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

Abstract

To provide a combination therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as pancreatic cancer and metastases thereof.SOLUTION: Provided herein is a method of treating or preventing pancreatic cancer in a patient, comprising administering to the patient (i) an antibody having the ability of binding to CLDN 18.2 and (ii) an agent for stabilizing or increasing the expression of CLDN 18.2.SELECTED DRAWING: None

Description

[Technical field]

[0001] Pancreatic cancer is one of the most lethal cancers. Mortality approaches 100% due to the tendency for early metastatic spread and the disease's high resistance to radiation and chemotherapy. Considering that 27,000 new cases are diagnosed in North America and 68,000 in Europe each year, there is an urgent need to develop new therapeutic strategies to reduce the mortality rate of pancreatic cancer patients. [Background technology]

[0002] The splice variant 2 of the tight junction molecule claudin 18 (claudin 18.2 (CLDN18.2)) is a member of the claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa transmembrane protein that contains four membrane-spanning domains and two small extracellular loops. In normal tissues, with the exception of the stomach, there is no detectable expression of CLDN18.2 by RT-PCR. Immunohistochemistry with a CLDN18.2-specific antibody reveals that the stomach is the only positive tissue. CLDN18.2 is a highly selective gastric lineage antigen that is expressed exclusively on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the process of malignant transformation and is therefore frequently presented on the surface of human gastric cancer cells. Furthermore, this pan-tumor antigen is ectopically activated at significant levels in esophageal, pancreatic and lung adenocarcinomas.

[0003] IMAB362, a chimeric IgG1 antibody against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and bundles four independent and highly potent mechanisms of action. After target binding, IMAB362 mediates cell killing by induction of apoptosis induced by ADCC, CDC and cross-linking of the target at the tumor cell surface as well as direct inhibition of proliferation. Thus, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo.

[0004] The toxicity and PK / TK profile of IMAB362 has been thoroughly investigated in mice and cynomolgus monkeys, including a dose range finding study, a 28-day repeat dose toxicity study in cynomolgus monkeys and a 3-month repeat dose toxicity study in mice. Repeated doses of IMAB362 iv are well tolerated in both mice (maximum treatment duration 3 months with once weekly dosing at the highest dose level of 400 mg / kg) and cynomolgus monkeys (up to 5 weeks with once weekly application up to a maximum of 100 mg / kg). No signs of systemic or local toxicity are induced. In particular, gastric toxicity has not been observed in any toxicity study. IMAB362 does not induce immune activation and cytokine release. No adverse effects on male or female reproductive organs were recorded. IMAB362 does not bind to tissues lacking targets. Biodistribution in mice indicates that the reason for the absence of gastric toxicity is likely compartmentalization of tight junctions at the luminal site of healthy gastric epithelium, which would greatly reduce the accessibility of the IMAB362 epitope.

[0005] IMAB362 is in early clinical trials. A Phase I clinical trial was conducted in humans. Five dose cohorts (33 mg / m 2, 100 mg / m 2 , 300 mg / m 2 , 600 mg / m 2 , 1000mg / m 2 ) received a single intravenous dose of IMAB362 and were observed for 28 days. IMAB362 was very well tolerated, with no relevant safety findings in patients. One patient experienced a significant decrease in all measured tumor markers within 4 weeks of treatment. Repeated doses of IMAB362 are being administered in an ongoing Phase IIa clinical trial. Summary of the Invention [Problem to be solved by the invention]

[0006] Herein, we present data demonstrating that chemotherapeutic agents can stabilize or increase the expression of CLDN18.2 on the surface of pancreatic cancer cells, resulting in enhanced druggability of CLDN18.2 by anti-CLDN18.2 antibodies, such as IMAB362. Synergism has been observed between certain chemotherapy regimens, particularly those used for pancreatic cancer treatment, and anti-CLDN18.2 antibodies, such as IMAB362. Human cancer cells pretreated with chemotherapy are more sensitive to antibody-induced target-specific killing. In mouse tumor models, tumor control by anti-CLDN18.2 antibodies plus chemotherapy is superior to that by anti-CLDN18.2 antibodies as single agents. [Means for solving the problem]

[0007] The present invention generally provides combination therapies for the effective treatment and / or prevention of diseases associated with cells expressing CLDN18.2, including cancer diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, e.g. non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer and metastases thereof, particularly gastric cancer metastases, e.g. Krukenberg tumors, peritoneal metastases and lymph node metastases.

[0008] In one embodiment, the present invention provides a method for treating or preventing pancreatic cancer in a patient, comprising administering to the patient (i) an antibody capable of binding to CLDN18.2 and (ii) an agent that stabilizes or increases the expression, i.e., level, of CLDN18.2. CLDN18.2 is preferably expressed on the cell surface of cancer cells. The agent that stabilizes or increases the expression of CLDN18.2 can be administered before, simultaneously with, or after the administration of the antibody capable of binding to CLDN18.2, or a combination thereof.

[0009] The agent that stabilizes or increases the expression of CLDN18.2 may be a cytotoxic and / or cytostatic drug. In one embodiment, the agent that stabilizes or increases the expression of CLDN18.2 includes an agent that induces cell cycle arrest or accumulation of cells in one or more phases of the cell cycle, preferably one or more phases of the cell cycle other than the G1 phase, such as the S phase, the G2 phase, a combination thereof, or a combination of the S phase or the G2 phase and the G1 phase. The agent that stabilizes or increases the expression of CLDN18.2 may include an agent selected from the group consisting of nucleoside analogs, platinum compounds, camptothecin analogs, and taxanes, prodrugs thereof, salts thereof, and combinations thereof. The nucleoside analogs may be selected from the group consisting of gemcitabine, 5-fluorouracil, prodrugs thereof, and salts thereof. The platinum compounds may be selected from the group consisting of oxaliplatin, cisplatin, prodrugs thereof, and salts thereof. Camptothecin analogs may be selected from the group consisting of irinotecan, topotecan, prodrugs thereof and salts thereof. Taxanes may be selected from the group consisting of paclitaxel, docetaxel, prodrugs thereof and salts thereof. Agents that stabilize or increase the expression of CLDN18.2 may include agents selected from the group consisting of gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, prodrugs thereof, salts thereof and combinations thereof. Agents that stabilize or increase expression of CLDN18.2 may include a combination of oxaliplatin and 5-fluorouracil or a prodrug thereof, a combination of cisplatin and 5-fluorouracil or a prodrug thereof, a combination of at least one taxane and oxaliplatin, a combination of at least one taxane and cisplatin, a combination of at least one taxane and 5-fluorouracil or a prodrug thereof, or a combination of at least one camptothecin analog and 5-fluorouracil or a prodrug thereof.Agents that stabilize or increase the expression of CLDN18.2 may include a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of oxaliplatin, 5-fluorouracil or its prodrug, and irinotecan. Thus, the method of the present invention may include administering a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of oxaliplatin, 5-fluorouracil or its prodrug, and irinotecan. In one embodiment, the method of the present invention includes administering folinic acid, 5-fluorouracil or its prodrug, irinotecan, and oxaliplatin. Agents that stabilize or increase the expression of CLDN18.2 may include agents that induce immunogenic cell death. Agents that induce immunogenic cell death may include oxaliplatin.

[0010] In a further aspect, the present invention provides a method for treating or preventing pancreatic cancer in a patient, comprising administering to the patient (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof. The cancer disease may be Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment, the cancer is pancreatic cancer.

[0011] In one embodiment, the method of the invention further comprises administering an agent that stimulates γδ T cells. In one embodiment, the γδ T cells are Vγ9Vδ2 T cells. In one embodiment, the agent that stimulates γδ T cells is a bisphosphonate, such as a nitrogen-containing bisphosphonate (aminobisphosphonate). In one embodiment, the agent that stimulates γδ T cells is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid, and salts thereof. In one embodiment, the agent that stimulates γδ T cells is administered in combination with interleukin-2.

[0012] The methods of the invention may further comprise administering at least one additional chemotherapeutic agent, which may be a cytotoxic agent.

[0013] The antibody capable of binding to CLDN18.2 may bind to a natural epitope of CLDN18.2 present on the surface of a living cell. In one embodiment, the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, the antibody capable of binding to CLDN18.2 mediates cell killing by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment, the antibody capable of binding to CLDN18.2 is a monoclonal, chimeric or humanized antibody, or a fragment of an antibody. In one embodiment, the antibody mediates cell killing when bound to CLDN18.2, particularly CLDN18.2 expressed by a cell on its cell surface, where the cell is preferably a cancer cell, such as a cell of a cancer described herein. In one embodiment, the antibody having the ability of binding to CLDN18.2 is an antibody selected from the group consisting of: (i) an antibody produced by and / or obtainable from the clone deposited under accession 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 which is a chimeric or humanized form of an antibody included in (i), (iii) an antibody having the specificity of an antibody included in (i), and (iv) an antibody which contains an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody included in (i) and preferably has the specificity of an antibody included in (i). In one embodiment, the antibody is linked to a therapeutic agent, such as a toxin, a radioisotope, a drug, or a cytotoxic agent.

[0014] In one embodiment, the method of the invention comprises administering an antibody having the ability of binding to CLDN18.2 at a concentration of 1000 mg / m 2In one embodiment, the method of the present invention comprises administering an antibody capable of binding to CLDN18.2 at a dose of 300 to 600 mg / m 2 This includes administering the compound repeatedly at a dose of

[0015] According to the present invention, CLDN18.2 preferably has an amino acid sequence according to SEQ ID NO:1.

[0016] In one embodiment, the cancers described herein are CLDN18.2 positive. In one embodiment, the cancer cells of the cancers described herein are CLDN18.2 positive. In one embodiment, the cancer cells of the cancers described herein express CLDN18.2 on their cell surface.

[0017] In one embodiment, the pancreatic cancer described herein includes primary cancer, advanced cancer, or metastatic cancer, or a combination thereof, such as a combination of pancreatic primary cancer and metastatic cancer. In one embodiment, the method of the present invention is directed to the simultaneous treatment of primary cancer and metastatic cancer, such as pancreatic primary cancer and pancreatic metastatic cancer. In one embodiment, metastatic cancer includes metastasis to lymph nodes, ovaries, liver, or lung, or a combination thereof. In one embodiment, the pancreatic cancer includes carcinoma of the pancreatic duct. In one embodiment, the pancreatic cancer includes adenocarcinoma or carcinoma, or a combination thereof. In one embodiment, the pancreatic cancer includes pancreatic ductal adenocarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma, or acinar cell carcinoma, or a combination thereof. In one embodiment, the pancreatic cancer is partially or completely refractory to gemcitabine treatment, such as gemcitabine monotherapy. In one embodiment, preventing pancreatic cancer includes preventing recurrence of pancreatic cancer.

[0018] In one embodiment, the patient to be treated according to the present invention has undergone surgery for pancreatic cancer.In one embodiment, the patient has precancerous pancreatic lesions, in particular precancerous pancreatic lesions that comprise early malignant histological changes in pancreatic duct.In these embodiments, the method of the present invention is preferably aimed at preventing the development of malignant pancreatic cancer.

[0019] In a further aspect, the present invention provides a medical preparation for treating or preventing pancreatic cancer, the medical preparation comprising (i) an antibody capable of binding to CLDN18.2 and (ii) an agent that stabilizes or increases the expression of CLDN18.2. The medical preparation of the present invention may further comprise an agent that stimulates γδ T cells. The antibody capable of binding to CLDN18.2 and the agent that stabilizes or increases the expression of CLDN18.2, and optionally the agent that stimulates γδ T cells, may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be present in the form of a kit comprising a first container containing an antibody capable of binding to CLDN18.2 and a second container containing an agent that stabilizes or increases the expression of CLDN18.2, and optionally a container containing an agent that stimulates γδ T cells. The medical preparation may further comprise printed instructions regarding the use of the preparation for treating or preventing pancreatic cancer, in particular the use of the preparation in the method of the present invention. Various embodiments of the medical preparations and, in particular, the antibodies capable of binding to CLDN18.2, the agents that stabilize or increase the expression of CLDN18.2 and the agents that stimulate γδ T cells are as described above with respect to the methods of the present invention.

[0020] In a particular aspect, the present invention provides a medical preparation comprising (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine. The medical preparation of the present invention may further comprise an agent that stimulates γδ T cells. The antibody capable of binding to CLDN18.2 and gemcitabine, and optionally the agent that stimulates γδ T cells, may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be intended for treating or preventing cancer, such as pancreatic cancer. The medical preparation may be present in the form of a kit comprising a first container containing an antibody capable of binding to CLDN18.2 and a second container containing gemcitabine, and optionally a container containing an agent that stimulates γδ T cells. The medical preparation may further comprise printed instructions regarding the use of the preparation for the treatment or prevention of cancer, such as pancreatic cancer, in particular the use of the preparation in the method of the present invention. The various embodiments of the medical preparation and in particular the antibody capable of binding to CLDN18.2, the agent that stabilizes or increases the expression of CLDN18.2 and the agent that stimulates γδ T cells are as described above for the method of the present invention.

[0021] The present invention also provides agents described herein, such as antibodies capable of binding to CLDN18.2 and / or agents that stabilize or increase expression of CLDN18.2, for use in the methods described herein. For example, the present invention also provides antibodies capable of binding to CLDN18.2 for administration in combination with agents that stabilize or increase expression of CLDN18.2, such as gemcitabine, and optionally with agents that stimulate γδ T cells.

