicity was performed by patient serum even two weeks after IMAB362 administration. The loss of CDC activity at 28–32 days was significant and most pronounced in patients treated with low doses of IMAB362 (Figure 4). High dose (0204; 600 mg / m²) 2 and 0205; 1000 mg / m² 2 In patients treated with ), CDC activity appeared to be better preserved throughout the period studied. Based on currently available data, the mechanism underlying this decline is not yet understood.

[0349] [Table 8]

[0350] Effects of serum components on IMAB362-induced cytotoxicity The ADCC activity of mAB may be impaired in the presence of human serum. The effect of patient serum on ADCC activity was investigated. For this purpose, serum from each patient was used, collected 7, 14, and 28-32 days after IMAB362 administration, and therefore corresponding to the patient's complement factors plus circulating IV-administered IMAB362. All patient serum samples were diluted to 25% (v / v) final serum concentration, 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 single experiments under 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, pre-infusion serum samples from individual patients were subjected to 200.0 μg / ml IMAB362 (in vitro EC for IMAB362 in this system).80~100 It spiked at (equivalent to) [this value].

[0351] The inventors observed that in all assays, the IMAB362 antibody present in the patient serum after administration was highly active and induced cytotoxicity (Figure 5). The biological activity of IMAB362 was maintained for 28–32 days post-administration, and specific cell death still exceeded 48% across all dose groups. The overall difference between dose groups was surprisingly small, suggesting a saturation effect. Lower doses (33–300 mg / m²) were also observed. 2 In patients treated with ), a moderate decrease in specific mortality from 77.7–87.4% to 48.3–66.8% was observed over time, correlated with a decrease in serum antibody concentration (upper panel of Figure 5). The highest activity maintained stably over the long term was at 600 or 1000 mg / m². 2 This was observed in patients treated with IMAB362 (bottom panel of Figure 5).

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

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

[0354] Dynamics of IMAB362-mediated CDC in patient serum To measure the dynamics of IMAB362's CDC capacity in serum from patients in various dose groups, serum samples were collected 1, 7, 14, and 28 days after IMAB362 administration.

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

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

[0357] Summary and Conclusion Patients with GEC appear to retain their ability to induce both ADCC and CDC in target cells expressing CLDN18.2 in conjunction with IMAB362. Notably, the maximum specific lysis observed in ADCC and CDC, as well as the EC measured in relation to ADCC, were not impaired. 50 The highest score was observed in patient 0204, who had the most marked clinical and serum tumor antigen response.

[0358] Ex vivo analyses by the CDC regarding circulating IMAB362 at various time points after IMAB362 administration showed that even two weeks after administration, sufficient active IMAB362 was still present in the patient's circulation to induce potent ADCC and CDC.

[0359] Patient CDC activity linked to circulating IMAB362 decreases over time for reasons currently unknown.

[0360] (Example 3) Cytokine Cytokine serum levels 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. The inventors examined cytokines within the scope of this auxiliary analysis from the standpoint of defining potential biomarker candidates.

[0361] Cytokine levels were measured one day before IMAB362 infusion and on days 3 and 5 of the treatment cycle. The 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 necessary for T cell proliferation and function (IL-2) and cytokines necessary for NK cell proliferation (IL-2, IL-15).

[0362] Cytokines were analyzed by ELISA and flow cytometry (Interlab). Cytokines were analyzed 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-β, IL12p70, IL8, IL17A, IL23" and SOP-MU-IMM.M.0151.02 "Humanes 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 in 14 out of 15 patients (Table 9). Patient 0403 (300 mg / m²) 2 Cytokine levels were not measured for the following groups. Only serum cytokine levels above the reference range were analyzed for their 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 were measured for all patients on days 1, 3, and 5. Reference ranges are shown for each cytokine. Values ​​below or above the detection limit were set to the respective detection limit for calculation purposes. [Table 9]

[0365] Pro-inflammatory cytokine levels (IL-1, IL-6, IL-12, IFNγ, TNFα) were elevated above the normal range in 9 out of 14 patients (0104, 0105, 0201, 0203, 0204, 0112, 1202, 0112, 0205). IFNγ levels were elevated in 2 patients (0201, 1202), and TNFα levels were elevated in one of these 2 patients (0201). In both patients, IFNγ and TNFα levels were high before IMAB362 administration and decreased in subsequent days. IL-6 levels were elevated in 8 patients (0104, 0105, 0203, 1101, 0204, 0112, 1202, 0205). No clear pattern of change in IL-6 levels in relation to IMAB362 administration and the dose-response relationship was observed. Patient 0204 (600mg / m 2 IL-6 levels in patient IMAB362 were not elevated before administration, but rose significantly two days after infusion, a pattern not seen in any of the other patients. IL-1 and IL-12 levels remained within the normal range for all patients.

[0366] Anti-inflammatory cytokine levels (IL-4, IL-10) were elevated above the reference range in 6 out 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). The changes in anti-inflammatory cytokine levels did not show a clear pattern with respect to IMAB362 administration and dose-response relationships.

[0367] The cytokine levels of IL-2 and IL-15, which are essential for the function and proliferation of T cells and NK cells, were above the normal range in 9 out of 14 patients (0104, 0201, 0202, 0203, 1101, 1201, 0204, 1202, 0205). IL-2 levels were above the normal range in 6 patients (0201, 0202, 0203, 1101, 1202, 0205), and IL-15 levels were above the normal range in 5 patients (0104, 0202, 1201, 0204, 1202). Of the nine patients (0104, 0201, 0202, 0203, 1201, 1202, 0205) who had high IL-2 / IL-15 levels before administration, seven showed a decrease in cytokine levels in the following days: IL-2 / IL-15 levels were above the 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 with respect to serum IL-2 concentration. In all five patients (0201, 0202, 0203, 1202, 0205) with high pre-infusion levels of IL-2, a decrease to less than 50% of the pre-infusion level was observed on day 4 after administration. This decrease was 33 mg / m². 2 This was also observed in one patient (0201) who had significantly high IL-2 levels (354 pg / mL) before administration of IMAB362.