[0022] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0023] [Figure 1]Lentiviral vector used for transduction of pancreatic cancer cell lines. Human CLDN18.2 was cloned downstream of the EF1α promoter. The expression cassette is integrated between long terminal repeats (5' and 3'-LTR) that allow packaging and reverse transcription of viral mRNA. RSV: Rous sarcoma virus allows Tat-independent production of viral mRNA. Amp: Ampicillin resistance gene. PGKp: Promoter of blasticidin. WPRE: Woodchuck posttranscriptional regulatory element enhances transgene expression. LTR: Long terminal repeats allow viral packaging. SV40A allows transcription termination and polyadenylation of mRNA. pUC: Bacterial vector backbone. Bla: Promoter of ampicillin. [Diagram 2] Metastasis analysis of pancreatic cells in mouse lungs. Dissection of mouse lung after iv injection of mice with pancreatic cancer cells. [Diagram 3] CLDN18.2 expression in normal and cancerous pancreatic tissues. Staining of normal pancreatic formalin-fixed paraffin-embedded (FFPE) tissue (A) and pancreatic adenocarcinoma tissue (B) with monoclonal mouse 35-22A antibody (0.2 μg / ml). Hematoxylin counterstain (2:00 min). Magnification 200×. [Figure 4] CLDN18.2 expression in normal and precancerous pancreatic tissues. 43-14A staining of various precancerous structures: (A) normal and PanIN1; (B) PanIN2; (C) PanIN3. Magnification 200x. [Diagram 5] Pilot study - correlation between CLDN18.2 signal intensity and amount of positive tumor cells for analyzed pancreatic primary tumors. Each point represents a case of pancreatic primary cancer analyzed by staining FFPE sections with monoclonal mouse 35-22A antibody (0.2 μg / ml). The dotted line indicates the 10% value. [Figure 6] Pilot study - CLDN18.2 expression in primary and metastatic pancreatic tumor tissues. Staining of FFPE tissue sections (3 μm) of (A) adenocarcinoma primary tumors and (B) lymph node metastases with mouse monoclonal 35-22A antibody. Counterstained with hematoxylin (Mayers). [Figure 7] Main study: correlation between CLDN18.2 signal intensity and amount of positive tumor cells for analyzed pancreatic primary tumors. Each point represents a case of pancreatic ductal adenocarcinoma primary tumor (black circle) or neuroendocrine primary tumor (white circle) analyzed by staining FFPE sections with monoclonal mouse 43-14A antibody (0.2 μg / ml). [Figure 8] Correlation between CLDN18.2 signal intensity and amount of positive tumor cells for analyzed pancreatic metastases. Each point represents a case of pancreatic lymph node metastasis (black circle) or liver metastasis (white circle) analyzed by staining FFPE sections with monoclonal mouse 43-14A antibody (0.2 μg / ml). The dotted line indicates the 10% value. [Figure 9] Expression of CLDN18.2 in primary and metastatic pancreatic tumor tissues. (A, C, E) Staining of FFPE tissue sections (3 μm) with mouse monoclonal 43-14A antibody for adenocarcinoma primary tumors and (B, D, F) lymph node metastases. Sections were counterstained with Mayers hematoxylin. [Figure 10] Graphical analysis--CLDN18.2 expression in matched pancreatic primary tumor tissues and lymph node metastasis tissues. [Figure 11] Expression of CLDN18.2 in matched pancreatic primary tumor and metastasis tissues. FFPE tissue sections (3 μm) of (A) primary adenocarcinoma, (B) liver metastasis, and (C) lymph node metastasis were stained with mouse monoclonal 43-14A antibody. Sections were counterstained with Mayers hematoxylin. 200x magnification. [Figure 12A] CLDN18.2 mRNA levels in pancreatic cancer cell lines. [Figure 12BCD]CLDN18.2 mRNA levels in pancreatic cancer cell lines. (A) Q-PCR expression analysis of various pancreatic CA cell lines, lentiviral transduced (LVT) cell lines (grey bars), gastric cancer cell line KATO-III (positive control) and breast cancer cell line SKBR-3 (negative control). CLDN18.2 transcripts were amplified using gene-specific primers. Endogenous cell lines showing relative expression levels above 1×105 were scored as CLDN18.2 positive (hatched bars). NTC: H2O control samples. Error bars: mean + SD. (B-D) Passage-dependent CLDN18.2 expression analysis in Patu8988S (B), Panc05.04 (C) and the indicated LVT cell lines (D). Passage numbers are indicated below each bar. [Figure 13] CLDN18.2 protein levels in cell lysates of pancreatic cancer cell lines. Proteins were separated by 12.5% ​​SDS-PAGE. Western blot analysis was performed using CLDN18 antibody (Zymed-MID) to detect the C-terminus of CLDN18.1 and CLDN18.2, and a loading control antibody to detect β-actin. Exposure times of 140 s (Pierce SuperSignal West Dura) and 20 s (Pierce SuperSignal West Pico) were used, respectively. (A) Detection of CLDN18 in pancreatic cell line lysates, positive control cell lysates (HEK293-p740) and negative control cell lysates (SKBR-3). (B) CLDN18.2 expression compared between non-transduced parental cell lysates and lentiviral-transduced (LVT) cell line lysates. Patu8988S and SKBR-3 were added as positive and negative controls, respectively. [Figure 14]Detection and cellular localization of CLDN18 expression in pancreatic cancer cell lines. Staining of pancreatic cancer cell lines grown on coverslips. Antibody: 35-22A (20x magnification, exposure times are indicated below each image). DAPI was used to stain nuclei (blue). (A: AsPC1; B: BxPC3; C: CFPAC; D: DANG; E: HPAF-II; F: HUP-T3; G: HUP-T4; H: KCI-MOH; I: Panc1; J: Panc05.04; K: Panc02.04; L: Panc04.03; M: Patu8902; N: Patu8988S; O: Su86.86: P: Suit-2; Q: SW-1990; R: YAPC; S: gastric cancer control cell line KATO-III). [Figure 15] Detection and cellular localization of CLDN18 expression in CLDN18.2 transduced pancreatic cancer cell lines. CLDN18 detection in lentiviral transduced (LVT) pancreatic cancer cell lines using 35-22A antibody after fixation and permeabilization. Alexa488 or Alexa555 labeled secondary antibodies were used for detection. A: BxPC3-LVT; B: CAPAN1-LVT; C: DANG-LVT; D: HPAC-LVT; E: MiaPaCa2-LVT; F: Patu8902-LVT; G: Suit-2-LVT; H: YAPC-LVT. [Fig. 16ABCDEF] Binding of IMAB362 to the cell surface of CLDN18.2-positive pancreatic CA cell lines (pharmacodynamics). [Figure 16GHIJKL] Binding of IMAB362 to the cell surface of CLDN18.2 positive pancreatic CA cell lines (pharmacodynamics). IF analysis of CLDN18.2 expressing pancreatic cancer cell lines (A,B,D,E), lentiviral transduced pancreatic cell lines (GL) and KATO-III gastric cancer control cells (C,F). Cells were stained with IMAB362 under native conditions (D-E) and for comparison with 35-22A after fixation and permeabilization of cells (A-C). DAPI was used to stain nuclei. Exposure times are indicated in each panel. G: BxPC3-LVT; H: CAPAN1-LVT; I: DANG-LVT; J: MiaPaCa2-LVT; K: Patu8902-LVT; L: Suit2-LVT. [Figure 17]CLDN18.2 expression in xenograft tumors of various cell lines. Expression of CLDN18.2 in CAPAN1-LVT (A, B), BxPC3-LVT (C, D), PATU8988S-LVT (E, F), MiaPaCa2-LVT (G, H), YAPC-LVT (J, K) and DANG-LVT (L, M) xenograft tumors. Histological staining was performed with Zymed-MID antibody. Lens magnification: 10x (A, C, E, G, J, L) and 20x (B, D, F, H, K, M). [Figure 18] Engraftment test of Suit-2 and MiaPaCa2 pancreatic cancer cell lines. Cells were injected into the tail vein of nude mice. Animals were sacrificed 45 (A), 52 (B), and 59 (C) days after application of Suit-2 (A–C), or 59 (D), 66 (E), and 73 (F) days after injection of MiaPaCa2 (D–F). Lungs were prepared and stained with MHC class I antibody (anti-human MHC I, clone EPR1394Y) to detect human cells in mouse tissues. [Figure 19] Metastatic engraftment analysis of Patu8988S. Patu8988S cells were injected iv at 1 x 106 or 2 x 106 cells in Nu / Nu mice, and mouse lungs (A) and livers (B) were isolated at various time points indicated below the x-axis. To calculate the % human DNA present in each tissue preparation, a standard curve was generated by mixing human and mouse DNA and preparing seven 5-fold dilutions resulting in 100% (1) to 0.0064% (7) human DNA. [Figure 20] IHC analysis of Patu8988S metastasis in mouse lung tissue. Mice injected with Patu8988S cells via tail vein were sacrificed at different time points (A-D = day 70, E-H = day 86) and lung tissue was isolated and stained with 1:1000 diluted MHC-I (EPR1394Y) antibody (A, B, E, F) or 0.2 μg / ml anti-claudin 18 (Zymed-Mid) (C, D, G, H). Magnification: A, C, E, G = 10x and B, D, F, H = 20x. [Figure 21]IMAB362-mediated apoptosis of gemcitabine-treated pancreatic tumor cells. Apoptosis induced by cross-linking of CLDN18.2 on BxPC3-CLDN18 after 48 hours. BxPC3-CLDN18 were cultured in medium or medium + 100 ng / ml gemcitabine. There was a shift in the proportion of apoptotic cells in mononuclear cells. A similar shift was obtained by incubation of tumor cells with camptothecin. [Fig. 22ABCD] Efficacy of IMAB362-induced ADCC activity towards pancreatic cancer cells. [Fig. 22EFGH] Potency of IMAB362-induced ADCC activity towards pancreatic cancer cells. (A) ADCC performed on CLDN18.2-positive pancreatic cancer cell lines using PBMCs from different donors. (B-F) ADCC performed on LVT pancreatic cell lines ectopically expressing CLDN18.2 and the corresponding parental cells. (G) Dot plots. [Fig. 23AB] Efficacy of IMAB362-induced CDC activity on pancreatic cancer cells. [Fig. 23CDE] Efficacy of IMAB362-induced CDC activity on pancreatic cancer cells. (A) CDC performed on healthy human serum pool as complement source, IMAB362 and CLDN18.2 positive pancreatic CDOK1-p740 control cells in four independent experiments. (B) CDC performed on CLDN18.2 positive (Patu8988S, DANG, Panc05.04) and CLDN18.2 negative (CAPAN1, Suit2, BxPC3, YAPC) pancreatic cell lines. (C) CDC on ectopically expressing LVT cell lines. (D) Dot plot showing IMAB362 concentration that produces half maximal lysis rate (EC50) on pancreatic cancer cell lines. (E) Maximum killing rate obtained with IMAB362 on pancreatic cancer cell lines. [Figure 24]Effect of IMAB362 treatment on subcutaneous MiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumors were inoculated by subcutaneous injection of 1e7 MiaPaCa2-LVT cells into the flank of 15 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment was initiated 3 days after tumor cell injection with 200 μg IMAB362 or control, respectively. Treatment continued twice weekly with alternating ip and iv injections until animals were sacrificed. (A) Effect of IMAB362 treatment on tumor growth. sc tumor size was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival plot. Mice were sacrificed when tumors reached a volume of 1400 mm3 or when tumors became ulcerated. [Diagram 25] IMAB362 treatment of subcutaneous BxPC3-LVT xenografts. BxPC3-LVT xenograft tumors were inoculated by subcutaneous injection of 1e7 BxPC3-LVT cells into the flank of 15 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment was initiated 3 days after tumor cell injection with 200 μg IMAB362 or control, respectively. Treatment continued twice weekly with alternating ip and iv injections until animals were sacrificed. (A) Effect of IMAB362 treatment on tumor growth. Sc tumor size was measured twice weekly (mean+SEM, *p<0.05). (B) Kaplan-Meier survival plot. Mice were sacrificed when tumors reached a volume of 1400 mm3 or when tumors became ulcerated. [Figure 26]Effect of IMAB362 treatment on the growth of Suit2-LVT pancreatic metastases. 2x106 Suit2-LVT tumor cells were injected intravenously into the tail vein of 12 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment was initiated 3 days after tumor cell injection with 200 μg IMAB362, 200 μg isotype control or an equal volume of PBS. Animals were sacrificed 42 days after implantation. (A) qPCR analysis determining the percentage of human DNA present in mouse lung samples (average of 2-4 reactions per sample). (B) The percentage of human cells covering the mouse lung surface was determined by planimetry. Human cells were immunohistochemically stained in tissue sections with an anti-human MHC class I antibody. *p<0.05 (Kruskal-Wallis test). Error bars: mean ± SD. [Diagram 27AB] Q-PCR and IHC analysis of Patu8988S lung metastases. Each mouse was injected with 2x106 Patu8988S cells. Animals were sacrificed after 65 days. White circle: mice sacrificed after 63 days. [Fig. 27CD] Q-PCR and IHC analysis of Patu8988S lung metastases. Each mouse was injected with 2x106 Patu8988S cells. Animals were sacrificed after 65 days. Open circles: mice sacrificed after 63 days. (A) Mice were treated twice weekly with 200 μg IMAB362 or saline control. The amount of human DNA (ng) detected by Q-PCR calculated from the Ct values. (B) Repeat of the Q-PCR experiment described in (A). Here, the percentage of human DNA present in the mouse DNA was calculated from the Ct values. (C) Mice were treated with IMAB362 and an isotype control antibody (rituximab). The percentage of human DNA present in the mouse lungs was calculated from the Ct values. One outlier was detected (open triangle) for the IMAB362 group. Significance was indicated by including or excluding outliers. (D / E) Same experiment as in (C). Here, the surface of metastases was determined using the Image J program. Dot plots show the significance of IMAB362 inhibition including (D) or excluding (E) outliers. P value: unpaired t-test. Error bars ± SD. [Figure 28]Dose-response curves for gemcitabine. Pancreatic cancer cell lines show very different sensitivity to gemcitabine. Cell lines were exposed to different concentrations of gemcitabine for 4 days and inhibition of proliferation was analyzed by viability assay. [Figure 29] Dose-response curves for oxaliplatin. Pancreatic cancer cell lines show very different sensitivity to oxaliplatin. Cell lines were exposed to different concentrations of oxaliplatin for 4 days and inhibition of proliferation was analyzed by viability assay. [Diagram 30] Effect of chemotherapy treatment on CLDN18.2 expression (RNA). RNA from untreated, pretreated DANG cells (2 days) with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) (A) or Patu8988S cells pretreated for 3 days with Gem (10 ng / ml) or GemOx (Gem 10 ng / ml + Ox 100 ng / ml) (B). RNA was converted to cDNA and CLDN18.2 transcript levels were analyzed by quantitative real-time PCR. Results are shown as relative units compared to the transcript levels of the housekeeping gene HPRT. [Diagram 31] Effect of chemotherapy on CLDN18.2 protein levels in pancreatic cancer cells. Proteins from whole cell lysates of DANG cells (A) or Patu8988S cells (B) pretreated with untreated (medium), Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) were analyzed for CLDN18.2 expression detected with Zymed C-terminal polyclonal antiserum. Actin was used to show equal loading of proteins. [Diagram 32] FACS analysis of CLDN18.2 cell surface expression. CLDN18 expression (black histogram) of media-cultured (left) and Gem-treated (right) Patu8988S is overlaid compared to isotype control. Patu8988S is treated with gemcitabine (10 ng / ml) for 3 days. [Diagram 33]Cell cycle analysis of DANG cells treated or not with either gemcitabine (Gem; 2 ng / ml) or gemcitabine + oxaliplatin (GemOx; 1 ng / ml + 10 ng / ml) for 2 days. (A) Gemcitabine treatment results in cell cycle arrest at S phase. The area of ​​each bar was divided to indicate the percentage of cells in G0 / G1, S and G2 phases. (B) Western blot analysis showed upregulation of CLDN18 after treatment with Gem. [Diagram 34] Effect of gemcitabine on cell cycle (A) and CLDN18.2 expression (B, C) in Patu8988S cells. Patu8988S cells were untreated or treated with gemcitabine (10 ng / ml) for 2 days. (A) The area of ​​each bar was divided to show the percentage of cells in G0 / G1, S and G2 phases. The density of CLDN18.2 (x-axis) was plotted against the number of cells (y-axis). (B) CLDN18.2 expression in untreated (dotted line) vs. Gem-treated (solid line). (C) CLDN18.2 expression in Gem-treated Patu8988S cells in G0 / G1 phase (dotted line) vs. cells in S phase (solid line). [Figure 35A] The effect of chemotherapy on gastric cancer cells. [Figure 35B] The effect of chemotherapy on gastric cancer cells. [Figure 35C] Effect of chemotherapy on gastric cancer cells. Cultivation of Kato III cells for 96 h results in cell cycle arrest at G0 / G1 phase (a) and downregulation of CLDN18.2 (c). Cytostatic compounds that cause cell cycle arrest at various phases of the cell cycle stabilize CLDN18.2 expression (c). [Diagram 36] Effect of chemotherapy on gastric cancer cells. Cytostatic compounds causing cell cycle arrest at different phases of the cell cycle: S / G2 (irinotecan) or G2 (docetaxel). The area of ​​each bar was divided to show the percentage of cells in G0 / G1, S and G2 phases. [Figure 37]Dose-response curves for IMAB362-mediated ADCC after chemotherapy treatment with DANG. (A) Dose-response curves for one representative donor after pretreatment of DANG pancreatic cancer cells with Gem or GemOx for 40 hours. (B) EC50 values ​​(mean) for IMAB362-mediated ADCC. P-values: unpaired t-test. [Fig. 38AB] The effect of chemotherapy on gastric cancer cells. [Figure 38C] The effect of chemotherapy on gastric cancer cells. [Figure 38D] Effect of chemotherapy on gastric cancer cells. (a) Cells treated with irinotecan, docetaxel or cisplatin show lower levels of viable cells compared to target cells cultured in medium. (b) CLDN18.2 expression is increased in cells treated with irinotecan, docetaxel or cisplatin compared to cells cultured in medium. (c / d) Treatment of cells with irinotecan, docetaxel or cisplatin increases the potency of IMAB362 to induce ADCC. [Figure 39] Effect of chemotherapeutic agents on IMAB362-mediated CDC of MiaPaCa2-LVT cells. Dose-response curves of two independent assays. MiaPaCa2-LVT were cultured in medium, Gem (10 ng / ml) or GemOx (10 ng / ml Gem + 100 ng / ml Ox) for 70 h. [Diagram 40] Effect of chemotherapy on IMAB362-induced CDC. [Diagram 41]Effect of IMAB362 treatment in combination with Gem or GemOx on BxPC3-LVT xenografts. BxPC3-LVT xenograft tumors were inoculated by subcutaneous injection of 8.5e6 BxPC3-LVT cells into the flank of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment with chemotherapy (50mg / kg gemcitabine ip, 50mg / kg gemcitabine + 5mg / kg oxaliplatin ip, respectively) was initiated 3 days after tumor cell injection and continued once a week for 6 weeks. 24 hours after chemotherapy injection, IMAB362 800μg or control was applied intravenously in the tail vein. IMAB362 treatment was continued until mice were sacrificed. (A) Growth curves of subcutaneous BxPC3-LVT xenograft tumors. sc tumor size was measured twice a week (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or became ulcerated. [Diagram 42] Enhanced antitumor efficacy by combination of IMAB362 and gemcitabine regimen. BxPC3-LVT xenograft tumors were inoculated by subcutaneous injection of 8.5e6 BxPC3-LVT cells into the flank of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment with chemotherapy (100mg / kg gemcitabine ip or 100mg / kg gemcitabine + 5mg / kg oxaliplatin ip) was initiated 3 days after tumor cell injection and continued once a week for 6 weeks. 24 hours after chemotherapy injection, IMAB362 200μg (1 / 2 dose) or 400μg (total dose) was applied intravenously into the tail vein. IMAB362 treatment was continued by alternating ip and iv injections twice a week until the mice were sacrificed. (A) Growth curves of subcutaneous BxPC3-LVT xenograft tumors. The size of the sc tumors was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or became ulcerated. [Diagram 43]Effect of IMAB362 treatment in combination with gemcitabine on MiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumors were inoculated by subcutaneous injection of 5e6 MiaPaCa2-LVT cells into the flank of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment with chemotherapy (50 mg / kg gemcitabine ip) was initiated 4 days after tumor cell injection and continued once a week for 6 weeks. 200 μg of IMAB362 or control was applied intravenously in the tail vein 24 hours after chemotherapy injection. IMAB362 treatment was continued twice a week with alternating ip and iv injections until mice were sacrificed. (A) Growth of subcutaneous xenograft tumors. Tumor size was measured twice a week (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or became ulcerated. [Diagram 44] Effects of IMAB362 treatment in combination with gemcitabine on established MiaPaCa2-LVT xenograft tumors. MiaPaCa2-LVT xenograft tumors were inoculated by subcutaneous injection of 1e7 MiaPaCa2-LVT cells into the flank of female Hsd:athymic nude-Foxn1nu mice. Nine days after subcutaneous tumor inoculation, tumor-bearing mice were regrouped into uniform treatment groups with 8 animals per group and treatment was initiated. Mice were treated with 150 mg / kg gemcitabine ip twice weekly for 4 weeks. 24 hours after gemcitabine injection, 200 μg IMAB362 or control was applied intravenously in the tail vein. Treatment with 200 μg IMAB362 was continued twice weekly, alternating between ip and iv injections, until mice were sacrificed. (A) Subcutaneous tumor size was measured twice weekly (mean + SEM; **=p<0.01). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or became ulcerated (Log-rank (Mantel-Cox) test; **=p<0.01). [Fig. 45AB] Effect of IMAB362 in combination with gemcitabine on lung metastases in the Patu8988S xenograft model. [Fig. 45CD]Effect of IMAB362 in combination with gemcitabine on lung metastases in Patu8988S xenograft model. 2x106 Patu8988S tumor cells were injected intravenously into the tail vein of 12 female Hsd:athymic nude-Foxn1nu mice per treatment group. Two weeks after intravenous tumor cell injection, treatment was initiated with a maintenance treatment of 200μg IMAB362 twice weekly (iv / ip) in combination with 100mg / kg gemcitabine ip twice weekly for 4 weeks. The control group was treated with 200μg isotype control antibody in combination with 100mg / kg gemcitabine twice weekly. Animals were sacrificed 70 days after implantation. (A) Quantitative PCR analysis of human DNA in lung samples of mice treated with IMAB362 and isotype antibody (mean of triplicate reactions for each sample). Significant difference versus isotype control (P=0.0035, Mann-Whitney test). (B) The percentage of stained human cells covering the mouse lung surface was determined by computer-assisted analysis. Immunohistochemical staining of paraffin-embedded lung tissue was performed with anti-human MHC-I antibody (clone EPR1394Y) (mean±SEM; P=0.0003, Mann-Whitney test). (C and D) Examples of immunohistochemical staining with anti-MHC-I antibody on Patu8988s lung metastases in IMAB362+gemcitabine-treated mice (C) or isotype antibody+gemcitabine-treated mice (D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] 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, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0025] In the following, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the embodiments expressly described. This description should be understood to support and encompass embodiments that combine the embodiments expressly described with many of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0026] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0027] The practice of the present invention is, unless otherwise indicated, based on the teachings of the art (e.g., Molecular Cloning: A Laboratory Manual, 2 nd Conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques are used, as described in The Genetics of the Invention (see, for example, J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0028] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to mean the inclusion of the described member, integer or step or group of members, integers or steps, but not the exclusion of any other member, integer or step or group of members, integers or steps, although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject matter resides in the inclusion of the described member, integer or step or group of members, integers or steps. The terms "a" and "the" and similar references used in connection with the description of the present invention (particularly in connection with the claims) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to be a shorthand way of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not pose limitations on the scope of the invention or what is claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0029] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0030] 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)).

[0031] The term "CLDN18.2" preferably relates to human CLDN18.2 and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence.

[0032] The term "CLDN18.1" preferably relates to human CLDN18.1 and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO:2 of the Sequence Listing or a variant of said amino acid sequence.

[0033] The term "variant" according to the present invention refers in particular to mutants, splice variants, conformational variants, isoforms, allelic variants, species variants and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences originating from a different species than that of a given nucleic acid or amino acid sequence. The term "variant" encompasses any post-translational modification variants and conformational variants.

[0034] According to the present invention, the term "CLDN18.2 positive cancer" refers to a cancer comprising cancer cells which express CLDN18.2, preferably which express CLDN18.2 on the surface of said cancer cells.

[0035] "Cell surface" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules. For example, a transmembrane protein that has one or more extracellular portions is considered to be expressed on the cell surface.

[0036] CLDN18.2 is expressed on the surface of a cell if it is located on the surface of the cell and accessible for binding by a CLDN18.2-specific antibody added to the cell.

[0037] According to the present invention, CLDN18.2 is not substantially expressed in cells when the level of expression is lower compared to the expression in gastric cells or gastric tissue.Preferably, the level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% or even lower than the expression in gastric cells or gastric tissue.Preferably, CLDN18.2 is not substantially expressed in cells when the level of expression is only 2-fold, preferably 1.5-fold, higher than the level of expression in non-cancerous tissue other than the stomach, and preferably does not exceed the level of expression in said non-cancerous tissue.Preferably, CLDN18.2 is not substantially expressed in cells when the level of expression is lower than the detection limit and / or when the level of expression is so low that it does not allow binding by CLDN18.2-specific antibody added to the cells.

[0038] According to the present invention, CLDN18.2 is expressed in a cell when the level of expression is more than twice, preferably 10 times, 100 times, 1000 times or 10000 times higher than the level of expression in non-cancerous tissues other than the stomach.Preferably, CLDN18.2 is expressed in a cell when the level of expression is higher than the detection limit and / or when the level of expression is high enough to allow binding by a CLDN18.2-specific antibody added to the cell.Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of the cell.

[0039] According to the present invention, the term "disease" refers to any pathological condition, including cancer, particularly the forms of cancer described herein. Reference herein to cancer or a particular form of cancer also encompasses cancer metastasis thereof. In a preferred embodiment, the disease to be treated according to the present application involves cells expressing CLDN18.2.

[0040] "Diseases associated with cells expressing CLDN18.2" or similar expressions means, according to the present invention, that CLDN18.2 is expressed in cells of diseased tissues or organs. In one embodiment, the expression of CLDN18.2 in cells of diseased tissues or organs is increased compared to the state in healthy tissues or organs. Increase refers to an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only observed in diseased tissues, while expression in the corresponding healthy tissues is suppressed. For example, CLDN18.2 is expressed in pancreatic cancer tissues, while expression is undetectable in non-cancerous pancreatic tissues. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those in which cancer cells express CLDN18.2.

[0041] As used herein, "cancer disease" or "cancer" encompasses diseases characterized by dysregulated cell growth, proliferation, differentiation, adhesion and / or migration. "Cancer cells" refers to abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow after the stimuli that initiated new growth have ceased. Preferably, the "cancer disease" is characterized by cells that express CLDN18.2, and the cancer cells express CLDN18.2. The cells that express CLDN18.2 are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0042] According to the present invention, a "carcinoma" is a malignant tumor originating from epithelial cells.

[0043] "Adenocarcinoma" is a cancer that arises from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands and various other tissues that line the cavities and organs of the body. Epithelia are embryologically derived from ectoderm, endoderm and mesoderm. To be classified as an adenocarcinoma, the cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated forms may not. By staining the cells from the biopsy, the pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinoma can arise in many tissues of the body due to the ubiquity of glands in the body. Although not every gland secretes the same substances, as long as there is an exocrine function to the cells, they are considered glands, and therefore the malignant form is named adenocarcinoma. Malignant adenocarcinomas often metastasize if given enough time to invade and spread to other tissues.

[0044] The pancreas, an organ of endodermal origin, is a crucial regulator of protein and carbohydrate digestion and glucose homeostasis. The exocrine pancreas (80% of the organ's tissue mass) consists of a branching network of acinar and ductal cells that produce and deliver digestive enzymes to the gastrointestinal tract. Acinar cells, structured into functional units along the ductal network, synthesize and secrete enzymes into the lumen of the duct in response to cues from the stomach and duodenum. Cardioatrial cells reside within the acinar units near the ducts. The endocrine pancreas, which regulates metabolism and glucose homeostasis through the secretion of hormones into the bloodstream, consists of four specialized endocrine cell types that assemble into clusters called islets of Langerhans.

[0045] Pancreatic cancer is a malignant neoplasm that originates from transformed cells that arise in the tissues that form the pancreas. It is the fourth leading cause of cancer-related deaths in the United States and the eighth leading cause worldwide. Early pancreatic cancer often does not cause symptoms, and later symptoms are usually nonspecific and variable. Therefore, pancreatic cancer is often not diagnosed until it is advanced. Pancreatic cancer has a poor prognosis: for all stages combined, the 1- and 5-year relative survival rates are 25% and 6%, respectively. For localized disease, the 5-year survival rate is about 20%, while the median survival times for locally advanced and metastatic disease, which together represent more than 80% of individuals, are about 10 and 6 months, respectively.

[0046] Pancreatic cancer includes adenocarcinomas (tumors exhibiting glandular structure) that arise within the exocrine component of the pancreas and neuroendocrine carcinomas that arise from islet cells.

[0047] Pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, is typically characterized by moderately to poorly differentiated glandular architecture on microscopic examination. Pancreatic ductal adenocarcinoma (PDAC) generally arises in the head of the pancreas, with invasion of surrounding tissues including lymphatics, spleen and peritoneal cavity, and metastasis to the liver and lung. PDAC exhibits a predominantly glandular pattern with duct-like architecture and variable degrees of cellular atypia and differentiation. Less common subtypes of PDAC include colloidal, adenosquamous or sarcomatoid histology. Localized differences in histology, tumor grade and degree of differentiation often exist within individual tumors. Even the smallest primary lesions commonly exhibit perineural and lymphovascular invasion, suggesting a propensity for early distant metastasis.

[0048] The second most common type of exocrine pancreatic cancer is mucinous. Mucinous adenocarcinomas produce large amounts of mucin, which gives them a cystic appearance on imaging studies.

[0049] Pancreatic neuroendocrine tumors form in the hormone-producing cells (islet cells) of the pancreas. Acinar cell neoplasms arise from the acinar cells of the pancreas.

[0050] According to the present invention, the term "cancer" also includes cancer metastases of a primary tumor, such as a primary pancreatic cancer. Thus, for example, when referring to pancreatic cancer, this also includes metastases of pancreatic cancer, for example metastases to the lungs, liver and / or lymph nodes.

[0051] "Metastasis" refers to the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process, which depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter the body cavities and vessels, and then invasion of the target organ after being carried by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, because tumor cells or components remain and can develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis", which refers to metastasis far from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the therapeutic method of the present invention is metastasis arising from pancreatic cancer as a primary site. In a preferred embodiment, such pancreatic cancer metastasis is metastasis to lymph nodes, metastasis to lungs and / or metastasis to liver.

[0052] Krukenberg tumor is a rare metastatic tumor of the ovary accounting for 1%-2% of all ovarian tumors. 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%). Cancer of the colon, appendix and breast (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from cancer of the gallbladder, bile duct, pancreas, small intestine, ampulla of Vater, cervix and bladder / urachus have been reported.

[0053] Refractory cancers are malignancies that are either initially refractory to treatment or become refractory over time, for which specific treatments are ineffective.

[0054] "Treating" means administering a compound or composition or combination of compounds or compositions to a subject to prevent or eliminate disease, including reducing tumor size or number of tumors in the subject; halting or slowing the progression of disease in the subject; preventing or slowing the onset of new disease in the subject; reducing the frequency or severity of symptoms and / or recurrences in a subject who currently has or has previously had the disease; and / or prolonging, i.e., increasing, the survival of the subject.

[0055] In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or arresting the progression or deterioration, or preventing or delaying the onset of the disease or its symptoms.

[0056] The term "patient" according to the present invention means a subject for treatment, in particular a diseased subject, including a human, a non-human primate or another animal, in particular a mammal, such as a cow, horse, pig, sheep, goat, dog, cat or rodent, such as a mouse and a rat. In a particularly preferred embodiment, the patient is a human.

[0057] The term "agent that stabilizes or increases the expression of CLDN18.2" refers to an agent or a combination of agents that, when provided to a cell, causes an increase in the RNA and / or protein level of CLDN18.2 in the cell, preferably an increase in the CLDN18.2 protein level on the cell surface, compared to the situation in which the cell is not provided with the agent or combination of agents. Preferably, the cell is a cancer cell, particularly a cancer cell that expresses CLDN18.2, and is therefore a target of the CLDN18.2-binding antibody, such as a cell of a cancer type described herein, particularly a cell of pancreatic cancer. The term "agent that stabilizes or increases the expression of CLDN18.2" refers in particular to an agent or a combination of agents that, when provided to a cell, causes a higher density of CLDN18.2 on the surface of the cell, compared to the situation in which the cell is not provided with the agent or combination of agents. "Stabilizing the expression of CLDN18.2" particularly includes a situation where an agent or a combination of agents prevents or reduces the decrease in the expression of CLDN18.2, e.g., when an agent or a combination of agents is not provided, the expression of CLDN18.2 would be reduced and providing said agent or a combination of agents prevents or reduces said decrease in CLDN18.2 expression. "Increasing the expression of CLDN18.2" particularly includes a situation where an agent or a combination of agents increases the expression of CLDN18.2, e.g., when an agent or a combination of agents is not provided, the expression of CLDN18.2 would be reduced, essentially remain constant or increase and providing said agent or a combination of agents increases CLDN18.2 expression compared to a situation where an agent or a combination of agents is not provided, so that the resulting expression is higher compared to a situation where an agent or a combination of agents is not provided, the expression of CLDN18.2 would be reduced, essentially remain constant or increase.

[0058] 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 growth inhibitors. Chemotherapeutic agents can affect cells in one of the following ways: (1) damage the DNA of the cells, so that the cells can no longer replicate; (2) inhibit the synthesis of new DNA strands so that cell replication is impossible; (3) stop the mitotic process of the cells so that the cells cannot divide into two cells.

[0059] According to the present invention, the term "substance that stabilizes or increases the expression of CLDN18.2" preferably relates to a substance or combination of substances, such as a cell growth inhibitory compound or a combination of cell growth inhibitory compounds, provided to cells, particularly cancer cells, such that the cells are in one or more phases of the cell cycle, preferably other than G1 phase and G0 phase, preferably one or more phases of the cell cycle other than G1 phase, preferably G2 phase or S phase of the cell cycle, such as one or more of G1 / G2 phase, S / G2 phase, G2 phase or S phase of the cell cycle, resulting in the cells stopping or accumulating. The term "the cells stopping or accumulating in one or more phases of the cell cycle" means an increase in the percentage of cells in said one or more phases of the cell cycle. Each cell passes through a cycle that includes four phases to replicate itself. The first phase, called G1, is the stage where the cell prepares to replicate its chromosomes. The second stage is called S, during which DNA synthesis occurs and the DNA is replicated. The next phase is G2, where RNA and proteins are replicated. The final stage is M phase, which is the stage of actual cell division. In this final stage, the replicated DNA and RNA separate and move to separate ends of the cell, and the cell actually divides into two identical functional cells. Chemotherapeutic agents that are DNA-damaging agents usually cause accumulation of cells in G1 and / or G2 phases. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, usually cause accumulation of cells in S phase. Examples of these drugs are gemcitabine, 6-mercaptopurine and 5-fluorouracil.

[0060] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" includes nucleoside analogues, such as gemcitabine, 5-fluorouracil or its prodrugs, platinum compounds, such as oxaliplatin and cisplatin, taxanes, such as paclitaxel and docetaxel, and camptothecin analogues, such as irinotecan and topotecan, as well as drug combinations, such as drug combinations comprising one or more of gemcitabine, oxaliplatin and 5-fluorouracil, such as drug combinations comprising gemcitabine and oxaliplatin, gemcitabine and 5-fluorouracil, oxaliplatin and 5-fluorouracil, or other drug combinations described herein. According to the present invention, reference to an agent that stabilizes or increases the expression of CLDN18.2, such as a nucleoside analogue, a platinum compound, a camptothecin analogue or a taxane, such as gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan or paclitaxel, is intended to include any prodrug, such as an ester, salt, or derivative, such as a conjugate, of said agent. Examples are conjugates of said agent and a carrier substance, such as protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salt of said agent is pharma-ceutically acceptable.

[0061] In one preferred embodiment, the "agent that stabilizes or increases the expression of CLDN18.2" is or contains an "agent that induces immunogenic cell death."

[0062] In certain circumstances, cancer cells may enter a lethal stress pathway that leads to the release of a combination of spatiotemporally defined signals that are decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:798-804). In such circumstances, cancer cells trigger the release of signals that are sensed by innate immune effectors such as dendritic cells and trigger cognate immune responses, including CD8+ T cells and IFN-γ signaling, so that tumor cell death can elicit an effective anti-cancer immune response. These signals include pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) at the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1. Taken together, these steps constitute the molecular determinants of immunogenic cell death (ICD). Although anthracyclines, oxaliplatin and γ-irradiation are able to induce all of the signals that define ICD, cisplatin, for example, is deficient in the ability to induce CRT translocation from the ER to the surface of dying cells, a step that requires ER stress, and requires complementation by the ER stress inducer thapsigargin.

[0063] According to the present invention, the term "agent that induces immunogenic cell death" refers to an agent or combination of agents that, when provided to a cell, particularly a cancer cell, can induce the cell to enter a lethal stress pathway that ultimately produces a tumor-specific immune response.In particular, an agent that induces immunogenic cell death, when provided to a cell, induces the cell to release a combination of spatiotemporally defined signals, including, in particular, pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) at the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1.

[0064] According to the present invention, the term "agent that induces immunogenic cell death" encompasses anthracyclines and oxaliplatin.

[0065] The term "nucleoside analog" refers to structural analogs of nucleosides, a category that includes both purine and pyrimidine analogs.

[0066] The term "gemcitabine" has the following formula: [ka] It is a compound which is a nucleoside analogue of the formula:

[0067] In particular, the term refers to the compound 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidin-2-one.

[0068] According to the present invention, gemcitabine is preferably administered by the intravenous route. Preferably, gemcitabine is administered at a dose of 0.5 to 2 g / m 2 , preferably 0.8 to 1.5 g / m 2 , more preferably 1 to 1.2 g / m 2 It is administered in a range of body surface area doses. For example, gemcitabine is administered at 1000 mg / m2 once weekly for 7 out of 8 weeks. 2 and then once weekly for three out of four weeks.

[0069] The term "nucleoside analogues" encompasses fluoropyrimidine derivatives such as fluorouracil and its prodrugs. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U), commercially available under the trade names Adrucil, Carac, Efudix, Efudex and Fluoroplex, has the following formula: [ka] It is a compound which is a pyrimidine analogue of the formula:

[0070] In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0071] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapy agent that is a prodrug that is converted to 5-FU in tissues. Capecitabine, which can be administered orally, has the following formula: [ka] has.

[0072] In particular, this term refers to the compound pentyl [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

[0073] 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.

[0074] The term "cisplatin" or "cisplatinum" has the formula: [ka] This refers to the compound cis-diamminedichloroplatinum(II) (CDDP).

[0075] The term "carboplatin" refers to a compound of the formula: [ka] This refers to the compound cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II).

[0076] The term "oxaliplatin" has the following formula: [ka] This refers to a compound that is a platinum compound complexed to a diaminocyclohexane carrier ligand.

[0077] In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II). Oxaliplatin for injection is also commercially available under the trade name Eloxatine.

[0078] Taxanes are a class of diterpene compounds initially derived from natural sources such as plants of the genus Taxus, although some are synthetically produced. The main mechanism of action of drugs in the taxane class is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0079] According to the present invention, the term "docetaxel" refers to the following formula:

Chemical formula

[0080] In particular, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-tritil 4-acetate 2-benzoate 13-{ (2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropanoate}.

[0081] According to the present invention, the term "paclitaxel" refers to the following formula:

Chemical formula

[0082] In particular, the term "paclitaxel" refers to the compound (2α,4α,5β,7β,10β,13α)-4,10-bis-(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate.

[0083] According to the present invention, the term "camptothecin analog" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog" refers to a derivative of the following structure: [ka] It refers to a compound containing:

[0084] According to the invention, preferred camptothecin analogues are inhibitors of the DNA enzyme topoisomerase I (topo I). Preferred camptothecin analogues according to the invention are irinotecan and topotecan.

[0085] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. In chemical terms, it has the following formula: [ka] Camptothecin is a semisynthetic analogue of the natural alkaloid camptothecin, having the formula:

[0086] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo 1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0087] Topotecan has the formula: [ka] It is a topoisomerase inhibitor.

[0088] In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0089] Anthracyclines are a class of drugs commonly used in cancer chemotherapy that are also antibiotics. Structurally, all anthracyclines share a common four-ring 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[0090] Anthracyclines preferably exert one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by intercalating between the base pairs of DNA / RNA strands, thus preventing replication of rapidly proliferating cancer cells, 2. Inhibit topoisomerase II enzyme, preventing relaxation of supercoiled DNA, thus blocking DNA transcription and replication, 3. Generate iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0091] According to the present invention the term "anthracycline" relates to an agent, preferably an anti-cancer agent, for inducing apoptosis, preferably by inhibiting the DNA rebinding of topoisomerase II.

[0092] Preferably, according to the present invention, the term "anthracycline" generally refers to a compound having the following ring structure: [ka] and includes analogues and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof.

[0093] Examples of anthracyclines and anthracycline analogues include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pirarubicin, valrubicin, N-trifluoro-acetyldoxorubicin-14-valerate, aclacinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-imino-daunomycin, mitoxantrone and aclacinomycin A (aclarubicin). Mitoxantrone is a member of the anthracenedione class of compounds, which are anthracycline analogues that lack the sugar moiety of anthracyclines but retain a planar polycyclic aromatic ring structure that allows for intercalation into DNA.

[0094] Particularly preferred anthracyclines according to the invention are those of the formula: [ka] [In the formula, R 1 is selected from the group consisting of H and OH; R 2 is selected from the group consisting of H and OMe; R 3 is selected from the group consisting of H and OH, and R 4 is selected from the group consisting of H and OH. It is a compound of the formula:

[0095] In one embodiment, R 1 is H and R 2 is OMe, and R 3 is H, and R 4 is OH. In another embodiment, R 1 is OH and R 2 is OMe, and R 3 is H, and R 4 is OH. In another embodiment, R 1 is OH and R2 is OMe, and R 3 is OH, and R 4 is H. In another embodiment, R 1 is H and R 2 is H and R 3 is H, and R 4 is OH.

[0096] Particularly contemplated as an anthracycline in the context of the present invention is epirubicin, which has the following formula: [ka] It is an anthracycline drug having the formula: ##STR1## and is marketed in the United States under the trade name Ellence, and elsewhere under the trade name Pharmorubicin or Epirubicin Ebewe. In particular, the term "epirubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. Epirubicin is preferred in some chemotherapy regimens over doxorubicin, the most common anthracycline, because it is believed to cause fewer side effects.

[0097] According to the present invention, the agent that stabilizes or increases the expression of CLDN18.2 can be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, and can be part of a drug combination, for example a drug combination established for use in cancer treatment.Such a drug combination can be a drug combination used in chemotherapy, and can be a drug combination used in FOLFIRINOX chemotherapy regimen.

[0098] The drug combination used in FOLFIRINOX chemotherapy includes leucovorin, fluorouracil, irinotecan (such as irinotecan hydrochloride), and oxaliplatin. Oxaliplatin is administered at a dose of 85 mg / m 2 Irinotecan can be administered at 180 mg / m 2 leucovorin 400 mg / m 2 and fluorouracil 400 mg / m as a bolus. 2 followed by 5-fluorouracil, preferably as a 46-hour continuous infusion, at 2400 mg / m2, preferably every 2 weeks. 2 It may be administered at

[0099] The term "folinic acid" or "leucovorin" refers to a compound useful in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Thus, when referring to the administration of 5-fluorouracil or a prodrug thereof herein, in one embodiment, said administration may include administration in combination with folinic acid. Folinic acid has the following formula: [ka] has.

[0100] In particular, the term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}pentanedioic acid.

[0101] γδ T cells (gamma delta T cells) are a small subset of T cells that have a unique T cell receptor (TCR) on their surface. The majority of T cells have a TCR consisting of two glycoprotein chains called α-TCR chain and β-TCR chain. In contrast, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is usually much rarer than αβ T cells. Human γδ T cells play an important role in stress surveillance responses such as infections and autoimmunity. It has been suggested that transformation-induced changes in tumors also give rise to stress surveillance responses mediated by γδ T cells, enhancing antitumor immunity. Importantly, after antigen engagement, activated γδ T cells at the lesion site provide cytokines (e.g., INFγ, TNFα) and / or chemokines that mediate the recruitment of other effector cells to display immediate effector functions such as cytotoxicity (via death receptor and cytolytic granule pathways) and ADCC.

[0102] The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cell receptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates and are speculated to play an early and essential role in sensing "danger" from invading pathogens, as they expand dramatically in many acute infections, e.g., tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria, and can outnumber all other lymphocytes within days.

[0103] γδ T cells respond to small non-peptide phosphoantigens (phosphoantigens), such as pyrophosphate synthesized in bacteria and isopentenyl pyrophosphate (IPP) produced in mammalian cells via the mevalonate pathway. IPP production in normal cells is not sufficient for the activation of γδ T cells, but dysregulation of the mevalonate pathway in tumor cells leads to the accumulation of IPP and the activation of γδ T cells. IPP can also be therapeutically increased by aminobisphosphonates, which inhibit the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). Among others, zoledronic acid (ZA, zoledronate, Zometa™, Novartis) is representative of such aminobisphosphonates and has already been administered clinically to patients for the treatment of osteoporosis and metastatic bone disease. After treatment of PBMCs in vitro, ZA is taken up specifically by monocytes. IPP accumulates in monocytes and differentiates into antigen-presenting cells that stimulate the expression of γδ T cells. In this situation, the addition of interleukin 2 (IL-2) as a growth and survival factor for activated γδ T cells is preferred. Finally, certain alkylating amines have been described to activate Vγ9Vδ2 T cells in vitro, but only at millimolar concentrations.

[0104] According to the present invention, the term "agent stimulating γδ T cells" relates to a compound which stimulates the expression of γδ T cells, in particular Vγ9Vδ2 T cells, in vitro and / or in vivo, in particular by inducing the activation and proliferation of γδ T cells. Preferably, the term relates to a compound which increases the production of isopentenyl pyrophosphate (IPP) in mammalian cells in vitro and / or in vivo, preferably by inhibiting the mevalonate pathway enzyme, farnesyl pyrophosphate synthase (FPPS).

[0105] One particular group of compounds that stimulate γδ T cells are the bisphosphonates, in particular the nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

[0106] For example, suitable bisphosphonates for use in the present invention may include one or more of the following compounds, including analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof: [1-Hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, e.g. zoledronate; (Dichloro-phosphono-methyl)phosphonic acids, such as clodronate; {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronic acid, e.g. ibandronate; (3-Amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic acids, e.g. Pamidronate; (1-Hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acids, risedronic acids, e.g. Risedronate; (1-hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, minodronic acid; [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid; [4-Amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic acids, alendronic acids, e.g. Alendronate; [(Cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid; (1-hydroxyethane-1,1-diyl)bis(phosphonic acid), etidronic acids, e.g., etidronate; and {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic acid.

[0107] According to the present invention, zoledronic acid (INN) or zoledronate (marketed by Novartis under the trade names Zometa, Zomera, Aclaster and Reclast) is a particularly preferred bisphosphonate. Zometa is used to prevent bone fractures in patients with cancer, such as multiple myeloma and prostate cancer, and to treat osteoporosis. It can also be used to treat hypercalcemia of malignancy, and can also be useful in treating pain from bone metastases.

[0108] In one particularly preferred embodiment, the agent that stimulates γδ T cells according to the invention is administered in combination with IL-2, such a combination having been shown to be particularly effective in mediating the proliferation and activation of γ9δ2 T cells.

[0109] Interleukin 2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. It is a lymphocyte-attracting protein and is part of the body's natural response to microbial infections and in distinguishing self from foreign (non-self). IL-2 mediates its actions by binding to the IL-2 receptor expressed by lymphocytes.

[0110] The IL-2 used according to the invention may be any IL-2 that supports or allows stimulation of γδ T cells and may be derived from any species, preferably human. IL-2 may be isolated, recombinantly produced or synthetic IL-2, and may be naturally occurring IL-2 or modified IL-2.

[0111] The term "antigen" relates to an agent such as a protein or peptide containing an epitope against which an immune response is and / or should be directed. In a preferred embodiment, the antigen is a tumor-associated antigen such as CLDN18.2, i.e. a component of a cancer cell that may originate from the cytoplasm, the cell surface and the cell nucleus, in particular an antigen that is produced, preferably in large amounts, intracellularly or as a surface antigen on the cancer cell.

[0112] In connection with the present invention, the term "tumor-associated antigen" preferably relates to a protein that is specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage and is expressed or abnormally expressed in one or more tumors or cancer tissues. In connection with the present invention, the tumor-associated antigen preferably relates to the cell surface of cancer cells and is preferably not expressed at all or only rarely expressed in normal tissues.

[0113] The term "epitope" refers to an antigenic determinant in a molecule, i.e., a part of the molecule that is recognized by the immune system, for example, recognized by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. An epitope usually consists of chemically active surface groups of a molecule such as amino acids or sugar side chains and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished by the fact that in the presence of a denaturing solvent, the binding to the former is lost but the binding to the latter is not lost. The epitope of a protein such as CLDN18.2 preferably includes a continuous or discontinuous portion of the protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length. For example, the epitope can preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

[0114] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies and antibody fragments or derivatives, including, but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, such as scFvs, and antigen-binding antibody fragments such as Fab and Fab' fragments, and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0115] The antibody described herein may be a human antibody. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0116] 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 remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused to a constant domain, or may comprise only the complementarity determining regions (CDRs) grafted into appropriate framework regions within the variable domain. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions, e.g., modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from mouse antibodies). Other forms have one or more CDRs that are altered compared to the original antibody.

[0117] The term "chimeric antibody" refers to an antibody in which a portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular class, and the remaining segments of the chains are homologous to the corresponding sequence in another antibody. Typically, the variable regions of both the light and heavy chains mimic the variable regions of an antibody from one species of mammal, and the constant portions are homologous to the sequences of an antibody from another species. One obvious advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, for example, using B cells or hybridomas from readily available non-human host organisms, in combination with constant regions from human cell preparations. The variable regions have the advantage of ease of preparation and their specificity is independent of the source, while the constant regions that are human are less likely to provoke an immune response from a human subject when the antibody is injected than constant regions from a non-human source. However, the definition is not limited to this particular example.

[0118] The term "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") of an antibody or similar terms refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) an F(ab') fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. 2(iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of one arm of an antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; (vi) isolated complementarity determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, optionally 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 recombinant methods by a synthetic linker that allows them to be made into a single-chain protein (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. A further example is a binding domain immunoglobulin fusion protein that includes (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0119] The term "bispecific molecule" is intended to encompass any agent, such as a protein, peptide, or protein or peptide complex, that has two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to encompass any agent, such as a protein, peptide, or protein or peptide complex, that has three or more different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Thus, the present invention encompasses, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules against CLDN18.2 and other targets, such as Fc receptors on effector cells. The term "bispecific antibody" also encompasses diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing them to pair with the complementary domains on another chain and create two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0120] The antibody may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells, and in particular kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Suitable therapeutic agents for forming antibody conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), and / or cyclosporine (e.g., cyclosporine ... Examples of therapeutic agents include, but are not limited to, cyclosporine, cyclosporine, cyclopentasiloxane, cyclosporine, cyclopentasiloxane, cyclohex ...

[0121] The antibodies can also be coupled to radioactive isotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals.

[0122] The antibody conjugates of the present invention can be used to modulate a given biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon gamma; or biological response modifiers, such as lymphokines, interleukin 1 ("IL-1"), interleukin 2 ("IL-2"), interleukin 6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[0123] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies" in 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).

[0124] As used herein, an antibody is "derived" from a particular germline sequence if it is obtained by immunizing an animal or by screening an immunoglobulin gene library, and the antibody selected in said screen is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a particular germline sequence exhibits no more than 10 amino acid differences, more preferably no more than 5, or even more preferably no more than 4, 3, 2 or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0125] As used herein, the term "heteroantibody" refers to two or more antibodies, derivatives or antigen-binding regions thereof linked together, at least two of which have different specificities, including a binding specificity for an Fc receptor on an effector cell and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

[0126] The antibody described herein can be a monoclonal antibody. The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, monoclonal antibodies are produced by hybridomas that include B cells obtained from non-human animals, such as mice, fused to immortalized cells.

[0127] The antibody described herein may be a recombinant antibody. The term "recombinant antibody" as used herein encompasses all antibodies that are produced, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes or hybridomas produced therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies produced, expressed, created or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences.

[0128] The antibodies described herein may be derived from various species, including, but not limited to, mouse, rat, rabbit, guinea pig and human.

[0129] The antibodies referred to herein encompass polyclonal and monoclonal antibodies, and include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, kappa or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ) or an IgG4 antibody (e.g., IgG4, κ, λ).

[0130] The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungal cells, including yeast cells.

[0131] As used herein, a "heterologous antibody" is defined with respect to the transgenic organism producing such an antibody. The term refers to an antibody that is not constructed from the transgenic organism and generally has an amino acid sequence or encoding nucleic acid sequence that corresponds to one found in an organism derived from a species other than the transgenic organism.