[0368] Different IL-2 concentration profiles were observed in patient 1101 (300 mg / m²). 2As shown in IMAB362), IL-2 levels were within the normal range before injection and on day 2 after injection, but high IL-2 concentrations were observed on day 4 after injection.

[0369] IL-15 levels decreased on day 4 post-administration in all four patients (0104, 0202, 1201, 1202) who had high pre-infusion IL-15 levels. This concentration profile was very similar to the observed IL-2 concentration profile, although the relative decrease in levels was not as pronounced.

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

[0371] In summary, the pre-treatment level analysis of patients showed that IL-6, IL-10, IL-2, and IL-15 were elevated in a substantial proportion of late-stage patients with gastroesophageal disease. In contrast, none of the patients had elevated levels of IL-1, IL-12, IL-4, IFNγ, and TNFα, or only one patient had elevated levels.

[0372] Analysis of cytokine level changes within the first 5 days after IMAB362 treatment yielded the following observations: In all 5 patients with high IL-2 levels, these levels decreased significantly, with 4 of the 5 patients reaching normal levels. Similarly, in all 4 patients with high IL-15 levels, a moderate decrease was observed after IMAB362 administration. Significant post-treatment decreases were also observed in one patient with high levels of IFNγ and TNFα, respectively. In contrast, IL-6 levels increased after IMAB362 administration, with 4 patients showing elevated IL-6 levels before treatment and 7 of the 14 patients showing elevated IL-6 levels on day 5 after treatment.

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

[0374] Patients underwent screening to measure the presence of the IMAB362 target, CLDN18.2, in their tumors. CLDN18.2 status was determined by immunohistochemistry using 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+ (2x intensity) staining intensity were enrolled in this trial. Inclusion and exclusion criteria were examined during the screening visit (V1). Patients were recruited from university hospitals specializing in the treatment of gastroesophageal cancer.

[0375] The patient had to meet all of the following selection criteria: • Metastatic, resistant, or recurrent advanced adenocarcinoma of the stomach or lower esophagus as confirmed by histological examination. • CLDN18.2 expression confirmed by immunohistochemistry in paraffin-embedded tumor tissue samples with a staining intensity of at least 2+ (0 to 3+ on a scale) in at least 50% of tumor cells. • At least one measurable site of the disease according to RECIST criteria (CT scan or MRI within two weeks prior to the second visit) • Age ≥ 18 • Written informed consent • ECOG Performance Status (PS) 0-1 or Karnovsky Index 70-100% • Average life expectancy > 3 months ·Platelet count ≧100,000 / mm 3 • Hemoglobin ≥ 10 g / dl • Bilirubin levels are normal. • AST and ALT levels < 2.5 times the upper limit of normal (ULN) (5 times ULN if liver metastases are present) • Creatinine <1.5 × ULN • For women with potential pregnancy (last menstrual period less than 2 years prior to registration): Negative pregnancy test (β-HCG) at baseline and use of two highly effective methods of contraception during the treatment period and for 8 weeks after the last infusion of the test drug. Male patients whose sexual partner is a woman who may become pregnant must use generally accepted methods of contraception during the treatment period and for 8 weeks after the last infusion of the study drug.

[0376] Patients who met one or more of the following exclusion criteria were not eligible for trial enrollment: • Pregnancy or breastfeeding • Prior severe allergic reaction or intolerance to monoclonal antibodies, including humanized or chimeric antibodies. • Less than 3 weeks after prior antitumor chemotherapy or radiation therapy • Other test drugs or devices used concurrently with or within 4 weeks prior to this test • Other combination anticancer therapies (not due to indications under the investigational treatment) • Known HIV infection or known active hepatitis (type A, B, or C) • Combination of vitamin K antagonists (e.g., coumadin, marcmar) and anticoagulants. • Therapeutic dose of heparin (prophylactic dose is acceptable) • Uncontrolled diseases, including but not limited to any of the following: - Progressive or active infection requiring parenteral antibiotics - Symptomatic congestive heart failure - Unstable angina - Uncontrolled hypertension - Clinically important arrhythmias - Myocardial infarction within the past 6 months - Gastric bleeding within the past 4 weeks -Symptomatic peptic ulcer - Clinical symptoms of brain metastases • A mental illness or social condition that makes compliance with the examination impossible.

[0377] All patients in all cohorts received repeated doses of IMAB362 every two weeks at their 2nd, 5th, 6th, 7th, and 8th visits (5 doses). The dose escalation procedure included the following cohorts with two different doses (antibody / body surface area) of IMAB362: Cohort 1: 300 mg / m² 2 Cohort 2: 600 mg / m² 2 Cohort 3: 600 mg / m² 2

[0378] The antibody solution was administered as a 2-hour intravenous infusion every two weeks. It was important that the infusion time was at least 2 hours. An infusion system (e.g., Infusomat® fmS) had to be used to control the infusion time. Infusion sets containing the test drug, which had been inspected for suitability by the manufacturer, had to be used for drug administration. The infusion of the test drug had to take place in the morning. A qualified physician had to be available during and for 24 hours after the infusion.

[0379] 37 patients received at least one treatment. Unfortunately, documentation for three of these patients is incomplete in the database; therefore, 34 patients will be included in the all-patient treatment set (APT set) for safety analysis. Patients 4, 6, and 24 received 300 mg / m², respectively. 2 IMAB362 Cohort 1, 600 mg / m² 2 IMAB362 Cohort 2 and 600 mg / m² 2 I was assigned to cohort 3 of IMAB362.

[0380] During the treatment period, one patient from Cohort 1, three patients from Cohort 2, and twelve patients from Cohort 3 discontinued the study before receiving five infusions of IMAB362 and completed their ninth visit (including a second tumor imaging) two weeks after the fifth infusion. These patients were replaced.