[0132] As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain combined with a murine light chain is a heterohybrid antibody.

[0133] The present invention includes all antibodies and antibody derivatives described herein that are encompassed by the term "antibody" for purposes of the present invention. The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another agent or antibody, or an antibody fragment.

[0134] The antibodies described herein are preferably isolated. As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind to antigens other than CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform or variant of human CLDN18.2 may, however, have cross-reactivity to other related antigens, such as related antigens from other species (e.g., CLDN18.2 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, an "isolated" monoclonal antibody combination relates to antibodies that have different specificities and are combined in a well-defined composition or mixture.

[0135] The term "binding" according to the present invention preferably relates to specific binding.

[0136] According to the present invention, an antibody is capable of binding to a given target if it has significant affinity for and binds to the given target in a standard assay. "Affinity" or "binding affinity" is often measured using the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or a lower dissociation constant (K D ) to bind to a specific target.

[0137] If an antibody does not have significant affinity for a target in a standard assay and does not bind significantly, particularly not detectably, to said target, said antibody cannot (substantially) bind to said target. Preferably, said antibody does not bind detectably to said target when present at a concentration of up to 2 μg / ml, preferably up to 10 μg / ml, more preferably up to 20 μg / ml, particularly up to 50 μg / ml or 100 μg / ml or more. Preferably, an antibody has a K D At least 10 times, 100 times, or 10 times 3 Double, 10 4 Double, 10 5 Double or 10 6 Twice as high as K D For example, if an antibody binds to a target at a K D 10 -7 M, the K for binding to a target for which the antibody does not have significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3M, 10 -2 M or 10 -1 It's M.

[0138] An antibody is specific for a given target if it can bind to the given target but cannot bind to other targets, i.e. it has no significant affinity for other targets in standard assays and does not significantly bind to other targets. According to the present invention, an antibody is specific for CLDN18.2 if it can bind to CLDN18.2 but cannot (substantially) bind to other targets. Preferably, an antibody is specific for CLDN18.2 if its affinity and binding to such other targets is not significantly higher than its affinity or binding to proteins unrelated to CLDN18.2, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins, such as MHC molecules or transferrin receptors, or any other specific polypeptide. Preferably, an antibody has a K for binding to a target for which the antibody is not specific. D At least 10 times, 100 times, or 10 times 3 Double, 10 4 Double, 10 5 Double or 10 6 Twice lower K D An antibody is specific for a given target if it binds to the given target at D 10 -7 M, the K D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It's M.

[0139] Binding of an antibody to a target can be experimentally determined using any suitable method, for example, see Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, for example, by equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NYAcad.ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may differ if measured under different conditions, for example, under different salt concentrations, pH. Thus, affinity and other antigen binding parameters, such as K D ,I C 50 The measurement is preferably carried out using standard solutions of antibody and antigen and standard buffers.

[0140] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0141] As used herein, "isotype switching" refers to the phenomenon in which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0142] The term "naturally occurring" as used herein when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0143] The term "rearranged" as used herein refers to a configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned immediately adjacent to a DJ or J segment in a conformation that essentially encodes a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA, and rearranged loci have at least one recombined heptamer / 9amer homology element.

[0144] The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to a configuration in which the V segment has not been recombined so that it is immediately adjacent to a D or J segment.

[0145] According to the present invention, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, preferably an epitope located within the extracellular domain of CLDN18.2, particularly within the first extracellular domain, preferably within amino acid positions 29 to 78 of CLDN18.2. In certain embodiments, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NO:3, 4 and 5; (ii) an epitope located on CLDN18.2-loop1, preferably SEQ ID NO:8; (iii) an epitope located on CLDN18.2-loop2, preferably SEQ ID NO:10; (iv) an epitope located on CLDN18.2-loop D3, preferably SEQ ID NO:11; (v) an epitope encompassing CLDN18.2-loop 1 and CLDN18.2-loop D3; or (vi) a non-glycosylated epitope located on CLDN18.2-loop D3, preferably SEQ ID NO:9.

[0146] According to the present invention, the antibody capable of binding to CLDN18.2 is preferably an antibody capable of binding to CLDN18.2 but not to CLDN18.1. Preferably, the antibody capable of binding to CLDN18.2 is specific for CLDN18.2. Preferably, the antibody capable of binding to CLDN18.2 is an antibody capable of binding to CLDN18.2, preferably expressed on the cell surface. In a particular preferred embodiment, the antibody capable of binding to CLDN18.2 binds to a native epitope of CLDN18.2 present on the surface of a living cell. Preferably, the 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, the antibody capable of binding to CLDN18.2 is specific for the aforementioned proteins, peptides or immunogenic fragments or derivatives thereof. An antibody capable of binding to CLDN18.2 may be obtained by a method comprising the step of immunizing an animal with a protein or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46 and 48-50, or a nucleic acid or host cell expressing said protein or peptide. Preferably, the antibody binds to cancer cells, in particular cells of the aforementioned cancer types, and preferably does not substantially bind to non-cancerous cells.

[0147] Preferably, the binding of the antibody capable of binding to CLDN18.2 to cells expressing CLDN18.2 induces or mediates the death of the cells expressing CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells, in particular selected from the group consisting of tumorigenic gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer cells. Preferably, the antibody induces or mediates the death of the cells by inducing one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis and inhibition of proliferation of cells expressing CLDN18.2. Preferably, the ADCC-mediated lysis of the cells occurs in the presence of effector cells, which in a particular embodiment are selected from the group consisting of monocytes, mononuclear cells, NK cells and PMNs. Inhibition of cell proliferation can be measured in vitro by quantifying cell proliferation in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside that is an analogue of thymidine and can be incorporated into newly synthesized DNA of replicating cells (during the S phase of the cell cycle), replacing thymidine during DNA replication. Detecting the incorporated chemical, for example using an antibody specific for BrdU, indicates cells that were actively replicating their DNA.

[0148] In a preferred embodiment, the antibodies described herein have the following characteristics: a) specificity for CLDN18.2; b) a binding affinity to CLDN18.2 of about 100 nM or less, preferably about 5-10 nM or less, more preferably about 1-3 nM or less; c) the ability to induce or mediate CDC on CLDN18.2-positive cells; d) the ability to induce or mediate ADCC on CLDN18.2 positive cells; e) the ability to inhibit proliferation of CLDN18.2 positive cells; f) Ability to induce apoptosis in CLDN18.2 positive cells The compound may be characterized by one or more of the following:

[0149] In a particularly preferred embodiment, the antibody having the ability of binding to CLDN18.2 is produced by a hybridoma deposited at the DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) with the following name and accession number: a.182-D1106-055, Accession No. DSM ACC2737, deposited on October 19, 2005 b.182-D1106-056, Accession No. DSM ACC2738, Deposited October 19, 2005 c.182-D1106-057, Accession No. DSM ACC2739, Deposited October 19, 2005 d.182-D1106-058, Accession No. DSM ACC2740, deposited October 19, 2005 e.182-D1106-059, Accession No. DSM ACC2741, Deposited October 19, 2005 f.182-D1106-062, Accession No. DSM ACC2742, Deposited October 19, 2005 g.182-D1106-067, Accession No. DSM ACC2743, Deposited October 19, 2005 h.182-D758-035, Accession No. DSM ACC2745, deposited on November 17, 2005 i.182-D758-036, accession number DSM ACC2746, deposited on November 17, 2005 j.182-D758-040, Accession No. DSM ACC2747, deposited on November 17, 2005 k.182-D1106-061, Accession No. DSM ACC2748, deposited on November 17, 2005 l.182-D1106-279, Accession No. DSM ACC2808, deposited on October 26, 2006 m.182-D1106-294, Accession No. DSM ACC2809, Deposited October 26, 2006 n.182-D1106-362, accession number DSM ACC2810, deposited October 26, 2006.

[0150] Preferred antibodies according to the invention are those produced by and obtained from the above mentioned hybridomas, namely 37G11 for 182-D1106-055, 37H8 for 182-D1106-056, 38G5 for 182-D1106-057, 38H3 for 182-D1106-058, 39F11 for 182-D1106-059, 43A11 for 182-D1106-062, 43A12 for 182-D1106-063, 43A13 for 182-D1106-064, 43A14 for 182-D1106-065, 43A20 for 182-D1106-066, 43A15 for 182-D1106-067, 43A21 for 182-D1106-068, 43A16 for 182-D1106-069, 43A22 for 182-D1106-070, 43A17 for 182-D1106-071, 43A18 for 182-D1106-072, 43A19 for 182-D1106-073, 43A23 for 182-D1106-074, 43A24 for 182-D1106-075, 43A25 for 182-D1106-076, 43A26 for 182-D1106-077, 43A27 for 182-D1106-078, 43A28 for 61C2 for D1106-067, 26B5 for 182-D758-035, 26D12 for 182-D758-036, 28D10 for 182-D758-040, 42E12 for 182-D1106-061, 125E1 for 182-D1106-279, 163E12 for 182-D1106-294, and 175D10 for 182-D1106-362; and chimeric and humanized forms thereof.

[0151] Preferred chimeric antibodies and their sequences are shown in the table below.

[0152] TIFF0007681061000016.tif140170 In a preferred embodiment, the antibody, particularly an antibody in chimeric form according to the invention, encompasses an antibody comprising a heavy chain constant region (CH) having an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof. In a further preferred embodiment, the antibody, particularly an antibody in chimeric form according to the invention, encompasses an antibody comprising a light chain constant region (CL) having an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In a particular preferred embodiment, the antibody, particularly an antibody in chimeric form according to the invention, encompasses 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.

[0153] In one embodiment, the antibody capable of binding to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising a mouse kappa variable light chain, a human kappa light chain constant region allotype Km(3), a mouse heavy chain variable region, a human IgG1 constant region, allotype G1m(3).

[0154] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and fragments thereof, and / or a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28 and fragments thereof.

[0155] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a heavy and light chain combination selected from the following possibilities (i) to (ix): (i) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof; (ii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof; (iii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof; (iv) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof; (v) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof; (vi) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof; (vii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof; (viii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof; and (ix) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19, or a fragment thereof, and the light chain comprises the amino acid sequence represented by SEQ ID NO: 28, or a fragment thereof.

[0156] "Fragment" or "fragment of an amino acid sequence" as used above relates to a part of an antibody sequence, i.e. an antibody sequence truncated at the N-terminus and / or C-terminus, which when it replaces said antibody sequence in an antibody retains the binding of said antibody to CLDN18.2 and preferably the function of said antibody as described herein, e.g. CDC-mediated lysis or ADCC-mediated lysis. Preferably, a fragment of an amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the amino acid residues from said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 relates to said sequence, preferably with 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus removed.

[0157] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a heavy chain variable region (VH) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and fragments thereof.

[0158] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a light chain variable region (VL) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, and fragments thereof.

[0159] In certain preferred embodiments, the antibody capable of binding to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (ix): (i) the VH comprises the amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof; (ii) the VH comprises the amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof; (iii) the VH comprises the amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof; (iv) the VH comprises the amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof; (v) the VH comprises the amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof; (vi) the VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof; (vii) the VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof; (viii) the VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof; (ix) the VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof, and the VL comprises the amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

[0160] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VH containing a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi): (i) CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, (ii) CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, (iii) CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, (iv) CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, (v) CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, and (vi) CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19.

[0161] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VL containing a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix): (i) CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20, (ii) CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21, (iii) CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22, (iv) CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23, (v) CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24, (vi) CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (vii) CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26, (viii) CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0162] In a preferred embodiment, the antibody having the ability of binding to CLDN18.2 comprises a VH and VL combination, each of which contains a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix): (i) VH: CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14; VL: CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21; (ii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15; VL: CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20; (iii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16; VL: CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22; (iv) VH: CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, VL: CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17; VL: CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24; (vi) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19; VL: CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23; (vii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19; VL: CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26; (viii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27, and (ix) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19; VL: CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0163] In a further preferred embodiment, the antibody having the ability of binding to CLDN18.2 preferably comprises one or more, preferably at least the CDR3 variable region, of the complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or the light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 as described herein, and preferably comprises one or more, preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and / or the light chain variable region (VL) as described herein. In one embodiment, one or more of said complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3 as described herein. In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, and preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2 described herein.

[0164] In one embodiment, an antibody comprising one or more CDRs, sets of CDRs or combinations of sets of CDRs described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion comprises at least about 50% of either or both of the first and fourth framework regions, said 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies performed by recombinant DNA techniques may result in the introduction of residues at the N- or C-terminal end of the variable region that are introduced by linkers to facilitate cloning or other engineering steps, including the introduction of linkers for linking the variable regions of the invention to further protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the creation of diabodies) or protein tags.

[0165] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs described herein comprises said CDRs within a human antibody framework.

[0166] Reference herein to an antibody comprising a particular chain or a particular region or sequence in relation to its heavy chain preferably relates to the situation where all heavy chains of said antibody comprise said particular chain, region or sequence, and this applies correspondingly to the light chains of the antibody.

[0167] The term "nucleic acid" as used herein is intended to encompass DNA and RNA. Nucleic acids can be single-stranded or double-stranded, but preferably are double-stranded DNA.

[0168] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA or the production of RNA and proteins / peptides. This term also includes partial expression of a nucleic acid. Furthermore, expression can be performed transiently or stably.

[0169] The teachings given herein with respect to specific amino acid sequences, such as those shown in the sequence listing, should also be interpreted as relating to sequences that are functionally equivalent to the specific sequences, such as variants of the specific sequences that result in amino acid sequences that exhibit the same or similar properties as those of the specific amino acid sequences. One important property is to retain the binding of the antibody to its target or to maintain the effector function of the antibody. Preferably, a sequence that is variant with respect to a specific sequence, when it replaces the specific sequence in an antibody, retains the binding of the antibody to CLDN18.2 and preferably the function of the antibody as described herein, such as CDC-mediated lysis or ADCC-mediated lysis.

[0170] It will be appreciated by those skilled in the art that the sequences of the CDRs, hypervariable regions and variable regions in particular may be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions are identical or highly homologous to regions of the antibodies specified herein. By "highly homologous" it is contemplated that 1-5, preferably 1-4, such as 1-3 or 1 or 2 substitutions may be made within the CDRs. In addition, the hypervariable and variable regions may be modified to show substantial homology with regions of the antibodies specifically disclosed herein.

[0171] For the purposes of the present invention, a "variant" of an amino acid sequence includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and / or an amino acid substitution variant. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0172] Amino acid insertion variants include the insertion of one or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

[0173] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0174] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be in any position in the protein.

[0175] An amino acid substitution variant is characterized in that at least one residue within the sequence has been removed and another residue inserted in its place. Modifications are preferably present at positions within the amino acid sequence that are not conserved between homologous proteins or peptides and / or the amino acid is substituted with another amino acid having similar properties. Preferably, the amino acid changes in the protein variant are conservative amino acid changes, i.e., substitutions of amino acids with similar charge or uncharged amino acids. Conservative amino acid changes include substitutions of one member of an amino acid family with a side chain related amino acid. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids.

[0176] Preferably, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example using Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, cap open 10.0, gap extension 0.5.

[0177] "Sequence similarity" indicates the percentage of amino acids that are identical or which are conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0178] The term "percent identity" is intended to represent the percentage of amino acid residues that are identical between the two sequences to be compared, obtained after the best alignment, and this percentage is purely statistical, the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment, said comparison being performed segment by segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be created manually or by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0179] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared to obtain the percent identity between the two sequences, and multiplying the result by 100.

[0180] The term "transgenic animal" refers to an animal having a genome that includes one or more transgenes, preferably heavy and / or light chain transgenes, or transchromosomes (integrated or not integrated into the animal's native genomic DNA), and preferably capable of expressing the transgenes. For example, a transgenic mouse 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 CLDN18.2 antigen and / or cells expressing CLDN18.2. The human heavy chain transgene may be integrated into the mouse's chromosomal DNA, as in the case of transgenic mice such as HCo7 or HCol2 mice, e.g., HuMAb mice, or the human heavy chain transgene may be maintained extrachromosomally, as in the case of transchromosomal (e.g., KM) mice described in WO 02 / 43478. Such transgenic and transchromosomal mice are capable of producing multiple isotypes of human monoclonal antibodies against CLDN18.2 (eg, IgG, IgA and / or IgE) by undergoing VDJ recombination and isotype switching.

[0181] As used herein, "reduce" or "inhibit" refers to an ability to cause an overall decrease or an overall decrease in a level, e.g., the level of expression or proliferation of a cell, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0182] Terms such as "increase" or "enhance" relate to an increase or enhancement, preferably 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%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

[0183] Mechanism of action of mAbs The following provides a discussion of the mechanisms underlying the therapeutic effect of the antibodies of the invention, but should not be construed as a limitation on the invention in any way.

[0184] The antibodies described herein preferably interact with components of the immune system, preferably via ADCC or CDC.The antibodies described herein can also be used to target payloads (e.g., radioisotopes, drugs or toxins) to directly kill tumor cells, or can be used synergistically with traditional chemotherapeutic agents to attack tumors via complementary mechanisms of action, which may include anti-tumor immune responses that may be compromised due to the cytotoxic side effects of chemotherapeutic agents on T lymphocytes.However, the antibodies described herein can also act simply by binding to CLDN18.2 on the cell surface, thus, for example, blocking cell proliferation.

[0185] Antibody-dependent cell-mediated cytotoxicity ADCC refers to the cell killing capacity of effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells are marked by antibodies.

[0186] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain engages with the 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 as a mechanism that directly induces various degrees of immediate tumor destruction, leading to antigen presentation and induction of T cell responses against the tumor. Preferably, in vivo induction of ADCC leads to T cell responses and host-derived antibody responses against the tumor.

[0187] Complement-dependent cytotoxicity 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 both very effective at directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of the antigen-antibody complex results in the exposure of a number of closely adjacent C1q binding sites on the C H 2 domains (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previous low-affinity C1q-IgG interaction into a high-affinity interaction, which initiates a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector chemotactic / activator proteins C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex that creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0188] Production and testing of antibodies The antibodies described herein can be made by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization techniques of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are generally preferred in principle, other techniques for making monoclonal antibodies, such as viral transformation or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, can also be used.

[0189] A preferred animal system for making hybridomas that secrete monoclonal antibodies is the mouse system. The production of hybridomas in mice is a very well-established procedure. Techniques for isolating immune spleen cells for immunization protocols and fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0190] Other preferred animal systems for producing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (described, e.g., in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0191] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Production of human antibodies in such transgenic mice can be performed as detailed for CD20 in WO 2004 / 035607.

[0192] Yet another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding genes from lymphocytes that produce antibodies with defined specificities, see e.g. Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0193] To generate antibodies, mice can be immunized with carrier-bound peptides derived from the antigen sequence, i.e., the sequence against which the antibody is to be directed, enriched preparations of recombinantly expressed antigen or fragments thereof, and / or cells expressing the antigen, as described above. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. If immunization with a purified or enriched preparation of the antigen does not produce antibodies, mice can also be immunized with cells expressing the antigen, e.g., a cell line, to stimulate an immune response.

[0194] Immune responses can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. To enhance the percentage of hybridomas secreting specific antibodies, mice can be boosted intraperitoneally or intravenously with antigen-expressing cells 3 days before sacrifice and splenectomy.

[0195] To generate hybridomas that produce monoclonal antibodies, spleen cells and lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for production of antigen-specific antibodies. Individual wells can then be screened for antibody-secreting hybridomas by ELISA. Antibodies with specificity for the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. Antibody-secreting hybridomas can be replated, screened again, and subcloned by limiting dilution if still positive for monoclonal antibodies. Stable subclones can then be cultured in vitro to generate antibodies in tissue culture medium for characterization.

[0196] Antibodies can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection techniques well known in the art (Morrison, S. (1985) Science 229:1202).

[0197] For example, in one embodiment, the gene of interest, e.g., an antibody gene, can be ligated into an expression vector, such as a eukaryotic expression plasmid as used by the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems known in the art. The purified plasmid containing the cloned antibody gene can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells, such as plant-derived cells, fungi, or yeast cells. The method used to introduce these genes can be methods described in the art, such as electroporation, lipofectin, lipofectamine, and others. After introduction of these antibody genes into the host cells, cells expressing the antibody can be identified and selected. These cells are transfectomas, which can then be amplified for expression levels and scaled up to produce the antibody. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0198] Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli. Furthermore, antibodies can be produced in transgenic non-human animals, e.g., milk from sheep and rabbits or eggs from hens, or in transgenic plants; see, e.g., Verma, R., et al. (1998) J. Immunol. Meth. 216:165-181; Pollock, et al. (1999) J. Immunol. Meth. 231:147-157; and Fischer, R., et al. (1999) Biol. Chem. 380:825-839.

[0199] Chimerization 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 applied repeatedly, resulting in reduced therapeutic efficacy. The main immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided if the respective antibodies are chimerized or humanized. Chimeric antibodies are antibodies whose different portions originate from different animal species, e.g., those with variable regions originating from a mouse antibody and human immunoglobulin constant regions. Antibody chimerization is achieved by linking the heavy and light chain variable regions of a mouse antibody with human heavy and light chain constant regions (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are made by linking a human kappa light chain constant region to a mouse light chain variable region. In an equally preferred embodiment, chimeric antibodies can be made by linking a human lambda light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for making chimeric antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain constant regions for making chimeric antibodies are IgG2, IgA, IgD and IgM.

[0200] Humanization Antibodies interact with target antigens primarily through amino acid residues located within six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector that contains the CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases that contain germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B cell maturation. The germline gene sequences also differ individually from the sequences of high affinity secondary repertoire antibodies uniformly throughout the variable regions.

[0201] The ability of the antibody to bind the antigen can be determined using standard binding assays, such as ELISA, Western blot, immunofluorescence and flow cytometric analysis.

[0202] To purify the antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysis-based bioreactors. The supernatant can be filtered and concentrated if necessary, and then subjected to affinity chromatography with protein G-sepharose or protein A-sepharose. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0203] To determine whether a selected monoclonal antibody binds to a unique epitope, site-directed or multi-site directed mutagenesis can be used.

[0204] To determine the antibody isotype, an isotype ELISA can be performed with various commercially available kits (e.g. Zymed, Roche Diagnostics). The wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with monoclonal antibodies or purified isotype controls for 2 hours at ambient temperature. The wells can then be reacted with either mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM specific peroxidase-conjugated probes. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at an OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) can be used as described by the manufacturer.

[0205] Flow cytometry can be used to reveal the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. Cell lines expressing the antigen 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 supernatants or in PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, APC- or Alexa647-labeled anti-IgG antibodies can be bound to the antigen-bound monoclonal antibodies under the same conditions as the primary antibody staining. Samples can be analyzed by flow cytometry in a FACS machine utilizing side light scatter properties to gate on single live cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, a method of co-transfection can be used. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescent channel than antibody-stained cells. Since the majority of transfected cells express both transgenes, the antigen-specific monoclonal antibody will selectively bind to the fluorescent marker-expressing cells, while the non-specific antibody will bind to the non-transfected cells in equal proportions. A selective assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells may be stained exactly as described above and examined by fluorescence microscopy.

[0206] Immunofluorescence microscopy can be used to reveal the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. For example, cell lines expressing the antigen naturally or after transfection and negative controls lacking antigen expression are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin and 100 μg / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen for 30 minutes at 25°C. After washing, the cells can be reacted with an Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0207] Cell extracts from cells expressing the antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

[0208] The antibody can be further tested for reactivity with the antigen by immunohistochemistry in a manner 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 bearing xenograft tumors inoculated with cell lines expressing the antigen naturally or after transfection. For immunostaining, the antibody 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.

[0209] Antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CLDN18.2. Testing monoclonal antibody activity in vitro provides an initial screen prior to testing in in vivo models.

[0210] Antibody-dependent cell-mediated cytotoxicity (ADCC) Briefly, polymorphonuclear cells (PMN), NK cells, monocytes, mononuclear cells or other effector cells from healthy donors can be purified by Ficoll Hypaque density gradient centrifugation followed by lysis of contaminating red blood cells. Washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum and purified to identify cells expressing CLDN18.2. 51 Cr-labeled target cells can be mixed with various ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence enhancing ligand (BATDA). Highly fluorescent chelates of europium with the enhancing ligand released from dead cells can be measured by fluorometer. Another alternative technique may utilize transfection of target cells with luciferase. Added lucifer yellow can then only be oxidized by live cells. Purified anti-CLDN18.2 IgG can then be added at various concentrations. An irrelevant human IgG can be used as a negative control. The assay can be performed at 37°C for 4-20 hours depending on the effector cell type used. In the culture supernatant, 51 Samples can be assayed for cell lysis by measuring Cr release or the presence of EuTDA chelate. Alternatively, luminescence resulting from the oxidation of Lucifer Yellow can be a measure of viable cells. Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine whether cell lysis is enhanced with multiple monoclonal antibodies.

[0211] Complement-dependent cytotoxicity (CDC) Monoclonal anti-CLDN18.2 antibodies can be tested for their ability to mediate CDC using a variety of known techniques. For example, serum for complement can be obtained from blood by methods known to those skilled in the art. To measure the CDC activity of mAbs, various methods can be used. For example, 51 Cr release can be measured, or propidium iodide (PI) exclusion assays can be used to assess membrane permeabilization. Briefly, target cells were washed and 5×10 5 100 / ml can be incubated with various concentrations of mAb for 10-30 min at room temperature or 37°C. Serum or plasma can then be added to a final concentration of 20% (v / v) and the cells can be incubated for 20-30 min at 37°C. All cells from each sample can be added to the PI solution in a FACS tube. The mixtures can then be analyzed immediately by flow cytometry analysis using a FACSArray.

[0212] In an alternative assay, induction of CDC can be measured in adherent cells. In one embodiment of this assay, cells are plated at 3×10 in tissue culture flat-bottom microtiter plates 24 hours prior to the assay. 4 Cells are seeded at a density of 1000 μg / well. The next day, growth medium is removed and cells are incubated in triplicate with antibodies. Control cells are incubated with growth medium or growth medium containing 0.2% saponin for measurement of background lysis and maximum lysis, respectively. After 20 min incubation at room temperature, the supernatant is removed and 20% (v / v) human plasma or serum in DMEM (pre-warmed to 37° C.) is added to the cells and incubated for another 20 min at 37° C. All cells from each sample are added to a propidium iodide solution (10 μg / ml). The supernatant is then replaced with PBS containing 2.5 μg / ml ethidium bromide and the fluorescence emission at 520 nm excitation is measured at 600 nm using a Tecan Safire. The percentage of specific lysis is calculated as follows: % specific lysis=(sample fluorescence−background fluorescence) / (maximum lysis fluorescence−background fluorescence)×100.

[0213] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies To test for the ability to initiate apoptosis, monoclonal anti-CLDN18.2 antibodies can be incubated with, for example, CLDN18.2 positive tumor cells, such as SNU-16, DAN-G, KATO-III or CLDN18.2 transfected tumor cells, at 37° C. for about 20 hours. Cells can be harvested, washed in Annexin-V binding buffer (BD biosciences), and incubated with Annexin-V conjugated with FITC or APC (BD biosciences) for 15 minutes in the dark. All cells from each sample can be added to 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, Cat. No. AD0200) is a nonisotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis of cells growing in microplates. Incorporated BrdU is detected using a europium-labeled monoclonal antibody. To enable antibody detection, cells are fixed with Fix solution and DNA is denatured. Unbound antibody is washed away and DELFIA inducer is added to dissociate europium ions from the labeled antibody into solution where they form highly fluorescent chelates with components of the DELFIA inducer. Detection utilizes time-resolved fluorometry and the measured fluorescence is proportional to DNA synthesis in the cells of each well.

[0214] Preclinical trials Monoclonal antibodies that bind to CLDN18.2 can also be tested in in vivo models (e.g., in immunodeficient mice bearing xenograft tumors inoculated with cell lines expressing CLDN18.2, such as DAN-G, SNU-16, or KATO-III, or cell lines expressing CLDN18.2 after transfection, such as HEK293) to measure their effectiveness in controlling the growth of tumor cells expressing CLDN18.2.

[0215] In vivo testing after xenografting of CLDN18.2-expressing tumor cells into immunocompromised mice or other animals can be performed using the antibodies described herein. Tumor-free mice can be administered the antibodies and then injected with tumor cells to measure the effect of the antibodies in preventing tumor formation or tumor-related symptoms. Tumor-bearing mice can be administered the antibodies and the therapeutic effect of each antibody in reducing tumor growth, metastasis or tumor-related symptoms can be measured. Application of the antibodies can be combined with application of other substances, such as cytostatics, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies, to measure synergistic effects and potential toxicity of the combination. To analyze toxic side effects mediated by the antibody, animals can be inoculated with the antibody or a control agent and thoroughly examined for symptoms that may be related to CLDN18.2 antibody treatment. Possible side effects of in vivo application of CLDN18.2 antibodies include toxicity in CLDN18.2-expressing tissues, including the stomach, among others. 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.

[0216] Mapping of epitopes recognized by antibodies can be performed as detailed in "Epitope Mapping Protocols (Methods in Molecular Biology)" by Glenn E. Morris ISBN-089603-375-9 and "Epitope Mapping: A Practical Approach" by Olwyn MRWestwood, Frank C. Hay, Practical Approach Series, 248.

[0217] The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

[0218] The pharmaceutical compositions will usually be presented in unit dosage form and may be prepared in a manner known per se The pharmaceutical composition may, for example, be in the form of a solution or suspension.

[0219] Pharmaceutical compositions may contain salts, buffer substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharma- ceutically acceptable. The term "pharmaceutical acceptable" refers to the non-toxicity of substances that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0220] Pharmaceutically unacceptable salts can be used to prepare pharma- ceutically acceptable salts and are encompassed by the present invention. Such pharma- ceutically 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. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0221] Suitable buffering substances for use in the pharmaceutical compositions include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

[0222] Suitable preservatives for use in the pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0223] Injectable formulations may contain pharma- ceutically acceptable excipients, such as lactated Ringer's solution.

[0224] The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

[0225] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, particularly, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

[0226] The term "excipient" as used herein is intended to indicate any substance that may be present in a pharmaceutical composition and which is not an active ingredient, such as a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent or coloring agent.

[0227] The agents and compositions described herein may be administered by any conventional route, for example, parenteral administration, including injection or infusion. Administration is preferably parenteral, for example, intravenous, intraarterial, subcutaneous, intradermal or intramuscular routes.

[0228] Compositions suitable for parenteral administration usually include sterile aqueous or non-aqueous preparations of active compounds, preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are usually used as a medium for solution or suspension.

[0229] The agents and compositions described herein are administered in effective amounts. "Effective amount" refers to an amount that achieves a desired response or a desired effect, either alone or together with further administrations. In the case of treating a particular disease or a particular condition, the desired response is preferably related to the prevention of disease progression. This includes slowing disease progression, particularly preventing or reversing disease progression. In the treatment of a disease or condition, the desired response can also be slowing or preventing the onset of said disease or said condition.

[0230] The effective amount of the agent or composition described herein depends on the condition to be treated, the severity of the disease, the patient's individual parameters, including the age, physiological state, size and weight of the patient, the duration of treatment, the type of concomitant treatment (if any), specific route of administration and similar factors.Therefore, the dose of the agent described herein to be administered may depend on such various parameters.If the initial dose is insufficient in the patient's response, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0231] The agents and compositions described herein can be administered to a patient, for example in vivo, to treat or prevent various disorders as described herein. Preferred patients include human patients with disorders that can be corrected or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by an altered expression pattern of CLDN18.2.

[0232] For example, in one embodiment, the antibodies described herein can be used to treat a patient having a cancer disease, such as a cancer disease as described herein, characterized by the presence of cancer cells that express CLDN18.2.

[0233] The aforementioned pharmaceutical compositions and methods of treatment according to the present invention may also be used for immunization or vaccination to prevent the diseases mentioned herein.

[0234] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Below, examples of reference forms are given. 1. A method for treating or preventing pancreatic cancer in a patient, comprising administering to the patient (i) an antibody capable of binding to CLDN18.2 and (ii) an agent that stabilizes or increases the expression of CLDN18.2. 2. The method according to 1, wherein CLDN18.2 is expressed on the cell surface of cancer cells. 3. The method of 1. or 2., wherein the agent that stabilizes or increases expression of CLDN18.2 comprises an agent that induces cell cycle arrest or accumulation of cells in one or more phases of the cell cycle, preferably one or more phases of the cell cycle other than G1 phase, more preferably G2 phase and / or S phase. 4. The method of any one of 1. to 3., wherein the agent that stabilizes or increases the expression of CLDN18.2 comprises an agent selected from the group consisting of nucleoside analogs, platinum compounds, camptothecin analogs, taxanes, prodrugs thereof, salts thereof, and combinations thereof. 5. The method of any one of 1. to 4., wherein the agent that stabilizes or increases expression of CLDN18.2 comprises an agent selected from the group consisting of gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, prodrugs thereof, salts thereof, and combinations thereof. 6. The method of any one of 1. to 5., wherein the agent that stabilizes or increases expression of CLDN18.2 comprises an agent that induces immunogenic cell death. 7. The method of claim 6, wherein the agent that induces immunogenic cell death comprises oxaliplatin. 8. The method of any one of 1. to 7., wherein the method comprises administering a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of oxaliplatin, 5-fluorouracil or a prodrug thereof, and irinotecan. 9. The method of any one of 1. to 8., wherein the method comprises administering folinic acid, oxaliplatin, 5-fluorouracil or a prodrug thereof, and irinotecan. 10. A method for treating or preventing cancer in a patient, comprising administering to the patient (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine. 11. The method according to claim 10, wherein the cancer is pancreatic cancer. 12. The method of any one of 1. to 11., wherein the method further comprises administering an agent that stimulates γδ T cells, the γδ T cells being preferably Vγ9Vδ2 T cells. 13. The method of claim 12, wherein the agent that stimulates γδ T cells is a bisphosphonate. 14. The method of 12. or 13., wherein the agent that stimulates γδ T cells is a nitrogen-containing bisphosphonate (aminobisphosphonate). 15. The method of any one of 12. to 14., wherein the agent that stimulates γδ T cells is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid, and salts thereof. 16. The method according to any one of claims 12 to 15, wherein the agent that stimulates γδ T cells is administered in combination with interleukin-2. 17. The method according to any one of 1. to 16., wherein the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. 18. A method according to any one of 1. to 17., wherein the antibody capable of binding to CLDN18.2 mediates cell killing by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. 19. The antibody having the ability to bind to CLDN18.2, (i) a polypeptide having the 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 19. The method according to any one of 1. to 18., wherein the antibody is selected from the group consisting of an antibody produced by and / or obtainable from the clone deposited under ACC2810, (ii) an antibody which is a chimeric or humanized form of the antibody included in (i), (iii) an antibody having the specificity of the antibody included in (i), and (iv) an antibody which contains an antigen-binding portion or antigen-binding site, particularly the variable region, of the antibody included in (i) and preferably has the specificity of the antibody included in (i). 20. The method comprises administering to the patient at least one of the antibodies capable of binding to CLDN18.2 at a concentration of 1000 mg / m 2 20. The method of any one of 1. to 19., comprising administering at a dose of up to 21. The method comprises administering to the patient an antibody capable of binding to CLDN18.2 at a concentration of 300 to 600 mg / m 2 21. The method according to any one of 1. to 20., comprising repeatedly administering at a dose of 22. The method of any one of 1. to 21., wherein the cancer is CLDN18.2 positive. 23. The method of any one of 1. to 22., wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO:1. 24. The method of any one of 1. to 9. and 11. to 23., wherein the pancreatic cancer includes primary cancer, advanced cancer or metastatic cancer, or a combination thereof, such as a combination of primary pancreatic cancer and metastatic cancer. 25. The method of claim 24, wherein the metastatic cancer comprises metastasis to lymph nodes, ovaries, liver or lungs, or a combination thereof. 26. The method of any one of 1. to 9. and 11. to 25., wherein the pancreatic cancer comprises pancreatic ductal cancer. 27. The method of any one of 1. to 9 and 11 to 26, wherein the pancreatic cancer comprises an adenocarcinoma or carcinoma, or a combination thereof. 28. The method of any one of 1. to 9 and 11 to 27, wherein the pancreatic cancer comprises pancreatic ductal adenocarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma or acinar cell carcinoma, or a combination thereof. 29. The method of any one of 1. to 9 and 11 to 28, wherein the pancreatic cancer is partially or completely refractory to gemcitabine treatment, such as gemcitabine monotherapy. 30. A method according to any one of 1. to 9 and 11 to 29, wherein preventing pancreatic cancer comprises preventing recurrence of pancreatic cancer. 31. A method according to any one of 1. to 9 and 11. to 30, wherein the patient has undergone surgery for pancreatic cancer. 32. The method according to any one of 1. to 9. and 11. to 31., wherein the patient has a precancerous pancreatic lesion, particularly a precancerous pancreatic lesion comprising early malignant histological changes in the pancreatic duct. 33. A medical preparation for treating or preventing pancreatic cancer, comprising (i) an antibody capable of binding to CLDN18.2 and (ii) an agent that stabilizes or increases the expression of CLDN18.2. 34. The medical preparation according to 33, which is in the form of a kit comprising a first container containing the antibody capable of binding to CLDN18.2 and a second container containing the agent that stabilizes or increases the expression of CLDN18.2. 35. The medical preparation described in 33. or 34., further comprising printed instructions for using the preparation for the treatment or prevention of pancreatic cancer. 36. A medical preparation comprising (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine. 37. The medical preparation according to 36, for treating or preventing cancer, particularly pancreatic cancer. 38. The medical preparation according to 36. or 37., which is in the form of a kit comprising a first container containing the antibody capable of binding to CLDN18.2 and a second container containing gemcitabine. 39. The medical preparation according to any one of claims 36 to 38, further comprising printed instructions for use of said preparation for the treatment or prevention of cancer, particularly pancreatic cancer. EXAMPLES

[0235] [Example 1] Experimental Materials and Methods 1. Antibodies [Table 1]

[0236] 2. Immunohistochemistry (IHC) Tissue sections (4 μm thick) were stored at 2 to 8°C until use.

[0237] Prior to the deparaffinization step, the sections were incubated in a drying oven at 58-60°C for 1 h in order to melt the paraffin and quantitatively remove the water, thereby improving adhesion of the tissue to the glass slides ("baking").

[0238] Deparaffinization After melting and drying, slides were deparaffinized using two xylol steps (5 min) and rehydrated using a decreasing alcohol array (at ambient temperature 20–27 °C): ·Xylene bath for 5 (± 1) minutes; This process was repeated once in a fresh bath; · Excess fluid; · Absolute ethanol for 5 (± 1) minutes; Repeat this process once with a fresh bath; · Remove excess liquid; · 96% ethanol for 5 (± 1) minutes; Repeat this process once with a fresh bath; · Remove excess liquid; · 80% ethanol for 5 (± 1) min; · Remove excess liquid; · 70% ethanol for 5 (± 1) min; · Remove excess liquid; - Distilled or deionized water for 5 minutes.

[0239] Epitope Retrieval and Quenching After paraffin removal, the target epitopes were retrieved using a heat-induced epitope retrieval procedure. For this, slides were placed in a staining jar filled with 200 ml of retrieval buffer (10 mM citrate buffer; 0.05% Tween-20; pH 6) and incubated at 120°C for 10 min in a pressure cooker (PASCAL, Dako). The jar was then removed from the cooker and allowed to cool in the epitope retrieval solution for 10 (±1) min at room temperature. Slides were washed in washing buffer (1x PBS).

[0240] After cooling, sections were transferred to staining jars filled with 200 ml of quenching solution (0.3% peroxidase in 1× PBS), incubated for 15 min at room temperature, and then subjected to 2×5 min washing steps in fresh wash buffer.

[0241] Blocking and antibody incubation Excess wash buffer was removed and slides were covered with 200 μl of blocking buffer (10% goat serum in 1x PBS) and incubated for 30 min at room temperature. Blocking buffer was removed and replaced with 200 μl of diluted antibody solution (diluted in blocking buffer). Slides were incubated overnight at 2-8°C with primary antibodies: [Table 2]

[0242] The next day, the primary antibody solution was removed and the sections were washed 3x5 min in wash buffer. Excess wash buffer was then removed and 200 μl of ready-to-use secondary antibody solution was added (Power Vision HRP goat α-mouse; Immunologic; NL). Slides were incubated for 30 min at room temperature. Excess liquid was removed and slides were washed 3x5 min in fresh wash buffer.

[0243] Substrate reaction and counterstaining After removal of excess wash buffer, sections were covered with approximately 50-150 μL of freshly prepared substrate-chromogen solution (VectorRed; Vector Labs) for 2 min. Excess substrate was removed and slides were incubated in a jar with deionized water for 1-5 min.

[0244] Tissue counterstaining was then performed by immersing the sections in a jar containing 200 ml of Mayer's hematoxylin for 2 min, after which the sections were placed in tap water for 5-10 min to stain the nuclei blue.

[0245] Dehydration and mounting After counterstaining, sections were dehydrated using ascending alcohol arrays: - Immerse in 70% ethanol (for about 5 to 10 seconds) -Immerse in 80% ethanol (for about 5 to 10 seconds) - Immerse in 96% ethanol (for about 5 to 10 seconds) - Immerse in 96% ethanol (for about 5 to 10 seconds) -Immerse in absolute ethanol (for about 5 to 10 seconds) 5 minutes in xylene -Xylene for 5 minutes.

[0246] A non-aqueous mounting medium (X-TRA-Kit, Medite) was used for mounting the samples. Slides were mounted directly from the last xylene-filled jar and air-dried at room temperature. [Table 3]

[0247] 3.Culture All pancreatic cancer cell lines and additional control cell lines used for the experiments presented herein are cultured in media according to the data sheets of origin and by standard tissue culture procedures. Conditions are summarized in Table 4. For all newly obtained cell lines, cells were tested for mycoplasma contamination and master cell banks were prepared. [Table 4]

[0248] 4. Luciferase Transfection of Pancreatic Cell Lines For ADCC assays, pancreatic cancer cell lines were transiently transfected with luciferase RNA (pST1-luc2mut-2hBgUTR-A121-EciI vector (pST1-109)) with an ARCA cap and expressed in H 2 The RNA was stored in 22 μl aliquots at -80°C. For all pancreatic cell lines, optimal electroporation conditions were determined that yielded the highest transfection rate and cell viability. For each assay, cells were detached with PBS / 5 mM EDTA and 2.5 × 10 ng / ml of X-Vivo PBS were dissolved in 250 μl of PBS / 5 mM EDTA. 6 Cells were mixed with 10 μg of RNA in an ice-cold cuvette. Cells were immediately electroporated (GenePulser Xcell, Biorad) and resuspended in pre-warmed assay medium to give 5 × 10 5 The electroporation conditions tested for all cell lines were as follows: EP1: 250V, 475μF EP2: 200V, 300μF EP3: 150V, 300μF EP4: 200V, 400μF EP5: 250V, 950μF Control: 0V, 0μF.

[0249] Cell viability was determined directly after electroporation using CASY or by staining the cells with trypan blue and measuring the percentage of dead cells in a Neubauer chamber. Cells were plated in white 96-well plates (2.5 × 10 4 The cells were seeded in quadruplicates in 1000 x 1000 cells / well and incubated for 24 h. Luciferase activity was then measured in a luminometer (Tecan Infinite200) for 90 min after addition of the luciferin mixture. If an RLU value >1.000 was obtained, the transfection was successful and, consequently, ADCC was measurable.

[0250] 5. Quantitative Real-time PCR (Q-PCR) To isolate RNA from pancreatic cancer cell lines, cells were seeded in 10 cm dishes and grown for 2-3 days to 80% confluency. RNA was isolated using the RNeasy® Mini Kit (Qiagen) following the instructions provided. cDNA preparation was performed using the SuperScript® III First Strand Kit (Invitrogen) following the manufacturer's instructions provided. RNA and cDNA samples were stored at -80°C.

[0251] Quantitative analysis of CLDN18.2 transcripts was performed by amplifying oligo(dT)-primed cDNA in a 40-cycle PCR reaction using PCR primers No. 5054s (5'-AGAGAGCTCTGGCTTCACCGAGTG-3') and No. 5060as (5'-CCAGAAGTTAGTCACCAGCATGTTGG-3'), which distinguish between CLDN18.1 and CLDN18.2 isoforms. Reactions were prepared using SYBR Green (QuantiTect SYBR Green PCR Kit, Qiagen), which intercalates into double-stranded DNA. Reactions and measurements were performed using the ABI-PRISM7900 Sequence Detection System instrument and software (Applied Biosystems).

[0252] The relative expression levels of CLDN18 transcripts were computed using ΔΔCT calculations with respect to the housekeeping gene HPRT.