[0381] Two patients in Cohort 2 did not have measurable disease at baseline and were excluded from the efficacy analysis. Minor protocol deviations occurred, 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 levels (n=3; 8.8%), ALT or AST >2.5 ULN (>5 ULN in cases with liver metastases) (n=2; 5.9%), creatinine levels >1.5 ULN (n=1; 2.9%), and a long time window between the screening period and the start of treatment (>15 days) (n=2; 5.9%), but these did not result in exclusion from the analysis. One patient had a myocardial infarction within the past 6 months. Abandonment was observed.

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

[0383] Table 10 summarizes the results of cancer localization and histopathological malignancy classification. The mean time from initial diagnosis to screening visit for this study was 16 months (minimum 2.7 months / maximum 56 months). HER2 / neu expression status was 600 mg / m². 2 For most patients, the information was unavailable, excluding the five patients treated with [specific treatment method]. Of these five patients, one was HER2 / neu positive.

[0384] The TNM classification was identified for gastric (n=16) and esophageal or gastroesophageal junction (n=19) cancers. In the APT set, 25% of patients had primary gastric tumors classified as T1 or T2, 31% had T3, 25% had T4, and 19% were undetermined. At the time of diagnosis, 69% of patients in the APT set had at least one or two invasive lymph nodes as indicated by the N1 classification, and 56% had peritoneal metastases (M1). 69% of patients with esophageal or gastroesophageal junction cancer were diagnosed as ≥T3. At least one or two invasive lymph nodes (N1) were reported in 84% of patients. In addition, 84% of patients had peritoneal metastases.

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

[0386] Based on MedDRA SOC data, the most frequent clinically relevant medical history was surgery in 25 patients (73.5%), chemotherapy in 30 patients (88.2%), and radiation therapy in 7 patients (79.4%). In most cases, surgery consisted of surgical resection of organs (e.g., gastrectomy (72%), esophagectomy (16%), lymph node dissection (32%), cholecystectomy (20%)).

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

[0388] Of the 34 patients, a total of 30 (88.2%) had at least one comorbidity, i.e., a disease that was progressing on the day of infusion of the study drug. Based on MedDRA SOC data, the most common diagnoses were "gastrointestinal disorders" in 19 patients (56%), "general / 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%). Combination therapies were primarily drugs for acid-related disorders (17 patients; 50%), analgesics (12 patients; 35.3%), and drugs for GI disorders (10 patients; 29.4%).

[0389] A. Safety Evaluation Overall compliance with the study protocol was ensured because the investigational drug was administered by the investigator at the study site, and patients had to remain in the hospital for observation for at least 24 hours and up to 72 hours. Assignment of eligible subjects to dose cohorts was carried out precisely as specified by the study protocol (administered by DSMB). The time from the first screening visit to the last trial day and the defined trial period ranged from a minimum of 18 days to a maximum of 355 days. The average trial period was 106 days. Sixteen patients terminated the trial early before the ninth target visit.

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

[0391] 300 mg / m² 2 (n=4) or 600 mg / m² 2A safety analysis was performed on the APT set (n=30), including all 34 patients who received at least one dose. 241 physician-reported adverse events were coded according to the MedDRA dictionary and replaced with base terms. Adverse events using base terms were counted only once per patient (even if the same adverse event occurred more than once for that patient during the study period). The highest NCI-CTC grade for each patient was recorded. 32 patients (94%) had at least one adverse event during the study period (regardless of relationship). No adverse events were recorded for 2 patients. Overall, 6 patients (18%) did not experience any potentially drug-related adverse events. 104 drug-related adverse events using base terms were reported for 28 patients. Eight potentially drug-related serious adverse events were reported for 4 patients. Low-dose group (300 mg / m²) 2 Due to the small number of patients, a detailed comparison between the two dose groups was not possible. 300 mg / m² 2 Cohort and 600 mg / m² 2 The incidence of patients experiencing related adverse events in the groups (cohorts 2 and 3) was 75% and 83%, respectively.

[0392] Overall, the most frequently reported AEs from SOC were "gastrointestinal disorders" (27 / 34 patients, 79.4%) and "general / systemic disorders and administration site conditions" (26 / 34 patients, 76.5%). Based on MedDRA PT, 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 being related to the study drug. These treatment-related AEs were categorized into 104 different basic terms and were observed in 28 / 34 patients.

[0393] Most adverse events were mild to moderate. Eight patients (23.5%) experienced moderate drug-related events during treatment, and twelve patients (35.3%) experienced serious drug-related events during treatment.

[0394] Severe drug-related adverse events include vomiting at 300 mg / m². 2 Two patients in the dose group experienced nausea, with one patient experiencing complications. 600 mg / m² 2 In the dose group, 10 patients experienced serious drug-related adverse events. Of the 6 patients who experienced vomiting, 3 also experienced nausea. One patient experienced hypersensitivity (allergic reaction), one experienced excessive salivation, one experienced dehydration, and one experienced hypoalbuminemia. The last two patients also reported vomiting and nausea. Two patients experienced hypersensitivity (allergic reaction) during the infusion of the study drug, one of which was classified as moderate and the other as severe. Both patients recovered after discontinuation of the infusion.

[0395] Of all reported adverse events occurring under treatment, 12 out of 34 patients required the investigational drug to be effective due to adverse events (AEs). In 7 cases (21%), AEs resulted in permanent discontinuation of the study. Underlying adverse events were drug-related in 3 patients (hypersensitivity (allergic reaction) (n=2), vomiting, and abdominal pain) and non-drug-related in the other 4 patients (general deterioration of health (n=3), pneumonia). One patient required dose reduction, and another patient delayed drug administration for 4 days due to severe vomiting with nausea. Infusion was interrupted / extended in 3 patients. 27 patients (79%) received concomitant therapy for AEs. 11 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, four of which were study drug-related. Related adverse events such as vomiting, nausea, and GI bleeding and dehydration were determined by the investigators 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 patients. None of the deaths were classified by the investigators as study drug-related.

[0397] One patient was a 45-year-old Caucasian male with a lean diet (BMI 19.3) and good overall health (ECOG Performance Status Grade 1, Karnovsky Index 80%).