[0253] 6. Western Blot Analysis For protein isolation of pancreatic cancer cell lines, cells were seeded in 10 cm dishes and grown for 2-3 days to 80% confluency. Cells were lysed by adding 800 μl of 4x SDS sample buffer (34% glycine, 250 mM Tris pH 6.8, 5% β-mercaptoethanol, 8.2% SDS). To degrade genomic DNA, protein samples were sonicated under the following conditions: power control: level 1, duty cycle: 70% for 20-25 s. Protein concentration was measured by spectrophotometer (absorbance at 280 nm) and samples were stored at -80 °C until use.

[0254] To detect CLDN18.2 expression in Western blotting, a preparative 12.5% ​​polyacrylamide gel (for two small gels, 4.1 ml of 29:1 acrylamide / bis-acrylamide, 100 μl of 10% SDS, 2.5 ml of Tris pH 8.8, H 2 A mixture of 3.2 ml of HO, 100 μl of APS, and 10 μl of TEMED was prepared between two stationary glass plates. After polymerization, the gel was packed in a stacking gel (1.5 ml of 29:1 acrylamide / bis-acrylamide, 100 μl of 10% SDS, 2.5 ml of Tris pH 6.8, HO). 2The gel was overlaid with 5.8 ml of 0, 100 μl of APS, and 10 μl of TEMED, and the gel comb was placed between the glass plates. After polymerization, 75 μg of each protein sample prepared by addition of (1:20) 4×SDS sample buffer (Tris-HCL 250 mM, 34% glycerol, 8.2% SDS, pH 6.8) and 7.5 μl of size marker mixture (Magic Mark XP Western Standard 1.5 μl mixed with SeaBlue Plus2 Prestained Standard 6 μl) were added to the gel. The gel was run in 1×SDS running buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS) at 80 V for 30 min and 180 V for 60 min. Semi-dry blotting of the gels onto nitrocellulose membranes was performed in 1x transfer buffer (25 mM Tris, 0.192 mM glycine, 20% MeOH) at 160 mA for 90 min. Blots were first blocked in 5% milk powder / PBS and primary antibodies (0.25 μg / ml anti-claudin 18 (C-term) or 0.1 μg / ml anti-β-actin) were added in a solution of 1% milk powder / PBS. Blots were incubated overnight at 4°C, washed three times for 10 min each in 1x PBS / 0.05% Tween 20, and then incubated with labeled secondary antibodies (goat anti-rabbit IgG (FC) diluted 1:1000) in 1% milk powder / PBS at room temperature for 1 h. Blots were again washed three times for 10 min each in 1× PBS / 0.05% Tween 20, and detection was performed by addition of 1–3 ml of detection solution (Pico and Dura Detection System (Pierce)) for 1 min and scanning of the blots in an LAS-3000 detection box (increment: 10 s, time interval: 10 s, sensitivity: high) according to GA_056_Chemolumineszentwickler LAS3000.

[0255] 7. Flow Cytometry (FACS) Cells were harvested from exponentially growing cultures at 70-85% confluence using PBS / 5mM EDTA or trypsin / EDTA. Cells were counted, centrifuged (468g) for 5 min, and the pellet was resuspended in FACS buffer (2% FCS, 0.1% sodium azide in PBS) to a concentration of 2 × 10 6 The antibody was adjusted to 0.1 μg / ml. 100 μl of cells were plated in a round-bottom 96-well plate and centrifuged again (5 min, 468 g). IMAB362 (or isotype control rituximab) was serially diluted from 0.1 to 200 μg / ml (11 dilution steps + no antibody control) in 50 μl of FACS buffer and added to the cells for 30 min at 4 °C. Then, 200 μl of FACS buffer was added to each well and the plate was centrifuged (5 min, 468 g). The supernatant was removed and the wash repeated. Secondary goat anti-human antibody (FC specific, F(ab')2 conjugated to APC (Dianova)) was diluted (1:100) in FACS buffer and 30 μl was added to each well. The plate was incubated for 30 min at 4 °C. After incubation, the plates were washed again twice with 200 μl of FACS buffer and the pellets were finally resuspended in 100 μl of FACS buffer for FACS Array Bioanalyzer (BD) measurement according to GA_018_BD FACS Array Bioanalyzer.

[0256] 8. Lentiviral Transduction Construction of lentiviral vector: Lentiviruses belong to the RNA viruses that stably integrate into human genomic DNA in both dividing and non-dividing cells. The vector pLenti6.4 (Invitrogen) was used as a backbone. The vector contains the blasticidin gene for the selection of reliably transduced cells. CLDN18.2 fused to the EF1α promoter was cloned into the recombination region of the vector to generate pL64B42E(EF1α-hClaudin18.2)-blasticidin (Figure 1).

[0257] Cell line selection: Cell lines were selected according to literature data or according to data previously tested in vivo. Selection criteria included homogeneous subcutaneous growth in nude mice and a therapeutic window of 20-100 days. Three cell lines that already showed weak expression of CLDN18.2 mRNA (DANG, YAPC and BxPC3) and three cell lines that according to the literature are capable of metastasizing (MiaPaCa-2, Patu8902 and Suit-2) were incorporated. Two other cell lines, known to grow as homogeneous subcutaneous tumors in vivo, were randomly selected (HPAC, CAPAN1).

[0258] Determination of blasticidin selection conditions: For all cell lines, the blasticidin concentration required for selection of cells after lentiviral transduction was determined before performing the transduction. Pancreatic cancer cells were seeded at high density in 6-well plates, resulting in 80-90% confluency after 24 h. Blasticidin (stock solution: 10 mg / ml, Invitrogen) was added to the wells at increasing concentrations ranging from 0.5 to 12 μg / ml (5 dilution steps + control without blasticidin). The medium was changed every 3-4 days and cells were analyzed under a microscope, after which the medium was removed. The amount of dead cells and the status of live cells were recorded. The cells were cultured for 14 days. The minimal blasticidin concentration resulting in 100% apoptotic cells after 14 days was preferred for selection of lentiviral transduced cells. The required blasticidin concentrations for each of the established LVT cell lines are shown in Table 4.

[0259] Envelope selection: For lentiviral transduction, the GFP-lentiviral control vector pL64B42E-(EF1a-GFP)-blasticidin was packaged into various enveloped particles (VSV-G, GALV, RD114, Mokola-G and Rabies-G). Depending on the composition of proteins and cell membranes present in the envelope, attachment to the target cancer cells is largely efficient. For all pancreatic cancer cell lines, the VSV-G envelope showed the highest transduction efficiency (68.5-91.2%) (Table 5). As a result, the CLDN18.2 expression vector pL64B42E(EF1α-hClaudin18.2)-blasticidin was packaged into the VSV-G envelope. Producer cells were infected and the virus was expressed at high titers (3.86 × 10 7 Viral supernatants were stored at -80°C. [Table 5]

[0260] Lentiviral transduction of pancreatic cancer cell lines: For infection of pancreatic cancer target cell lines, 24-well plates were coated with 200 μl of 1× RetroNectin® (20 μg / ml, Takara Inc.), the plates were sealed with Parafilm® and incubated at 4°C for 3-16 h. Plates were washed with 200 ml of PBS and blocked with PBS / 2% BSA for 30 min at room temperature. Plates were washed again and loaded with 300 μl of viral supernatant by centrifugation at 2500 rpm for 25 min at 15°C. The supernatant was removed and loading was repeated three times. Plates were finally washed once with PBS and low passage target cells were inoculated into each well. For all pancreatic cancer cell lines, 5 × 10 per 24-well 5 ~1×10 7 Cells were seeded. Plates were incubated at 37°C for 2 days. Cells were then separated and transduction efficiency was determined by FACS using FITC-labeled IMAB362 antibody. Cells were expanded and master cell banks were prepared for each cell line.

[0261] 9. ADCC Assay Pancreatic cancer target cells were seeded into flasks 2 days prior to initiating ADCC to obtain 80-90% confluent cultures. Pancreatic cancer cells were transfected with luciferase RNA and cultured at 1 × 10 in 50 μl of assay medium (medium described in Table 4 supplemented with 20 mM HEPES). 4 Cells were seeded into white 96-well plates at a density of 1000 cells / well. In addition, NUGC-4 sub 10cH11 subE10 Luci#2 cells (8000 cells / well) were seeded as a positive control in all assays. Cells were cultured for 4–6 h before the addition of antibodies and purified PBMCs.

[0262] PBMCs were prepared from fresh human buffy coats obtained from healthy donors. Approximately 3 × 20–25 ml blood was diluted (1:2) with PBS and carefully loaded onto 4 × 15 ml Ficol-Paque Plus (GE Healthcare) in a 50 ml Falcon tube. The gradient was centrifuged (25 min, 700 g). After centrifugation, peripheral blood mononuclear cells (PBMCs) were collected from the interphase, washed in PBS / 2 mM EDTA, centrifuged (5 min, 468 g), resuspended again in PBS / 2 mM EDTA, and centrifuged (10 min, 208 g) to remove platelets. The pellet was resuspended in 50 ml PBS / 2 mM EDTA and cells were counted. PBMCs were centrifuged (5 min, 468 g) and 1.6 × 10 cells were collected for addition to the pancreatic cells. 7 at a concentration of 1.28 × 10 cells / ml for addition to NUGC-4 sub 10cH11 subE10 Luci#2 cells 7 The cells were resuspended in X-Vivo-15 medium at a concentration of 100 cells / ml.

[0263] Antibodies (IMAB362 and isotype control antibody ch78H11 1H6) were serially diluted 10 times (4.5-fold) to yield a concentration range of 200 μg / ml to 0.26 ng / ml. 25 μl of each dilution was added to target cells in quadruplicate. PBS without antibody was added to media and lysis control wells. 25 μl of PBMC were then added to each well (E:T ratio = 40:1) and the plate was incubated at 37°C, 5% CO 2The mixture was incubated at RT for 24 hours ± 1 hour.

[0264] The next day, 10 μl of 8% TritonX100 / PBS solution was added to the lysis control wells and 10 μl of PBS was added to all other wells. Finally, 50 μl of freshly prepared luciferin stock solution (160 mM HEPES, 1×PBS, 3.84 mg / ml D-luciferin (BD Biosciences)) was added to each well and the plate was incubated for 80 min at room temperature in the dark. Luminescence resulting from the oxidation of Lucifer Yellow by luciferase in live cells was measured using a microplate reader (Infinite200, Tecan, Switzerland). The percentage of cytotoxicity was calculated according to the following formula:

number

[0265] 10.CDC CDC was performed as follows.

[0266] Target cells (CHO-K1 p740 MACS / FACS(24H5)p3151 Luci#2A5) were seeded in 50 μl of assay medium in 96-well white assay plates (10,000 cells / well) and incubated at 37°C, 7.5% CO 2Samples were added after 24 hours + 20 minutes growth at 37 °C and 95% relative humidity. Each 96-well assay plate contained a total of three different negative controls (heat-inactivated serum, serum with and without IMAB362 and serum with isotype control antibody (rituximab)) as well as a positive control of healthy human serum pool (lot no. 31032011) containing 500 ng / ml IMAB362. An additional positive control was generated at the end of the reaction by adding 0.8% Triton X100 to a second medium control well to produce total lysis. One of the 96-well assay plates contained a functional positive control generated by seven serial 3.16-fold dilutions of IMAB362 (10000-31.8 ng / ml). This control produced a sigmoidal dose-dependent lysis of the target cells. All samples were prepared simultaneously (200 μl each) in a 96-well deep-well dilution plate. Samples were taken from each well three times by reverse pipetting to generate triplicates in the assay plate. 50 μl of each test article and control was added to the assay plate, after which the plate was incubated at 37° C., 7.5% CO 2 and incubated at 95% relative humidity for 80+5 minutes.

[0267] 10 μl of PBS was added to each well, except for the Triton lysis control wells. 10 μl of 0.8% Triton / PBS solution was added to each Triton lysis control well. Luciferin substrate solution was prepared (6114 μl Aqua bidest, 2496 μl HEPES (1M), 1998 μl 1×DPBS, 4992 μl D-luciferin stock solution (12 mg / ml)). 50 μl of luciferin substrate solution was added to each well. Plates were incubated at 37°C, 7.5% CO. 2 and 95% relative humidity for 45 minutes. The plate is read in a microplate reader. Complement dependent lysis is determined by the formula:

number

[0268] Modifications for testing pancreatic cancer cell lines: Pancreatic cancer cells were transfected with luciferase RNA using optimized conditions. For each cell line tested, 1.5 x 104 cells were seeded per well. · Trypsin was used on day 1 because most pancreatic cancer cell lines are difficult to dissociate and homogenize. Pancreatic cancer cells in assay plates were incubated at 37°C with 5% CO 2 and cultured. CDC assays on chemotherapy pre-treated cells were performed with the following IMAB362 concentrations or ch78H11 1H6 antibody concentrations as isotype control antibody: 640000, 160000, 40000, 10000, 2500, 625, 156 and 39ng / ml.

[0269] 11. Inhibition of proliferation Proliferation assays were performed to analyze the dose-response curves of each chemotherapeutic agent. [Table 6]

[0270] Cells were seeded in 96-well plates and 4–6 h later gemcitabine or oxaliplatin was added at the following concentrations: 1000, 500, 250, 100 and 20 ng / ml. Proliferation assays were performed at 37°C and 5% CO 2 The cells were incubated at 4°C for 4 days. 50 μl of XTT complete reagent (mixed 50 parts XTT + 1 part coupling reagent) was added and incubated at 37°C. Absorbance (cells + supernatant) measurements were taken on a Tecan Safire after 3 and 4 hours. Growth inhibition was calculated relative to the median value, which was set at 100%. EC 50 Values ​​were calculated with the GraphPad Prism program.

[0271] 12. Cultivation of Pancreatic Cancer Cell Lines with Chemotherapeutic Agents for ADCC or CDC For DANG 4~6E+06, cells were seeded and cultured for 2 days in medium or medium + 1ng / ml gemcitabine or 1ng / ml gemcitabine + 10ng / ml oxaliplatin. 1~1.4E+07 Patu8988S were seeded and cultured without or with 10ng / ml gemcitabine or with or without 10ng / ml gemcitabine combined with 100ng / ml oxaliplatin.

[0272] On the day ADCC was initiated, following the protocol described above, cell surface expression of CLDN18 was determined in FACS analysis as described above.

[0273] 13. Cell cycle analysis Cells were plated in 6-well plates and 5-6 hours later chemotherapy drugs were added for 24 h, 48 h or 3 days. Cells floating in medium were combined with the adherent cell layer, which was trypsinized. Cells were washed. Cell cycle analysis was started directly or cell surface staining was performed as described above before. Cells were resuspended in 1 ml PBS and added to 3 ml 4% PFA. Cells were fixed for 15 min at room temperature, after which cells were pelleted and washed. For RNase treatment cells were resuspended in 200 μl RNase (10000 U / ml) plus 0.05% Triton X-100 and incubated for 30 min at 37 °C. 1 ml PBS was added, samples were centrifuged and resuspended in 200 μl PBS / PJ 50 μg / ml. After at least 30 min samples were ready to be analyzed by flow cytometry. Cell cycle distribution was determined using FlowJo software analyzing DNA content histograms.

[0274] 14. Apoptosis Assay After the indicated treatments, apoptosis was measured by Annexin V binding (Detection Kit I) or DNA fragmentation assay (Apo-Direct) as recommended by the manufacturer (PharMingen, San Diego, CA). Briefly, cells floating in the supernatant were combined with the adherent fraction, which was trypsinized and then washed. An aliquot of 5E+05 cells was incubated with Annexin V-APC and PI for 15 min at room temperature in the dark. Cells were immediately analyzed by flow cytometry. Live cells exclude both Annexin V-APC and PI. Early apoptotic cells are Annexin V-APC positive and PI negative, whereas cells that are no longer viable due to apoptotic or necrotic cell death stain positively by both Annexin V and PI. The percentage of stained cells in each quadrant was quantified using FlowJo software (BD Biosciences, Franklin Lakes, NJ).

[0275] The apoptosis assay based on DNA fragmentation was performed as follows: Treated cells (adherent and floating) were fixed overnight in 70% ice-cold EtOH. After washing, 10 6 Fixed cells were incubated with terminal deoxynucleotidyl transferase enzyme (TdT) and FITC-dUTP for 90 min at 37°C to label DNA fragments. Cells were washed and incubated in RNase A / propidium iodide for 30 min at room temperature in the dark to stain total DNA and then analyzed by flow cytometry. Cell doublets and clumps were excluded from the analysis by gating.

[0276] 15. In Vivo Testing All in vivo experiments were performed in accordance with national regulations and ethical guidelines for laboratory animal testing.

[0277] 15.1 Xenograft Treatment Xenograft tumors were cultured in female Hsd:athymic nude-Foxn1 nuMice were inoculated by subcutaneous injection of tumor cells in 200 μl PBS into the flank. Tumor-bearing mice were treated with 0 μg, 200 μg, 400 μg or 800 μg antibody by iv injection once a week or alternating iv / ip injections twice a week. Chemotherapy agents were applied ip once or twice a week. Tumor size and animal health were monitored twice a week. At the end of chemotherapy treatment, tumors >1400 mm 3 Antibody application was continued until the tumor reached a volume of 100 μg / ml or became ulcerated. Tumor samples were either frozen for subsequent analysis or fixed in 4% formalin.

[0278] 15.2 Metastasis Assays Various pancreatic cancer cell lines were first analyzed for their ability to form metastases after i.v. application of cells in nude mice. For these engraftment analyses, 1 × 10 cells were administered in groups of 5–10 mice. 6 cells and / or 2 x 10 6 Cells were injected and one mouse was sacrificed at various time points to find the time points of metastatic engraftment and growth.

[0279] Translocation treatment was performed on 10–12 Hsd:athymic nude-Foxn1 mice per treatment group. nu These mice were then treated with 2 × 10 6 Cells (Patu8988S or Suit2-LVT) were injected intravenously. All mice were sacrificed at the same time point, as soon as the first symptoms of metastatic disease appeared (weight loss, weakness, shortness of breath) or the first mouse died.

[0280] Tissue preparation: For engraftment studies, mice were sacrificed at various time points or as soon as they showed obvious physiological signs of metastatic disease (weight loss, weakness, shortness of breath). All organs of mice were analyzed macroscopically for metastases. Only for Patu8988S and Suit-2 cells, lungs and lungs / liver, respectively, showed macroscopic metastases. These organs were cut into four equal pieces, and two (lungs: right upper lobe and left lower lobe) were kept for genomic DNA isolation. The other two pieces were formalin fixed and kept for IHC analysis (Figure 2).

[0281] Genomic DNA preparation and Q-PCR methods: Genomic DNA was extracted from lung or liver tissue. As a control, genomic DNA was also isolated from human pancreatic cancer cells Patu8988S, as well as from uninjected negative control mice.

[0282] The Q-PCR method is based on the amplification of human DNA present within metastases. The relative detection level of human DNA in mouse lung samples directly correlates with the amount and / or size of metastases. Since this method is biased by the fact that metastases do not spread uniformly in the lungs and sometimes one lobe is more heavily affected than the other, two different areas of the lung were mixed in one DNA preparation (Figure 2).

[0283] Q-PCR reactions were performed with the primer pair No. 5861 5'-GGGATAATTTCAGCTGACTAAACAG-3' and No. 5862 5'-TTCCGTTTAGTTAGGTGCAGTTATC-3', which specifically amplifies α-satellite DNA present in human chromosome 17 but not in mouse DNA. To generate a standard curve and as a positive control, Patu8988S DNA was mixed with mouse DNA and 5-fold dilutions were prepared to give 100%, 20%, 4%, 0.8%, 0.16%, 0.032% and 0.0064% human DNA in mouse DNA. This curve was used to calculate the amount of human metastatic DNA present in mouse lung tissue (linear regression). Q-PCR reactions were performed with 20 μl (200 ng) mouse lung DNA, 25 μl Sybr Green (Qiagen), 1.6 μl sense primer (10 μM) and 1.6 μl antisense primer, and H 2 The reaction was carried out in a final volume of 50 μl consisting of 1.8 μl of O.

[0284] [Example 2] CLDN18.2 expression in normal and neoplastic human pancreatic tissues To analyze the expression levels and patterns of CLDN18.2 in normal and pancreatic tumor tissues, histological staining of FFPE sections was performed with two mouse monoclonal antibody reagents (Figure 3).

[0285] Preliminary pilot experiments were performed by using the prototype antibody 35-22A on tissue microarrays (TMA). The main difficulties with TMA are the variability in the quality of the spotted tissues and the small size of the samples, which are therefore not representative of the sample characteristics. This, together with a suboptimal staining protocol, may have led to an underestimation of positive cases.

[0286] The main experiments were performed with antibody 43-14 A. These stainings were performed on larger (compared to TMA) tissue sections that had been pre-evaluated for the presence of tumor cells.

[0287] Precancerous lesions arising from the pancreatic duct can be ranked according to the International Pancreatic Intraepithelial Neoplasia (PanIN) system (PanIN-1A, PanIN-1B, PanIN-2, PanIN-3 subtypes).

[0288] PanIN-1 lesions (Figure 4A) consist of flat, tall columnar cells with basally located nuclei and abundant supranuclear mucin. The nuclei are small, round to oval in shape, and oriented perpendicular to the basement membrane. There is a histologic overlap between nonneoplastic flat hyperplastic lesions and flat neoplastic lesions without atypia.

[0289] PanIN-1B subtype lesions have a papillary, micropapillary or basally pseudostratified architecture and are otherwise identical to PanIN-1A (Hruban et al. Am J Surg Pathol. 2001 May;25(5):579-86.).

[0290] PanIN-2 lesions (Figure 4B) are flat or papillary and have typical nuclear abnormalities, including some loss of polarity, nuclear crowding, enlarged nuclei, pseudostratification, and hyperchromatism. Mitoses are rare, but when present, they are nonluminal (nonapical) and not atypic (Hruban et al. Am J Surg Pathol. 2001 May;25(5):579-86).

[0291] PanIN-3 lesions (Figure 4C) are usually papillary or micropapillary, but may rarely be flat. True cribriform, budding of small clusters of epithelial cells into the lumen, and luminal necrosis suggest the diagnosis of PanIN-3. The lesions are characterized by loss of nuclear polarity, dystrophic goblet cells (goblet cells with nuclei oriented toward the lumen and mucinous cytoplasm oriented toward the basement membrane), mitoses that may occasionally be abnormal, nuclear irregularity, and prominent (macro)nucleoli (Hruban et al. Am J Surg Pathol. 2001 May;25(5):579-86).

[0292] The expression of CLDN18.2 in precancerous tissues was analyzed with the 43-14A antibody using tissue samples from various sources.

[0293] CLDN18.2 was frequently detected in PanIN structures of PanIN-1, PanIN-2 and PanIN-3 subtypes, revealing early expression of CLDN18.2 in precancerous lesions (Figure 4), which is preserved at later stages, whereas no expression was observed in normal pancreatic tissue samples, including pancreatic ductal structures.

[0294] In conclusion, CLDN18.2 is an early marker of early malignant histological changes in the pancreatic duct.

[0295] Two studies were performed to evaluate the expression of CLDN18.2 in primary pancreatic cancer. For the pilot study, several TMAs of a total of 141 primary pancreatic cancer cases were stained with the monoclonal CLDN18.2-specific antibody 35-22A. The overall quality of the analyzed TMAs was unsatisfactory. Many spots were partially lost during retrieval, and the uneven counterstaining with hematoxylin suggested suboptimal tissue processing of the FFPE tissues.

[0296] Overall, >48.9% of stained cases were positive for CLDN18.2, including 49.2% (65 / 132) of pancreatic ductal adenocarcinomas, 50% (1 / 2) of acinar cell carcinomas, and 3 of 7 neuroendocrine carcinomas (Table 7). Tumor cell membranes stained without background for other cell types (Figure 6).

[0297] Furthermore, we observed a correlation between the intensity of CLDN18.2 expression and the percentage of stained tumor cells within the tumor (Table 8, FIG. 5). [Table 7] [Table 8] [Table 9]

[0298] The second study was performed with an optimized staining protocol for the highly sensitive antibody 43-14A using quality control tissue sections. [Table 10]

[0299] A total of 42 primary pancreatic ductal carcinoma samples were analyzed. Approximately 90% of these (38 of 42 cases) were positive for CLDN18.2 (Table 10), with the majority (>60%) showing strong signal intensity of +++ (Figure 7, Table 11). Again, a correlation was observed between CLDN18.2 expression levels and the percentage of positive tumor cells. The majority of cases analyzed (62%) had grade 3 tumors (Table 12). [Table 11] [Table 12]

[0300] Pancreatic cancer is diagnosed at an advanced stage in most patients, whose tumors have already metastasized to lymph nodes and other organs, especially the liver. In the main study, 79 FFPE tissue samples of lymph nodes and liver metastases of pancreatic cancer were analyzed in an immunohistochemical assay using the CLDN18.2-specific 43-14A antibody.

[0301] 70.5% (31 / 44 cases) of lymph node metastases and 68.6% (24 / 35 cases) of distant liver metastases showed clear tumor cell staining for CLDN18.2 (Table 13). The staining pattern of positive tumor cells was membranous, with additional weaker cytoplasmic signals in some cases (Figure 9). Consistent with the results of the primary tumor analysis, a correlation was found between CLDN18.2 expression levels and the percentage of CLDN18.2-positive tumor cells in metastatic samples (Figure 8).

[0302] No correlation was found between the grade of the analyzed tumors and the expression level of CLDN18.2 or the percentage of positive tumor cells. [Table 13]

[0303] To test whether CLDN18.2 expression in positive primary tumor cases was conserved in metastases from the same patients, matched primary cancer / lymph node metastasis doublets were screened with antibody 43-14A. [Table 14]

[0304] In 25 of 27 analyzed paired cases (92.5%), both the primary tumor and paired lymph node metastasis were positive for CLDN18.2, in one case both tissues were negative, and in the other case the primary tumor was CLDN18.2 positive but the metastasis was negative.

[0305] In 21 of 26 (80.7%) positive doublets, primary and metastatic tumor cell signal intensities were the same. In 5 cases, signal intensity decreased from +++ to ++.

[0306] In 11 of 25 paired tissues (44%), the number of positive tumor cells was lower in metastases compared to primary tumors (Table 14).

[0307] In summary, CLDN18.2 expression appears to be preserved when primary tumor cells progress to the metastatic stage: the overall intensity and percentage of positive tumor cells in lymph node metastases was only slightly lower compared to primary tumors (Figure 10).

[0308] For a small number of patient tissue samples derived from primary tumors, lymph node metastases and liver metastases were available. These matched triplets were stained to examine the preservation of CLDN18.2 expression in distant metastases. Six matched triplets were analyzed using antibody 43-14A. [Table 15]

[0309] In three of the six triplets, all three tissue specimens were comparable in terms of positive scores for CLDN18.2 (Figure 11). In three cases, a portion of the tumor cells in the primary lesions were CLDN18.2 positive, but the metastatic lesions did not show CLDN18.2 staining (Table 15).

[0310] [Example 3] Target expression in in vitro and in vivo models and human pancreatic cancer cell lines used to model pancreatic cancer Source of cell lines The primary objective of this preclinical evaluation study was to analyze the inhibitory effect of IMAB362 treatment in a suitable model system. To identify CLDN18.2-positive cell lines that could be used for in vitro and in vivo characterization of IMAB362 action, a set of 26 commercially available pancreatic cancer cell lines was screened and extensively characterized for CLDN18.2 expression. Cell banks for experimental use were prepared for each cell line immediately upon arrival. These were derived from primary pancreatic adenocarcinomas (10, of which 6 were mucinous adenocarcinomas), primary carcinomas (4), pancreatic adenocarcinoma metastases to the liver (5) or spleen (1), or isolated from ascites (5) (see Table 16). Some of these cell lines (8) were transduced with lentivirus to express CLDN18.2. [Table 16]

[0311] CLDN18.2 transcript expression in human pancreatic cancer cell lines To identify pancreatic cell lines expressing CLDN18.2, transcript levels were measured by quantitative real-time PCR (RT-PCR) using a forward primer binding to exon 1 of CLDN18.2 and a reverse primer binding to exon 3 of CLDN18. The human gastric cancer cell line KATO-III, which endogenously expresses CLDN18.2, and the CLDN18.2-negative breast cancer cell line SKBR-3 were included as positive and negative controls, respectively. RT-PCR was performed using 1 × 105 Interestingly, Patu8988S cells expressed CLDN18.2 at a level comparable to that of gastric cancer KATO-III cells (approximately 1 × 10 8 ) (Figure 12A). In conclusion, we detected robust CLDN18.2 expression in 5 of 22 pancreatic cancer cell lines.

[0312] In addition to the endogenous cell lines, LVT cell lines ectopically expressing CLDN18.2 were analyzed for transcript levels (Figure 12A). For six of the eight LVT cell lines, 1x10 8 We detected relative CLDN18.2 expression levels exceeding 1 × 10 in HAPC-LVT and Suit2-LVT cells only. 5 It was higher.

[0313] We investigated whether CLDN18.2 expression was stable during in vitro culture. Patu8988S, Panc05.04 cells and lentiviral transduced cell lines Suit2-LVT, MiaPaCa2-LVT and Patu8902-LVT were passaged up to 15 times and CLDN18.2 transcripts were analyzed (Figure 12B-D). We observed a loss of CLDN18.2 expression in both endogenous and transduced cells with increasing passage number. The loss of expression was highest in transduced cells. Therefore, early passages were used as much as possible for in vitro experiments, and the expression of CLDN18.2 in tumor xenografts was confirmed in the following engraftment experiments.

[0314] CLDN18.2 protein expression in human pancreatic cancer cell lines Detection of CLDN18.2 in whole cell lysates In addition to the transcript analysis, the expression of CLDN18.2 was analyzed at the protein level by Western blotting and IF. For Western blot analysis, cell lysates of 26 pancreatic cancer cell lines were examined by Western blotting (WB) using a CLDN18-specific antibody, anti-claudin 18 (C-term). Lysates of SKBR-3 cells were again used as a negative control, and lysates of HEK293 cells stably transfected with CLDN18.2 (HEK293-p740) were used as a positive control. Here, we detected high protein expression in Patu8988S, DANG and Panc05.04 cells, confirming the RNA data. Faint bands were detectable in Panc03.27 and BxPC3 cell lysates. YAPC cells identified as positive at the RNA level showed a faint band of smaller size in Western blotting. All other cell lines were negative (Figure 13).

[0315] Cellular expression of CLDN18 in pancreatic cancer cells To obtain supporting protein expression data, pancreatic cancer cell lines were examined by immunofluorescence (IF) after cell fixation and permeabilization using antibody 35-22A for detection. IF analysis confirmed the previous RNA and protein data and showed that the majority of pancreatic cancer cell lines were negative for CLDN18.2 staining (Figure 14). Nuclear dots were observed in a few cell lines (AsPC1, DANG, HUP-T3, HUP-T4, Panc01, etc.), which are likely staining artifacts. DANG, Panc03.27 and BxPC3 cells, identified as featuring low CLDN18.2 at the RNA and / or protein level, were negative in IF analysis, which has a lower detection sensitivity. In contrast, the membrane and cytoplasm of Panc05.04, Patu8988S and KATO-III gastric cancer control cells stained strongly positive for CLDN18.2. The staining intensity varied for each cell, and negative cells were also detected within the population (Figure 14J and N). In the LVT cell line, we observed strong membrane staining in more than 80% of all cells.

[0316] Confirmation of CLDN18.2 expression in pancreatic cancer cells To confirm the expression of CLDN18.2 and to evaluate the amount of this target on the cell surface, the endogenous cell lines Panc05.04 and Patu8988S as well as the LVT cell lines were stained with IMAB362 using a native staining protocol. Although staining of Patu8988S, Panc05.04 and KATO-III gastric cancer control cells with IMAB362 was weaker and the percentage of positive cells was lower compared to cells stained with 35-22A (Figure 16A-F), IF analysis confirmed that CLDN18.2 is expressed on the surface of pancreatic cancer cells. For the eight LVT pancreatic cancer cell lines ectopically expressing CLDN18.2, clear membrane staining was observed on almost all cells (as shown for six LVT cell lines in Figure 16G-L).

[0317] In conclusion, CLDN18.2 expression analysis identified the endogenously expressing pancreatic cancer cell lines Panc05.04 and Patu8988S and all eight lentiviral-transduced cell lines BxPC3-LVT, CAPAN1-LVT, DANG-LVT, MiaPaCa-2-LVT, Suit-2-LVT, Patu8902-LVT and YAPC-LVT as suitable CLDN18.2-positive cell model systems.

[0318] Development of pancreatic cancer xenograft and metastasis models Engraftment Assay for Identification of Suitable Subcutaneous Pancreatic Cancer Tumor Models A total of 37 engraftment studies on various pancreatic cancer cell lines were performed to identify suitable subcutaneous xenograft models for testing the in vivo efficacy of IMAB362. Among all cell lines tested, BxPC3-LVT, CAPAN1-LVT, MiaPaCa-2-LVT, HPAC-LVT, DANG-LVT and YAPC-LVT cell lines, which ectopically express CLDN18.2, were selected for subcutaneous xenograft models, which showed high engraftment rates and uniform tumor growth. In addition, the subcutaneous xenograft models Patu8988S and DANG cell lines, which endogenously express CLDN18.2, were selected to test the IMAB362 efficacy in vivo. Sc injection of Panc05.04 cells did not result in the formation of subcutaneous tumors. [Table 17A] [Table 17B]

[0319] Engraftment studies for identification of suitable metastatic models To investigate the effect of IMAB362 on metastasis formation, a model of metastatic cancer was established in nude mice. Pancreatic cancer cell lines were analyzed for their ability to metastasize after iv application. CAPAN1-LVT, MiaPaCa-2, Patu8988S, Patu8902 and Suit-2 cells were injected into the tail vein of nude mice as described by Mohanty and Xu 2010. Mice were sacrificed at various time points to measure the time of metastatic engraftment and growth rate (Table 18). [Table 18]

[0320] Engraftment analysis of Patu8902 cells and CAPAN1-LVT was not feasible because most mice died almost immediately. No macroscopic metastases were detected in the lungs and liver of the five surviving mice challenged with CAPAN-LVT cells after 72 days. Suit-2 and MiaPaCa2 cell injections, in contrast, were well tolerated. Lung tissues of these mice were analyzed in IHC analysis at various times after injection. In mice challenged with MiaPaCa-2 cells, no metastases were detected in the lungs until 73 days, so this cell line was not selected as an IMAB362-treated model. Suit-2 cancer cells metastasized to the lungs of mice. Numerous foci were detected throughout the tissue. Therefore, the Suit-2-LVT cell line, lentivirally transduced with CLDN18.2, was selected as a model system to analyze the effect of IMAB362 treatment on metastasis formation.

[0321] In addition to Suit-2, we also analyzed the ability of Patu8988S cells, which endogenously express CLDN18.2, to form metastases. Engraftment assays were performed with two different cell numbers per mouse (1 × 10 6 , 2×10 6 ) was injected iv. Lungs and livers were isolated at various time points as shown in Table 18. Initially, the various tissues obtained were analyzed using Q-PCR. Lungs and livers obtained up to day 70 were analyzed by amplifying human α-satellite DNA of chromosome 17. Lung results showed a clear increase in the percentage of human DNA in mouse lungs over time, which was independent of the number of cells injected. 1×10 6 or 2×10 6 With iv application of cells, 5.8% and 3.7% human DNA could be detected after 70 days, respectively (Figure 19). In the liver, human DNA was barely amplified. After 70 days, the percentage increased slightly, but was still less than 0.005%.

[0322] To confirm CLDN18.2 expression in Patu8988S metastasis, lung tissue was immunohistochemically stained using anti-human MHC class I antibody for detection of human cells in mouse tissue as well as anti-claudin 18 (center) antibody. MHC-I staining showed that obvious metastatic foci were detectable in mouse lung tissue sections but not in liver sections (Figure 20). Furthermore, the membranes of cells in these foci were stained with anti-claudin 18 (center) antibody, showing obvious expression of IMAB362 target protein in these cells. Therefore, this endogenous metastasis model was selected for the study of IMAB362 treatment in addition to the Suit2-LVT model.

[0323] [Example 4] IMAB362-mediated cell killing IMAB362 cross-linking induces efficient apoptosis Antibody binding to cell surface targets can initiate aberrant signaling that directly results in cell death. Such signaling events may depend on the target epitope, the valency of binding, and whether binding is associated with cross-linking of the target. For some CD20-positive lymphoma cell lines, induction of apoptosis by, for example, rituximab is only observed under cross-linking conditions. Such cross-linking may occur in vivo when high-affinity Fc receptor-positive immune cells interact with antibody-coated tumor cells.

[0324] Crosslinking of IMAB362 induces direct apoptosis within 18-42 hours in human gastric cancer cells NUGC-4 and KATO-III as measured by TUNEL assay. The degree of apoptosis correlates with the dose of antibody and the level of target expression on the cancer cells. Treatment with gemcitabine leads to cell cycle arrest of tumor cells followed by apoptotic cell death. Apoptosis of pancreatic tumor cells treated with gemcitabine is shown in Figure 21.

[0325] IMAB362-mediated ADCC activity against pancreatic cancer cells IMAB362 is highly potent in recruiting and activating Fcγ receptor-positive immune effector cells, such as natural killer cells. Binding of IMAB362 to target cells induces antibody-dependent cellular cytotoxicity (ADCC) by granzymes and perforin, which are secreted by effector cells when their Fcγ receptors bind to antibodies. The impact of this mechanism of action was previously shown for luciferase-positive and CLDN18.2-positive gastric cancer cells (such as NUGC-4 and KATO-III) by incubation with IMAB362 for 24 h (effector-to-target ratio = 40:1) in the presence of human peripheral blood mononuclear cells (PBMCs). Application of up to 200 μg / ml of IMAB362 produced a maximum lysis rate of 80-100%.

[0326] Here, we measured the ADCC activity of IMAB362 against pancreatic cancer cell lines. Increasing concentrations of IMAB362 were incubated with different cell lines at an E:T ratio of 40:1. PBMCs of different donors were added in each experiment. The results for all cell lines are summarized in Table 19. Of the five initially identified CLDN18.2-positive pancreatic cell lines, only Patu8988S, Panc05.04 and DANG were efficiently killed by the addition of IMAB362 and PBMCs (Figure 22A). Although CLDN18.2 surface expression was not detectable by FACS for Panc05.04 and DANG, the expression levels were significant enough to cause effector cell-dependent killing (EC 50 : Patu8988S: 0.01-1.4 μg / ml, DANG / Pan05.04: 0.1-38 μg / ml). These data indicate that the relative RNA levels are >5.5×10 5 It can be concluded that only cells expressing the .ALPHA.-specific lysate are efficiently lysed.

[0327] ADCC analysis was also performed on LVT pancreatic cancer cell lines and their corresponding parental cell lines (Figure 22B-F). ADCC strictly depends on the specific binding of IMAB362 to the target, since only CLDN18.2-positive target cells were killed by IMAB362 and PBMC. The half-maximal and maximum killing rates induced by IMAB362 in human pancreatic cancer cells varied among PBMC donors and also depended on the passage number of the cells, which affects the expression level of CLDN18.2.

[0328] The half-maximal killing rate of target cells as well as the IMAB362 concentration causing the maximum killing rate are shown in Figure 22G-H. The LVT pancreatic cancer cell line was killed at a high rate after the addition of small amounts of antibody, but for DANG and Panc05, the highest antibody concentrations are required to reach a maximum killing rate of about 50%. For Panc05.04, the figures include the results obtained for subclone 15D3 (a CLDN18.2-positive clone selected by limiting dilution of Panc05.04 and by FACS), which shows a comparable ADCC lysis rate to the LVT cell line. Unfortunately, CLDN18.2 expression in this clone was rapidly silenced in vitro after passaging the cells, and therefore this clone was not used for further experiments.

[0329] IMAB362-mediated CDC activity against pancreatic cancer cells Pancreatic cancer cells killed by IMAB362 in the ADCC assay were analyzed for sensitivity to the complement-dependent lytic activity of IMAB362. In addition, LVT cell lines and parental lines were tested in the CDC.

[0330] CDC activity is activated by complexes of antigens and IgM or IgG antibodies (classical pathway) or by microbial surfaces (alternative pathway). In the classical pathway, complement C5 is converted to C5b. The anaphylatoxins C3a, C4a and C5b are released, and the sequential binding of C5b, C7, C8 and C9 leads to the formation of the membrane attack complex (MAC). This pathway is inhibited by soluble but membrane-bound proteins (e.g. CR1, DAF, MCP, CD59, CD55, CD46) that protect self-tissues.

[0331] CHO-K1 cells stably transfected with CLDN18.2 (p740) and luciferase were used as assay positive controls in each assay (Figure 23A). Cell lines DANG, BxPC3, YAPC, Patu8988S, Panc05.04, CAPAN1 and Suit2 were not lysed by the addition of IMAB362 and healthy human serum pool (Figure 23B). Although DANG, Patu8988S and Panc05.04 cells are CLDN18.2 positive as shown in all previous experiments, these cells were not lysed in a complement-dependent manner. This is most likely due to the fact that tumor cells overexpress one or more membrane-bound complement inhibitory proteins, such as CD46, CD55 and CD59 (Geis et al., Curr Cancer Drug Targets, 2010 10:922-931). However, whether the expression of these inhibitory proteins on tumor cells influences the clinical outcome of antibody therapy remains controversial (Dzietczenia et al. Med. Oncol. 2010, 27:743-6; Weng and Levy at al., Blood 2001 98:1352-7).

[0332] In addition to the endogenous cell lines, all LVT cell lines were tested in the CDC assay. As shown in Figure 23, the addition of IMAB362 and serum to MiaPaCa-2-LVT, Suit2-LVT and CAPAN1-LVT resulted in an EC 50 values ​​resulted in dose-dependent lysis.

[0333] Overview of CLDN18.2 expression in human pancreatic cancer cell lines [Table 19]

[0334] [Example 5] Effect of IMAB362 on pancreatic cancer xenograft models Ten of the 41 tested pancreatic cancer xenograft models were selected to study the effect of IMAB362 in vivo. Using a pancreatic xenograft model with high expression of CLDN18.2, IMAB362 treatment showed high antitumor activity. This was studied by treatment of mice bearing BxPC3-LVT or MiaPaCa-2-LVT xenografts subcutaneously in the left flank. Treatment was initiated 3 days after tumor inoculation with twice weekly injections of IMAB362 200 μg. IMAB362-treated mice showed significantly inhibited tumor growth compared to mice treated with saline control. In addition, tumor growth suppression of IMAB362-treated mice resulted in an increased mean survival time (Figure 24 and Figure 25). The effect of IMAB362 correlates with the duration of treatment. Initiation of IMAB362 treatment at an early time point had an increased effect on tumor growth inhibition than initiation of treatment at a later time point to study the effect on established tumors. Furthermore, the antitumor effect of IMAB362 was dependent on the amount of CLDN18.2 target expression: IMAB362-mediated growth inhibition of tumors with low CLDN18.2 expression, such as DANG and Patu8988S xenografts, was lower compared to inhibition of tumor growth using xenograft tumors with high CLDN18.2 expression.

[0335] [Example 6] Treatment of pancreatic metastasis mouse model [Table 20]

[0336] Suit2-LVT Metastasis Model: 2x10 on mouse 6Suit2-LVT cells were intravenously injected and treated with 200 μg of IMAB362, isotype control antibody (IMAB027), or PBS as shown in Table 20. After 35 days, the first mouse (isotype control group) died. As a result, all mice were sacrificed on day 42, and the liver was harvested for IHC and Q-PCR analysis.

[0337] Q-PCR analysis of human DNA in mouse lungs was repeated at least twice in triplicate. Calculation of the resulting Ct values and percentage of human DNA revealed a significant decrease (P < 0.05) in Suit2-LVT metastases detected in the lungs when mice were treated with IMAB362 compared to both PBS and isotype control treatment (Figure 26A). To confirm these results, tissue sections of lung samples were prepared and stained with MHC-I antibody. The surface of positive-stained cells in lung sections was calculated using the ImageJ Program. A significant inhibition (P < 0.05) was observed for IMAB362 treatment compared to PBS treatment, confirming the results obtained by Q-PCR. However, for the isotype control antibody, the difference was not significant (Figure 26B). This discrepancy is likely due to differences in tissue processing: IHC treatment of tissue sections provides insight only into very small sections of the lung compared to Q-PCR analysis, where genomic DNA is extracted from half of the tissue.