[0398] The patient received 300 mg / m² every two weeks on November 4, November 22, 2011, and as a third dose on December 6. 2 The patient received an infusion of IMAB362. Prior to the trial, the patient had already experienced grade 1 nausea and vomiting. On November 7, 2010, grade 3 vomiting was diagnosed. This was assessed as severe, and the patient had to be hospitalized. The vomiting became grade 1 on November 17, 2010, and eventually stopped completely, allowing the patient to be discharged on the same day. Prior to the second and third infusions of IMAB362, the patient was treated with potent premedication (arizaprid, aprepitant, metoclopramide, dimehydrinate) as a preventative measure against nausea and vomiting, and therefore did not experience nausea or vomiting again. The investigator assessed the vomiting as being related to the study drug. The report was accepted by the organizer on January 19, 2011, and the SAE was determined to be unexpected but related to the study drug, and was therefore reported as SUSAR.

[0399] One patient was a 77-year-old male from the Caucasus. At screening, he had a normal diet (BMI 24) and was in very good general health (ECOG Performance Status: Grade 0, Karnovski Index: 100%). Prior to the trial, the patient had already experienced nausea and was therefore treated with metoclopramide as needed. The trial had to be terminated early due to the patient's death, and he received a dose of 600 mg / m² on November 9, 2011. 2The patient received only one dose of IMAB362. Left lung pleural effusion was diagnosed by X-ray prior to infusion and reported as SAE. The following morning, hematemesis occurred. Following administration of pantoprazole and ondansetron iv 8 mg, vomiting decreased and hematemesis resolved on 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 investigators assessed this event as drug-related. The report was received by the organizer on November 10, 2011, and the event was determined to be unexpected but drug-related and therefore reported as SUSAR. The patient's general condition deteriorated, renal failure developed, and unfortunately, the patient died on December 6, 2011.

[0400] One patient was a 42-year-old Caucasian male with good nutritional status (BMI 26) and excellent overall health (ECOG Performance Status Grade 0; Karnovsky Index: 100%). This patient was taking 600 mg / m². 2The patient received two infusions of IMAB362. On March 20, 2012, the patient received the first dose of the study drug. The patient developed nausea and severe vomiting, requiring a reduction in the infusion rate 35 minutes after the infusion. The symptoms were treated with pantoprazole 40 mg and granisetron 3 mg, as well as butylscopolamine IV (2 vials) and aprepitant IV (80 mg). This serious adverse event resulted in prolonged hospitalization. The investigators assessed this event as being related to the study drug. The SAE report was accepted by the organizer on March 21, 2012, and the event was determined to be expected and related to the study drug. A few days later, on March 24, 2012, the patient had to be hospitalized again due to severe dehydration caused by nausea and vomiting. Furthermore, the patient suffered from upper stomach pain. The patient received metamisole IV (1 g), a buprenorphine patch, and an infusion for hydration. On March 30, 2012, the symptoms subsided and the patient recovered from dehydration. The investigator assessed this event as unrelated to the study drug. The SAE report was accepted by the organizer 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 metoclopramide po30 drops and dimenhydrinate iv1 vial. On April 5, 2012, the symptoms worsened and were assessed as severe. In addition, the patient suffered from dysphagia, and therefore food intake was significantly reduced. On April 15, 2012, the symptoms disappeared. The investigator assessed this event as related to the study drug. The SAE report was accepted by the organizer 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 male with good nutritional status (BMI 26) and good overall health (ECOG Performance Status Grade 1; Karnovsky Index: 90%). From November 8, 2011 to January 3, 2012, the patient received 600 mg / m² every two weeks. 2The patient received five scheduled administrations of IMAB362. The first administration of IMAB362 occurred on November 8, 2011. During and after this administration, the patient developed nausea and vomiting. The symptoms worsened on November 9, 2011. Following treatment with metoclopramide, the symptoms resolved one day later. The investigators assessed this event as being related to the investigational drug. A SAE report was accepted by the organizer on November 10, 2011, and the event was determined to be expected and related to the investigational drug. The third administration was performed on December 6, 2011. The patient experienced moderate vomiting and mild nausea and was treated with clemastine, ranitidine, and ondansetron. Vomiting lasted one day. Nausea persisted for seven days. The study was terminated on January 16, 2012, due to disease progression. No follow-up visits were performed.

[0402] In conclusion, IMAB362 was found to be safe and well-tolerated in a large number of previously treated patient populations with advanced adenocarcinoma of the stomach, esophagus, or gastroesophageal junction. Overall, the most frequently reported adverse events from the situation of care (SOC) were "gastrointestinal disorders" (27 / 34 patients, 79.4%) and "general / systemic disorders and administration site conditions" (26 / 34 patients, 76.5%).

[0403] Based on MedDRA PT data, the most frequently recorded adverse events (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 researchers as being related to the study drug. These treatment-related AEs were observed in 28 of the 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] Based on MedDRA PT data, most associated adverse events were mild to moderate, with nausea (50%), vomiting (47%), fatigue (27%), abdominal pain (15%), peripheral edema (15%), loss of appetite (12%), and diarrhea (12%) occurring in more than 10% of patients.

[0406] Two patients experienced hypersensitivity (allergic reactions) during the infusion of the test drug, one of which was classified as moderate and the other as severe. Both patients recovered after discontinuing the infusion.

[0407] No abnormal CTC grade 4 (life-threatening) or 5 (fatal) clinical laboratory values ​​related to the study drug have been reported.

[0408] Twelve patients (35.3%) experienced severe drug-related adverse events during treatment. Severe drug-related adverse events included vomiting at 300 mg / m². 2 Two patients in the dose group experienced nausea, with one patient experiencing complications. 600 mg / m² 2 In the dose group, 10 patients experienced severe drug-related adverse events. Of the 6 patients who experienced vomiting, 3 also experienced nausea. One patient experienced hypersensitivity (allergic reaction), one experienced excessive salivation, one experienced dehydration, and one experienced hypoalbuminemia. The last 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 the process of recovery, 11 patients had not recovered from at least one AE, and the status of 2 patients was unknown. Of the 11 patients who had not recovered from at least one drug-related adverse event, 9 had gastrointestinal disorders (4 with nausea, 2 with vomiting).

[0410] Thirteen patients had 31 recorded 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. In four patients, treatment-related gastrointestinal SAEs such as vomiting (n=4), nausea (n=2), dehydration (n=1), and GI bleeding (n=1) were determined to be treatment-related. One of these patients who experienced vomiting was on a 300 mg / m² dose. 2 One patient was treated with [medication type], while the other three received 600 mg / m². 2 They were treated with IMAB362. Three of these four patients recovered, with the exception of one who died due to unrelated renal failure.

[0411] The incidence of drug-related adverse events is 300 mg / m². 2 Dose groups and 600 mg / m² 2 The incidence of adverse events was similar between the dose groups, at 75% and 83% of patients, respectively. The frequency and severity of nausea, vomiting, and fatigue were also similar between the two dose groups. There was no clear relationship between dose and the frequency / severity of adverse events.

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

[0413] Generally speaking, 300 mg / m² 2 and 600 mg / m² 2 IMAB362, administered in multiple doses, was found to be safe, well-tolerated, and vomiting and nausea were the most common associated adverse events.

[0414] B. Evaluation of pharmacokinetics and immunogenicity The preliminary drug concentration data for repeated-dose administration of IMAB362 is 300 mg / m² each. 2 and 600 mg / m² 2Data is available for 4 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, 14 days, and 4–8 weeks after the end of the final infusion. No analytes were detected in the pre-administration samples from individual patients assigned to cohorts 1–3.

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

[0418] A time-dependent decrease in IMAB362 concentration was observed in serum samples collected at subsequent points in time (from V3 to V5) (Figure 8). At the fifth visit, prior to the second infusion, the minimum serum levels measured for Cohort 1 ranged from 11.3 μg / mL to 36.8 μg / mL (mean 22.5 ± 10.5 μg / mL), while for Cohorts 2 and 3 combined, the levels measured ranged from 17.0 μg / mL to 100.2 μg / mL (mean 54.5 ± 29.0 μg / mL).

[0419] At the 8th visit (day 57), prior to the 5th infusion, the minimum serum levels measured for Cohort 1 ranged from 32.4 μg / mL to 67.1 μg / mL (mean 46.1 ± 18.5 μg / mL), while levels ranging from 28.3 μg / mL to 301.6 μg / mL (mean 147.2 ± 93.1 μg / mL) were measured for Cohorts 2 and 3 (Table 12).

[0420] After the injection during the 8th visit, c max The 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, 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 The 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 level of IMAB362 was 300 mg / m². 2 In patients treated with 600 mg / m², serum concentrations of IMAB362 decreased to below the desired level of 50–100 μg / ml during a two-week cycle. 2 At this dose, however, serum levels of IMAB362 remained above 50 μg / ml in the majority of patients even two weeks after the initial administration. Dose levels exceeded 50 μg / ml 7–29 days (mean 15 days) after the fifth dose (mean 151.3 ± 90.1 μg / mL).

[0423] Table 12: 300 and 600 mg / m² 2 Descriptive pharmacokinetic data of repeated-dose IMAB362 300 mg / m² 2Four patients (cohort 1) treated with repeated doses of 600 mg / m² and 600 mg / m². 2 Mean ± sd serum concentration (μg / ml) of IMAB362 in up to 30 patients treated with repeated administrations (30 patients for the first infusion, 12 patients for the fifth infusion) (combined cohorts 2 and 3). [Table 12]

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

[0425] Table 13: Accumulation of IMAB362 after repeated injections To determine the accumulation rate, the IMAB362 concentration ratio was calculated before the 6th, 7th, 8th, and 9.x (responder treatment) visits and before the 2nd infusion (5th visit). [Table 13]

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

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

[0428] A time-dependent decrease in IMAB362 concentration was observed in serum samples collected at subsequent points in time (from V3 to V5). At the fifth visit, prior to the second infusion, the minimum serum levels measured for Cohort 1 ranged from 11.3 μg / mL to 36.8 μg / mL (mean 22.5 ± 10.5 μg / mL), while for Cohorts 2 and 3 combined, the levels measured ranged from 17.0 μg / mL to 100.2 μg / mL (mean 54.5 ± 29.0 μg / mL).

[0429] At the 8th visit (day 57), prior to the 5th infusion, the minimum serum levels measured for Cohort 1 ranged from 32.4 μg / mL to 67.1 μg / mL (mean 46.1 ± 18.5 μg / mL), while levels ranging from 28.3 μg / mL to 301.6 μg / mL (mean 147.2 ± 93.1 μg / mL) were measured for Cohorts 2 and 3.

[0430] After the 5th injection during the 8th visit, c max The 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, 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 The 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 level of IMAB362 was 300 mg / m². 2 In patients treated with 600 mg / m², serum concentrations of IMAB362 decreased to below the desired level of 50–100 μg / ml during a two-week cycle. 2At this dose, however, serum levels of IMAB362 remained above 50 μg / ml in the majority of patients even two weeks after the initial administration. Dose levels exceeded 50 μg / ml 7–29 days (mean 15 days) after the fifth dose (mean 151.3 ± 90.1 μg / mL).

[0433] C. Evaluation of antitumor activity Complete Analysis Set (FAS): The study included all subjects who had taken the investigational drug at least once and for whom post-treatment efficacy data was available.

[0434] At the time of analysis, 50 patients were taking 600 mg / m². 2 They were enrolled at the specified dose. Of these, nine were recently included, and due to their recent enrollment, further data is not currently available. Ten patients have not undergone a second tumor imaging and are therefore not included in the FAS set. The FAS set includes 31 patients.

[0435] The average age was 57 years, ranging from 35 to 77 years. 90% of patients in the FAS set had an average Karnovski index (ranging from 70-100%). The majority of patients (81%) had received prior treatment with at least one chemotherapy regimen. Six patients had not received prior chemotherapy regimens.

[0436] [Table 14]

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

[0438] Due to the terminal stage of the disease, patients had an average of 2.0 metastatic sites (ranging from 1.0 to 4.0). The most prominent were 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 was performed 8–11 weeks after the first infusion, except for two patients, for whom the first tumor re-evaluation was performed 6 weeks later.