[0338] In addition to tissue processing, the results may indicate an unexpected inhibitory effect of the isotype control antibody targeting CLDN6. To explore this option, Suit2-LVT cells were analyzed in FACS for CLDN6 expression and IMAB027 binding. Addition of 200 μg / ml of IMAB362 to Suit2-LVT cells confirmed strong binding to the cells, while addition of 200 μg / ml of IMAB027 resulted in weak binding of the antibody to these target cells, indicating that CLDN6 is actually weakly expressed in these cells. These results suggest that at least two factors (tissue processing and weak IMAB027 inhibition) contributed to the discrepancy observed for the isotype control antibody.

[0339] Patu8988S metastasis model To analyze the effect of IMAB362 treatment on the development and growth of Patu8988S metastases in vivo, 2 × 10 mice were treated with IMAB362 per group. 6 Patu8988S cells were injected into the 18 mice. The first experiment was performed by comparing IMAB362 treatment with PBS-treated mice. In each group, one mouse died immediately after the injection of the cells. In the other 18 mice, metastases developed very quickly compared to the engraftment experiment. After 63 days, the first two mice in the PBS group were sacrificed due to deteriorating health. All other mice were sacrificed after 65 days. Optical analysis of the lungs revealed large metastases throughout the lung tissue. The amount of metastases was analyzed in a Q-PCR experiment (Figure 27). The results show that IMAB362 inhibits the growth of metastases in the lung tissue.

[0340] A second experiment with 11 mice per group was performed by comparing IMAB362 treatment with isotype control (rituximab) treatment. In this experiment, metastases developed slowly as observed in the engraftment experiment. Nevertheless, for consistency, this second experiment was terminated after 65 days. Lung tissue was again analyzed in Q-PCR, and again IMAB362 reduced metastasis growth. One mouse in the IMAB362 group was identified as an outlier, and excluding this outlier resulted in a near significant (P=0.0588) inhibition. These data were confirmed by IHC surface analysis as described for the Suit2-LVT metastasis experiment. Here the same outlier could be identified, and excluding this value in a t-test, the inhibition of IMAB362 was again borderline significant (P=0.0691), said outlier being from the same mouse.

[0341] [Example 7] Primary pharmacodynamics of IMAB362 in combination with chemotherapy Sensitivity of pancreatic cancer cells to gemcitabine and oxaliplatin We investigated the mechanism of action of IMAB362 in combination with the chemotherapeutic agents oxaliplatin or gemcitabine using pancreatic cancer cell lines constitutively expressing CLDN18.2 (DANG, Patu8988S) and cells stably transduced with CLDN18.2 (MiaPaCa-2-LVT, BxPC3-LVT).

[0342] Chemically, gemcitabine (Gemzar, marketed by Eli Lilly & Co.) is a nucleoside analogue. Like 5-fluorouracil (5-FU) and other analogues of pyrimidines, the triphosphate analogue of gemcitabine replaces one of the building blocks of nucleic acid during DNA replication. This process halts tumor growth, leading to apoptosis, as only one additional nucleoside can bind to the "defective" nucleoside.

[0343] Oxaliplatin works by forming both interstrand and intrastrand crosslinks in DNA. The crosslinks in DNA interfere with DNA replication and transcription, leading to cell death (Graham, Joanne; Mushin, Mohamed; Kirkpatrick, Peter (January 2004). "Oxaliplatin". Nature Reviews Drug Discovery 3(1):11-2).

[0344] Dose-response curves for gemcitabine and oxaliplatin demonstrated the variable sensitivity of the pancreatic tumor cell lines tested (Figures 28 and 29). [Table 21]

[0345] High concentrations of gemcitabine (IC50>100ng / ml) or oxaliplatin (IC50>500ng / ml) are required to inhibit cell proliferation of Patu8988S. DANG and BxPC3-LVT are highly sensitive to gemcitabine but not to oxaliplatin. MiaPaCa-2-LVT cells are most sensitive to oxaliplatin but less sensitive to treatment with gemcitabine (Figure 28, Figure 29 and Table 21).

[0346] Effect of chemotherapeutic agents on CLDN18.2 expression in pancreatic cancer cell lines The mechanism of action triggered by IMAB362 binding is strictly dependent on the presence and cell surface density of its target, CLDN18.2. Pretreatment of DANG and Patu8988S cells with gemcitabine (Gem) and gemcitabine in combination with oxaliplatin (GemOx) resulted in an increase in CLDN18.2 mRNA and protein levels as shown by RT-PCR (Figure 30) and Western blot (Figure 31) analysis of untreated and chemotherapy-pretreated cells. As a result, the amount of CLDN18.2 protein targetable by IMAB362 on the surface of pancreatic cancer cell lines pretreated with Gem or GemOx was increased as shown by flow cytometry (Figure 32).

[0347] Treatment of DANG and Patu8988S with gemcitabine leads to upregulation of CLDN18.2. Patu8988S shows strong upregulation of CLDN18.2 by Gem and lesser upregulation by GemOx.

[0348] Effects of chemotherapeutic compounds on cell cycle and CLDN18.2 expression Cell cycle progression refers to the sequence of events between one mitosis and another in cells. A stationary phase (G0 / G1) is followed by a DNA synthesis phase (S), then a cell expansion phase (G2), and DNA replication (M) followed by cell division into two progeny cells. Any interference with cellular machinery can block all cycle progression in any phase of cell cycle. For example, certain chemotherapeutic agents can block progression in either G2 or M phase or both G2 and M phases (G2 / M).

[0349] Gemcitabine treatment of DANG or Patu8988S results in cell cycle arrest at S phase (Figure 33, Figure 34). Patu8988S cultured with Gem was analyzed. Gemcitabine treatment not only results in cell cycle arrest, but also alters the expression of CLDN18.2 (Figure 34B). The change in CLDN18.2 density after gemcitabine treatment is even greater when proliferating cells in S phase are compared to quiescent cells in G0 / G1 phase (Figure 34C). In Patu8988S cells, CLDN18.2 is expressed in all phases of the cell cycle. After treatment with gemcitabine, its expression is further increased, and the highest level of CLDN18.2 per cell is found in the S phase cell population.

[0350] This variation in tumor cell phenotype has a significant impact on the biological efficacy of therapeutic antibodies. ADCC and CDC are dose-related, and therefore an increase in the target structure, CLDN18.2, provides a synergistic benefit to standard chemotherapy regimens.

[0351] Human gastric tumor cell line, Kato III cells, were cultured in RPMI 1640 medium (Invitrogen) containing 20% ​​FCS (Perbio) and 2 mM Glutamax (Invitrogen) at 37°C and 5% CO. 2 The cells were cultured at 8×10 with or without cytostatic compounds. 5-FU (Neofluor from NeoCorp AG) was tested at a concentration of 10 or 100 ng / ml, and oxaliplatin (Hospira) was tested at a concentration of 50 or 500 ng / ml. 5Kato III cells were cultured for 96 h without medium change or for 72 h followed by 24 h in standard medium at 37°C in 5% CO in 6-well tissue culture plates. 2 Cells were released from cell cycle arrest by 500 mM NaCl, harvested with EDTA / trypsin, washed and analyzed.

[0352] For extracellular detection of CLDN18.2 cells, cells were stained with monoclonal anti-CLDN18.2 antibody IMAB362 (Ganymed) or an isotype-matched control antibody (Ganymed). Goat anti-human IgG-APC from Dianova was used as secondary reagent.

[0353] The cell cycle stage was determined based on the measurement of the DNA content of the cells. This makes it possible to distinguish cells in the G1, S or G2 phase of the cell cycle. In the S phase, DNA replication occurs, and in the G2 phase, the cells proliferate and prepare for mitosis. Cell cycle analysis was performed using the CycleTEST PLUS DNA Reagent Kit from BD Biosciences according to the manufacturer's protocol. Flow cytometry acquisition and analysis were performed with BD FACS CantoII (BD Biosciences) and FlowJo (Tree Star) software.

[0354] The columns in Figures 35a and b show the percentage of cells in the G1, S or G2 phase of the cell cycle, respectively. Kato III cells cultured in medium show cell cycle arrest mainly in the G1 phase. Cells treated with 5-FU are mainly blocked in the S phase. Kato III cells treated with oxaliplatin show enrichment of cells mainly in the G1 and G2 phases. As seen in Figure 35c, cell cycle arrest in the S or G2 phase leads to stabilization or upregulation of CLDN18.2. As soon as the cells are released from any stage of the cell cycle (Figure 35b), CLDN18.2 expression at the cell surface of Kato III cells is upregulated (Figure 35d).

[0355] Kato III cells were pretreated with irinotecan or docetaxel for 4 days and analyzed for CLDN18.2 expression and cell cycle arrest. Treatment of cells with irinotecan caused dose-dependent inhibition of cell proliferation and cell cycle arrest at S / G2 phase (Figure 36). Treatment of cells with docetaxel caused dose-dependent inhibition of cell proliferation and cell cycle arrest at G2 phase (Figure 36).

[0356] Effect of chemotherapy on IMAB362-induced antibody-dependent cellular cytotoxicity (ADCC) A series of experiments were performed on the pancreatic cancer cell lines Patu8988S and DANG, which constitutively express CLDN18.2, to investigate the effect of gemcitabine (Gem) or gemcitabine plus oxaliplatin (GemOx) on IMAB362-mediated ADCC. Dose-response curves for IMAB362-mediated cytolysis of pretreated cells were compared with culture medium.

[0357] The dose-response curve of DANG (2 days) pretreated with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) shifts upward and leftward compared to untreated target cells (Figure 37A). Treatment of tumor cells with Gem or GemOx leads to upregulation of CLDN18.2 and higher sensitivity to IMAB362-mediated ADCC. We could observe a lower EC50 value and higher maximum cell lysis for IMAB362-mediated ADCC in DANG cells after treatment with chemotherapeutic agents (Figure 37B).

[0358] Peripheral blood mononuclear cells (PBMCs), including NK cells, monocytes, mononuclear cells or other effector cells, from healthy human donors were purified by Ficoll Hypaque density centrifugation. Washed effector cells were inoculated in X-Vivo medium. Kato III cells, which endogenously express CLDN18.2 and are of gastric origin, were used as target cells in this setting. Target cells stably expressed luciferase, Lucifer Yellow, which is only oxidized by viable cells. Purified anti-CLDN18.2 antibody IMAB362 was added at various concentrations, and an irrelevant chimeric human IgG1 antibody was used as an isotype control antibody. Samples were assayed for cytolysis by measuring the luminescence resulting from the oxidation of Lucifer Yellow, a value for the amount of viable cells remaining after IMAB362 induced cytotoxicity. IMAB362-induced ADCC was quantified in Kato III cells pretreated for 3 days with irinotecan (1000 ng / ml), docetaxel (5 ng / ml) or cisplatin (2000 ng / ml) compared to untreated, medium-cultured target cells.

[0359] Kato III cells pretreated with irinotecan, docetaxel or cisplatin for 3 days showed lower levels of viable cells compared to media-cultured target cells (Figure 38a), and claudin 18.2 expression was increased in cells pretreated with irinotecan, docetaxel or cisplatin compared to media-cultured cells (Figure 38b).

[0360] Furthermore, pretreatment of Kato III cells with irinotecan, docetaxel or cisplatin increased the potency of IMAB362 to induce ADCC (FIG. 38c, d).

[0361] Effect of chemotherapy on IMAB362-induced CDC The CDC efficacy of IMAB362 was characterized by incubation with target cells in the presence of human serum as a source of complement.

[0362] Media-cultured MiaPaCa-2-LVT exhibit an EC50 value for IMAB362-specific lysis of 7665 ng / ml. Treatment with Gem results in a decrease in the EC50 to 4677 ng / ml compared to the increase in maximum lysis (Figure 39).

[0363] The effect of chemotherapeutic agents on IMAB362-induced CDC was analyzed by pretreating KATO III gastric cancer cells with 10 ng / ml 5-FU and 500 ng / ml oxaliplatin (5-FU+OX) for 48 hours. A representative dose-response curve of IMAB362-induced CDC using chemotherapeutic agent-pretreated KATO III cells is shown in Figure 40. Pretreatment of tumor cells for 48 hours increased the potency of IMAB362 to induce CDC, resulting in higher maximal cell lysis of pretreated tumor cells compared to untreated cells.

[0364] [Example 8] Efficacy of IMAB362 in combination with chemotherapy in mouse tumor models The antitumor activity of IMAB362 in combination with Gem or GemOx was examined in a subcutaneous pancreatic cancer xenograft model previously used to test the efficacy of IMAB362 as a single agent.

[0365] BxPC3-LVT or MiaPaCa-2-LVT tumor-bearing nude mice treated with IMAB362 showed significant tumor growth delay compared to control mice treated with saline control. Chemotherapy with gemcitabine up to 100 mg / kg without additional IMAB362 treatment showed no significant therapeutic effect on BxPC3-LVT or MiaPaCa-2-LVT xenografts. In contrast, combined treatment with 50-100 mg / kg gemcitabine plus IMAB362 resulted in a significant increase in tumor growth inhibition and prolonged survival of tumor-bearing mice compared to mice treated with chemotherapy alone (Figure 41, Figure 42, Figure 43). These findings indicate the presence of a synergistic therapeutic effect of combined gemcitabine and IMAB362 immunotherapy.

[0366] Using a high dose of gemcitabine at 2×150 mg / kg per week, established MiaPaCa-2-LVT xenograft tumors were potently inhibited in tumor growth independent of IMAB362 treatment (FIG. 44A). However, mice treated with the combination of IMAB362 and gemcitabine showed highly significant survival advantage compared to mice treated with gemcitabine as a single agent (FIG. 44B).

[0367] [Example 9] ZA / IL-2 treatment leads to high proliferation of Vγ9Vδ2 T cells PBMCs were cultured in RPMI medium supplemented with 300 U / ml IL-2 with or without 1 μM zoledronic acid (ZA) for 14 days. The percentage of Vγ9+Vδ2+ T cells within the CD3+ lymphocyte population and the percentage of CD16+ cells within the CD3+Vγ9+Vδ2+ T cell population were measured by multicolor FACS on days 0 and 14.

[0368] The addition of IL-2 to PBMC cultures is necessary for lymphocyte survival and proliferation. Lymphocytes proliferate efficiently in cultures supplemented with 300 U / ml IL-2. FACS analysis using Vγ9 and Vδ2 specific antibodies reveals that the addition of ZA / IL-2 specifically induces the accumulation of Vγ9Vδ2 T cells. After 14 days, the CD3+ lymphocyte population can contain up to 80% Vγ9Vδ2 T cells. Although a proportion of Vγ9Vδ2 T cells express CD16, the enrichment of these cells within the CD3+ lymphocyte population is 10-700-fold, depending on the donor. The enrichment of CD16+Vγ9+Vδ2+ T cells in cultures is 10-600-fold higher compared to cultures grown without ZA. We conclude that in vitro ZA / IL-2 treatment of PBMC results in upregulation of the ADCC-mediated FcγIII receptor CD16 in a significant proportion of γδ T cells.

[0369] Similar to NK cells, Vγ9Vδ2 T cells expanded by ZA / IL-2 are positive for the FcγRIII receptor, CD16, through which cell-bound antibodies initiate ADCC. A series of experiments were conducted to evaluate whether Vγ9Vδ2 T cells can induce potent ADCC in combination with IMAB362.

[0370] PBMCs from two different donors (No. 1 and No. 2) were cultured in medium supplemented with 300 U / ml IL-2 with or without 1 μM ZA. After 14 days, the cells were harvested and added to NUGC-4 cells expressing CLDN18.2 together with serially diluted concentrations (0.26 ng / ml to 200 μg / ml) of IMAB362. Specific killing was measured in a luciferase assay. The ADCC assay was performed on 27 donors expanded in 300 U / ml IL-2 with or without ZA, where NUGC-4 was used as the target cell. For each donor, the EC 50 values and the maximum specific killing rate at a dose of 200 μg / ml IMAB362 were evaluated in a scatter plot.

[0371] Potent IMAB362-dependent ADCC activity was observed against CLDN18.2-positive NUGC-4 cells using PBMCs cultured with ZA / IL-2 for 14 days. Using ZA / IL-2-treated PBMC cultures, ADCC is dependent on the presence of Vγ9Vδ2 T cells. When the cells were cultured without ZA, the ADCC activity decreased for most donors. In these cultures, the residual ADCC activity is NK cell-dependent. By testing over 20 donors, the ADCC assay reveals that treatment of PBMCs with ZA / IL-2 improves the EC 50 and the maximum specific killing rate compared to PBMCs cultured with IL-2 alone.

[0372] [Example 10] Effect of IMAB362 in combination with gemcitabine in a mouse metastasis model To analyze the effect of IMAB362 in combination with gemcitabine treatment on Patu8988S lung metastases in vivo, 12 Hsd:athymic nude-Foxn1 mice were used per group. nu Mouse, 2 x 10 6 Mice were treated with intravenous injection of Patu8988S cells into the tail vein. 14 days after tumor cell injection, mice were treated with IMAB362 200 μg twice weekly or PBS (iv / ip) as control plus gemcitabine ip once weekly at a dose of 100 mg / kg for 4 weeks. Treatment with IMAB362 or PBS was maintained until mice were sacrificed 70 days after tumor cell injection. Analysis of xenografted tumor burden in lungs was performed by QPCR of human DNA in lung preparations and by optical analysis of immunohistological staining with anti-human MHC-I antibody (clone EPR1394Y). Results show that mice treated with IMAB362 plus gemcitabine have significantly lower amounts of human DNA in their lungs (Figure 45A) and that the surface of lung sections stained for human MHC-I complexes is significantly smaller than in the lungs of mice treated with irrelevant antibody plus gemcitabine (Figure 45B). Both methods revealed lower tumor burden of Patu8988s xenografts in the lungs of mice treated with IMAB362 plus gemcitabine, indicating that the combination with IMAB362 is significantly superior to gemcitabine monotherapy.

Claims

1. Use of an agent for the manufacture of a medicament for treating or preventing pancreatic cancer, metastasis of said pancreatic cancer, or a precancerous lesion of said pancreatic cancer, comprising a cell expressing CLDN18.2, The treatment or prevention comprises administering a combination therapy of the agent and an antibody capable of binding to CLDN18.2; The agent comprises one selected from the group consisting of gemcitabine, a salt thereof, or an ester thereof; The antibody is Mediates the killing of cells expressing CLDN18.2; a heavy chain variable region (VH) and a light chain variable region (VL), each of which contains complementarity determining regions CDR1, CDR2 and CDR3; The VH comprises CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, and CDR3: positions 116 to 126 of SEQ ID NO: 17; The VL comprises CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, and CDR3: positions 115 to 123 of SEQ ID NO: 24; Use of active substances.

2. Use of an antibody for the manufacture of a medicament for treating or preventing pancreatic cancer, metastasis of said pancreatic cancer, or a precancerous lesion of said pancreatic cancer, comprising a cell expressing CLDN18.2, The treatment or prevention comprises administering a combination therapy of an agent and an antibody capable of binding to CLDN18.2, The agent comprises one selected from the group consisting of gemcitabine, a salt thereof, or an ester thereof; The antibody is Mediates the killing of cells expressing CLDN18.2; a heavy chain variable region (VH) and a light chain variable region (VL), each of which contains complementarity determining regions CDR1, CDR2 and CDR3; The VH comprises CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, and CDR3: positions 116 to 126 of SEQ ID NO: 17; The VL comprises CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, and CDR3: positions 115 to 123 of SEQ ID NO: 24; Use of antibodies.

3. The use according to claim 1 or 2, wherein the combined therapy comprises an agent that induces one or more of cell cycle arrest or cell accumulation.

4. 4. The use according to any one of claims 1 to 3, wherein the combination therapy comprises a substance selected from the group consisting of fluorouracil, platinum compounds, topotecan, taxanes, capecitabine, salts or esters thereof, and combinations thereof.

5. 5. The use according to any one of claims 1 to 4, wherein the combination therapy comprises a substance selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, albumin-bound paclitaxel, salts or esters thereof, and combinations thereof.

6. The use according to any one of claims 1 to 5, wherein the combined therapy comprises a substance that induces immunogenic cell death.

7. The use according to claim 6, wherein the substance that induces immunogenic cell death comprises oxaliplatin.

8. The combination therapy is Gemcitabine and oxaliplatin combination, Gemcitabine and cisplatin combination, Gemcitabine and carboplatin, or Combination of gemcitabine, oxaliplatin, 5-fluorouracil or a salt or ester thereof, and irinotecan 8. The use according to any one of claims 1 to 7, comprising administering

9. The use according to any one of claims 1 to 8, wherein the combined therapy comprises administering folinic acid, oxaliplatin, 5-fluorouracil or a salt or ester thereof, and irinotecan.

10. 10. The use according to any one of claims 1 to 9, wherein the combined therapy further comprises administering a substance that stimulates γδ T cells, said substance being a bisphosphonate (aminobisphosphonate).

11. The use according to claim 10, wherein the γδ T cells are Vγ9Vδ2 T cells.

12. 12. The use according to claim 10 or 11, wherein the substance that stimulates γδ T cells is a nitrogen-containing bisphosphonate (aminobisphosphonate).

13. 13. The use according to any one of claims 10 to 12, wherein the substance that stimulates γδ T cells is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid and salts thereof.

14. The use according to any one of claims 10 to 13, wherein the substance that stimulates γδ T cells is administered in combination with interleukin 2.

15. The use according to any one of claims 1 to 14, wherein the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.

2.

16. The use according to any one of claims 1 to 15, wherein the antibody capable of binding to CLDN18.2 mediates cell killing by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, induction of apoptosis and inhibition of proliferation.

17. The antibody having the ability to bind to CLDN18.2, (i) an antibody produced by and / or obtainable from the clone deposited under accession number DSM ACC2810; (ii) an antibody which is a chimeric or humanized form of the antibody of (i); and (iii) An antibody comprising an antigen-binding portion, or an antigen-binding site, or a variable region of the antibody described in (i). The use according to any one of claims 1 to 16, wherein the antibody is selected from the group consisting of:

18. The therapy comprises administering to a patient a dose of 1000 mg / m ２ 18. The use according to any one of claims 1 to 17, comprising administering at a dose of up to

19. The therapy comprises administering to the patient a dose of 300 to 600 mg / m ２ 19. The use according to any one of claims 1 to 18, comprising repeated administration at a dose of

20. The use according to any one of claims 1 to 19, wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO:

1.

21. 21. The use according to any one of claims 1 to 20, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

22. 21. The use of any one of claims 1 to 20, wherein the metastatic cancer comprises metastasis to lymph nodes, ovaries, liver or lungs, or a combination thereof.

Images