[0440] [Table 15]

[0441] In six of the twelve patients with clinically controlled disease, at least one tumor marker (CEA; CA19-9; CA125; CA15-3) that was elevated at baseline decreased by 35–76% throughout the study period. In three patients, all tumor markers fell below the cutoff values, and tumor marker results were unavailable for one patient.

[0442] Interestingly, the four patients with progressive disease, who represented the best response, also experienced a reduction in tumor markers between 29% and 54% during the trial period.

[0443] Partial responses were achieved after 2.3 months of treatment (2 patients), 6.5 months (1 patient), and 4.8 months (1 patient), respectively. A partial response (PR) was confirmed in one patient and lasted for a further 4.4 months, resulting in a PFS of 9.2 months for this patient. For the other three patients, confirmation was made at 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 is not yet available; nd undetectable. * -The event did not occur until November 2012, so it is unclear whether an examination was performed or the exact date is currently unknown. The last follow-up date was used for each case. #-Tumor markers are below the cutoff value. Therefore, they are 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 (minimum 4 weeks; maximum 40 weeks). Due to the limited availability of events, the mean progression-free survival for patients with clinical benefit (PR+SD), as shown in Figure 9, has limited value. Patients without clinical benefit (PD) had a mean progression-free survival of 9 weeks (minimum 4 weeks; maximum 11 weeks) (Figure 9).

[0446] There were no differences in age (mean 56 vs. 59 years), no prior chemotherapy regimen (mean 1.9 vs. 2.1), or Karnovski index (mean 89 vs. 88%) between patients with clinical benefit (PR or SD as best effect) and patients with progressive disease (PD as best effect). Only the number of metastatic sites was lower in the responder group, mean 1.9 compared to mean 2.3 in the non-responder group. This 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 and mean number of stained cells were higher in patients with clinical benefit, at 77% vs. 67% and 71% vs. 60%, respectively.

[0448] Differences were also observed in the location of metastases. In patients with clinical benefit, the frequency of pleural effusion (25% vs. 5%), peritoneal carcinomatosis (42% vs. 11%), and ascites (42% vs. 16%) was higher compared to patients with the best-case progressive disease. The presence of liver metastases was far less frequent in patients with clinical benefit (17% vs. 58%).

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

[0450] Of the 31 patients in the FAS group, two had significant protocol violations (no target lesions), and nine patients did not complete the trial protocol by the ninth visit, thus receiving fewer than five required infusions of IMAB362. The PP group included 20 patients.

[0451] The average age was 60 years, with patients ranging from 35 to 77 years. 90% of patients in the PP set had an average Karnovski index (ranging from 70-100%). The majority of patients (80%) had received prior treatment with at least one chemotherapy regimen. Four patients had not received prior chemotherapy regimens.

[0452] [Table 17]

[0453] The mean number of prior chemotherapy regimens was 2.0 (ranging from 0 to 5). Chemotherapy regimens for gastroesophageal cancer consisted mainly of various combinations of 5-FU derivatives, platinum compounds, taxanes, epirubicin, irinotecan, trastuzumab for HER2 / neu-positive patients, and other investigational drugs. During the PP set, 80% of patients had received at least one dose of 5-FU or capecitabine, and 75% had been treated at least once with a platinum compound prior to enrollment. Five patients (25%) had been prior to treatment with trastuzumab or other investigational drugs. Further details can be found in Table 17. Five patients (25%) had also received radiotherapy prior to the start of the study.

[0454] Due to the terminal stage of the disease, patients had an average of 3.0 metastatic sites (ranging from 1.0 to 4.0). The most prominent were 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 was performed 8–11 weeks after the first infusion, with the exception of one patient, for whom the first tumor re-evaluation was performed 6 weeks later (Table 18).

[0456] [Table 18]

[0457] In six of the ten patients with clinically controlled disease, at least one tumor marker (CEA; CA19-9; CA125; CA15-3) that was elevated at baseline decreased by 35–76% throughout the study period. In two patients, all tumor markers fell below the cutoff values, and tumor marker results were unavailable for one patient.

[0458] Interestingly, the three patients with progressive disease, who represented the best response, also experienced a 29 to 54% reduction in tumor markers during the trial period.

[0459] Partial responses were achieved after 2.3 months of treatment (2 patients), 6.5 months (1 patient), and 4.8 months (1 patient), respectively. A partial response (PR) was confirmed in one patient and lasted for a further 4.4 months, resulting in a PFS of 9.2 months for this patient. For the other three patients, confirmation was made at 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 is not yet available; nd undetectable. * -The study was either terminated because no events occurred until November 2012, or the exact date is currently unknown. The last day of follow-up was used for each case. #-Tumor markers were below the cutoff value. Therefore, they were 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 (minimum 9 weeks; maximum 40 weeks). Due to the limited availability of events, the mean progression-free survival for patients with clinical benefit (PR+SD), shown in Figure 10, has limited usefulness. Patients without clinical benefit (PD) had a mean progression-free survival of 10 weeks (minimum 9 weeks; maximum 10 weeks) (Figure 10).

[0462] There were no differences in age (mean 57 vs. 62 years), no prior chemotherapy regimen (mean 2.1 vs. 2.0), or Karnovski index (mean 88 vs. 88%) between patients with clinical benefit (PR or SD as best outcome) and patients with progressive disease (PD as best outcome). Only the number of metastatic sites was higher in patients with clinical benefit (mean 3.0) compared to patients without clinical benefit (mean 2.2). This 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 and mean number of stained cells were higher in patients with clinical benefit, at 76% vs. 66% and 70% vs. 66%, respectively.

[0464] Differences were also observed regarding the location of metastases. In patients with clinical benefit, the frequency of pleural effusion (30% vs. 10%), peritoneal carcinomatosis (40% vs. 0%), and ascites (40% vs. 20%) was higher compared to patients with the best-case progressive disease. The presence of liver metastases was far less frequent in patients with clinical benefit (20% vs. 70%).

[0465] In conclusion, tumor status (according to RECIST) was compared to baseline two weeks after the fifth IMAB362 infusion (V9). For 31 patients (FAS), at least one staging classification after baseline was available. Patients were enrolled in the terminal stage of the disease with an average of 2.0 prior chemotherapy doses and an average of 2.0 metastatic sites.

[0466] Confirmed partial responses were evaluated in four patients, resulting in an overall response rate of 13%. Of these, three are currently in the process of progression, 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 of these 12 patients with clinical benefit ranged from 6 to 40 weeks. The mean could not be calculated for seven of these patients as events have not been recorded to date. In nine of the patients with clinical benefit, at least one tumor marker was elevated at baseline, and in six of these, it simultaneously decreased by -35 to -76%. Interestingly, the four patients with progressive disease who had the best response also showed a decrease of -29 to -54% in at least one elevated tumor marker during the course of the trial. The mean overall progression-free survival 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, while 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 frequency of liver metastases.

[0468] Based on the current dataset, patients with clinical benefit appear to have had a higher number of cells with positive IHC staining.

[0469] Furthermore, supplementary data were collected in selected patients, and these data demonstrated that the patients' 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, given the antitumor activity (partial response, stable disease, and reduction of tumor markers), IMAB362 deserves further investigation.

[0471] D. Overall conclusion This clinical trial was designed as a Phase IIa, multicenter, non-randomized, patient-to-patient dose-escalation, open-label clinical trial involving three cohorts. Patients eligible for this clinical trial were required to be anti-treatment to standard care or not receiving widely accepted therapies.

[0472] For this interim report, 34 patients were eligible for evaluation regarding safety analysis (APT set), of which 4 were in Cohort 1 (300 mg / m²). 2 ), 6 people in Cohort 2 (600 mg / m²) 2 ) and 20 people in Cohort 3 (600 mg / m²) 2 IMAB352 was registered in the Phase IIa trial. IMAB362, administered on a multi-dose schedule, was safe and well-tolerated in many previously treated 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. No Grade 4 or Grade 5 adverse events (including laboratory parameters) were observed in this Phase IIa trial or the previous Phase I trial. It is noteworthy that IMAB352 has not caused any Grade 4 associated AEs to date, given that the majority of registered monoclonal antibodies are associated with life-threatening Grade 4 and Grade 5 side effects. The indication for bevacizumab in metastatic breast cancer was revoked by the FDA in November 2011, following initial preliminary approval in 2008. Bevacizumab did not extend life and caused severe hypertension and bleeding, including bowel and nasal septal perforation. Cetuximab can cause acne-like rashes and grade 3-4 infusion reactions, anaphylaxis, and cardiac arrest requiring prophylaxis with the antihistamine diphenhydramine before treatment. Trastuzumab is still widely used, but it causes symptomatic cardiac failure in 2-7% of patients, a fact that has been known for over 10 years.

[0473] The primary metric for evaluating potential antitumor activity was tumor status as assessed by RECIST. Thirty-one patients received at least one assessment of this metric after baseline, and therefore were included in the FAS. Among these 31 patients (RR 13%, DCR 39%) who had received numerous prior therapies, four partial responses (PR) and eight stable diseases (SD) are very favorably comparable to response results in other Phase II trials of targeted monotherapy approved as second-line or end-of-life treatment.

[0474] The EGFR antagonist cetuximab achieved a 3% response rate (plus a further 7% SD) in terminal GEC (mostly with two or more metastatic sites and prior treatment) measured at 8 weeks in a phase II trial involving 30 patients. In a second trial involving 55 terminal patients, cetuximab resulted in a 5% RR and a further 11% SD measured at 8 weeks. Similar response rates were achieved in EGFR-positive anti-mCRC patients, and cetuximab was subsequently approved for this indication.

[0475] Sunitinib and erlotinib were tested in various Phase II trials involving approximately 150 patients in total in patients with end-stage GEC. The disease control rate (DCR) at 6–8 weeks varied between 16–39%, and response rates were reported to be 3–7%, respectively.

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

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

[0478] (Example 5) IMAB362-induced nausea / vomiting IMAB362 has been shown to induce nausea / vomiting up to NCI-CTC grade 3. Its symptomatology can be described as follows: (i) non-dose-dependent, (ii) acute onset mainly within 5 minutes of infusion, which may persist after the end of infusion, (iii) beginning with upper gastric spasms and excessive salivation, (iv) vomiting may begin without warning, (v) rare in patients who have undergone total gastrectomy, (vi) reaction occurs at the first infusion, while symptoms worsen with each cycle.

[0479] The fact that these adverse reactions rarely occur in patients who have undergone total gastrectomy suggests that the underlying mechanism is on-target action. With regard to IMAB362, vomiting is more frequent than nausea and is often reported to occur without prior nausea. Onset can be both acute and delayed. We hypothesize that a small amount of IMAB362 binds to a tightly junctioned epitope that is only partially accessible. This leads to localized disruption of the tight junction and leakage of gastric acid into the submucosa. The resulting tissue reaction and spasms initiate a cascade of nausea / vomiting.

[0480] Therefore, the recommended measures are prevention with effective antiemetics and protection of the gastric mucosa.

[0481] For example, patients should receive prophylactic antiemetics before starting medication. For both prophylactic and curative intervention, a combination of an NK-1 receptor (e.g., aprepitant / Emend) and a 5-HT3 receptor blocker (e.g., ondansetron / Zofran) is recommended and may be expanded to include further compounds. Antiemetics should preferably be administered for at least the first three days of each cycle. Prophylactic administration of butylscopolamine / buscopan immediately before each IMAB362 infusion may be considered.

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

[0483] Therefore, prophylactic treatment with antiemetics and protection of the gastric mucosa may be initiated immediately before IMAB362 infusion. For example, the following combinations may be administered, preferably intravenously: • NK-1 RA: For example, aprepitant / Emend (150 mg IV) • 5-HT3 RA: For example, palonosetron (0.25 mg IV), ondansetron / Zofran (8 mg IV), granisetron (3 mg IV) Butylscopolamine / buscopan • Proton pump inhibitors: Pantoprazole

[0484] Depending on the circumstances, metoclopramide / MCP, lorazepam, and / or atropine may also be administered.

[0485] IMAB362 is an antibody that is highly dependent 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 when other compounds have failed.

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

[0487] Prevention with antiemetics and protection of the gastric mucosa can be continued, for example, until the third day of each cycle.

Claims

1. A method for treating or preventing cancer, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered in such a manner that it provides a serum level of at least 40 μg / ml.

2. The method according to claim 1, wherein the serum level provided is between 40 μg / ml and 700 μg / ml.

3. The method according to claim 1 or 2, wherein the serum level is provided for at least 7 days.

4. The above method provides at least 300 mg / m² ２ The method according to any one of claims 1 to 3, comprising administering a dose of the antibody.

5. A method for treating or preventing cancer, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein the antibody is administered at a dose of at least 300 mg / m². ２ A method of administration using the specified dosage.

6. A method for treating or preventing cancer, comprising administering to a patient an antibody having the ability to bind to CLDN18.2, wherein at least 50% of the patient's cancer cells are CLDN18.2 positive, and / or at least 40% of the patient's cancer cells are positive in terms of surface expression of CLDN18.

2.

7. The method according to any one of claims 1 to 6, wherein the treatment of the cancerous disease results in the achievement of a stable disease.

8. A method for achieving a stable disease in a cancer patient, comprising administering to the patient an antibody having the ability to bind to CLDN18.

2.

9. The method according to claim 7 or 8, wherein a stable disease is achieved for at least two months.

10. The method according to any one of claims 1 to 9, wherein the antibody is administered in a single dose or in multiple doses.

11. A method for treating or preventing cancer, comprising administering an antibody having the ability to bind to CLDN18.2 to a patient, wherein the antibody is administered in multiple doses.

12. The method according to claim 10 or 11, wherein the antibody is administered in at least three doses.

13. The method according to any one of claims 10 to 12, wherein the administration of the antibody is performed at a time interval of at least 7 days.

14. The method according to any one of claims 1 to 13, further comprising administering one or more substances selected from the group consisting of antiemetics, antispasmodics, parasympathetic blockers, and gastric mucosal protective agents.

15. A method for treating or preventing cancer, comprising administering to a patient one or more substances selected from the group consisting of an antibody having the ability to bind to CLDN18.2, and antiemetics, antispasmodics, parasympathetic blockers, and gastric mucosal protective agents.

16. The method according to claim 14 or 15, wherein the method comprises administering to the patient a neurokinin 1 (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 two or more combinations thereof, an antispasmodic such as butylscopolamine (e.g., Buscopan), and a proton pump inhibitor such as pantoprazole (e.g., Pantozol).

17. The method according to any one of claims 1 to 16, wherein the antibody is administered by i.v. injection.

18. The method according to claim 17, wherein the injection described in i.v. above lasts for 1 to 4 hours.

19. A method for determining the responsiveness of a cancer patient to the treatment or prevention of cancer, comprising administering an antibody having the ability to bind to CLDN18.2, the method comprising measuring the blood level of one or more markers in the patient, wherein the one or more markers are selected from the group consisting of CA125, CA15-3, CA19-9, CEA, IL-2, IL-15, IL-6, IFNγ, and TNFα.

20. The method according to claim 19, wherein the level is measured in blood, plasma, or serum.

21. The method according to claim 19 or 20, wherein the one or more markers are selected from the group consisting of CA125, CA15-3, CA19-9, CEA, IL-2, IL-15, IFNγ, and TNFα, and a decrease in the level of at least one of the markers after administration of the antibody indicates that the patient is responsive to the treatment or prevention of cancer.

22. The method according to claim 19 or 20, wherein the marker is IL-6, and an increase in the level of the marker after administration of the antibody indicates that the patient is responsive to the treatment or prevention of cancer.

23. A method for determining whether a cancer patient is suitable for the treatment or prevention of a cancerous disease, comprising administering an antibody having the ability to bind to CLDN18.2, the method comprising the step of measuring the percentage of CLDN18.2-positive cancer cells.

24. The method according to claim 23, wherein the level of at least 50% CLDN18.2-positive cancer cells indicates that the patient is suitable for treatment or prevention of cancer.

25. The method according to claim 23 or 24, wherein the level of at least 50% of cancer cells positive for surface expression of CLDN18.2 indicates that the patient is suitable for treatment or prevention of cancer.

26. The method according to any one of claims 1 to 25, wherein the antibody mediates cell death by one or more of complement-dependent cell-mediated lysis (CDC), antibody-dependent cell-mediated lysis (ADCC), induction of apoptosis, and inhibition of proliferation.

27. The antibody is (i) 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 The method according to any one of claims 1 to 26, wherein the antibody is selected from the group consisting of an antibody produced by and / or available from a clone deposited under ACC2810, an antibody that is a chimeric or humanized form of the antibody contained in (ii)(i), an antibody having the specificity of the antibody contained in (iii)(i), and an antibody containing an antigen-binding portion or antigen-binding site, particularly a variable region, of the antibody contained in (iv)(i), and preferably an antibody having the specificity of the antibody contained in (i).

28. The method according to any one of claims 1 to 27, wherein the cancer is gastroesophageal cancer.

29. The method according to any one of claims 1 to 28, wherein the cancer is metastatic, anti-treatment, or recurrent advanced gastroesophageal cancer.

30. The method according to any one of claims 1 to 29, wherein the patient has previously received treatment with at least one agent selected from the group consisting of pyrimidine analogs, platinum compounds, epirubicin, docetaxel, and antidotes for antitumor drug treatment.

31. The method according to any one of claims 1 to 30, wherein the patient has an ECOG performance status between 0 and 1 and / or a Karnovski index between 70 and 100%.

32. The method according to any one of claims 1 to 31, wherein the patient is a human patient.

33. The method according to any one of claims 1 to 32, wherein CLDN18.2 has an amino acid sequence according to Sequence ID No. 1.

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