Combination therapy involving antibodies against claudin 18.2 for treatment of cancer

Abstract

To provide a combination therapy for effectively treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as pancreatic cancer and metastases thereof.SOLUTION: Use of an antibody for the manufacture of a medicament for treating or preventing pancreatic cancer, metastasis of the pancreatic cancer, or a precancerous lesion of the pancreatic cancer, the medicament comprising cells expressing CLDN18.2, wherein the treatment or prevention comprises a combination therapy of the antibody and an active agent selected from the group consisting of gemcitabine, a salt thereof, or an ester thereof, wherein the antibody comprises a heavy chain variable region (VH) having a specific amino acid sequence and a light chain variable region (VL) having a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Pancreatic cancer is one of the most lethal cancers. The tendency for early metastatic spread and the disease's high resistance to radiation and chemotherapy result in a mortality rate approaching 100%. 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 in 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 containing 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 using a CLDN18.2-specific antibody reveals that the stomach is the only positive tissue. CLDN18.2 is a highly selective gastric lineage antigen expressed exclusively on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the process of malignant transformation and is therefore often 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 combines four independent and highly potent mechanisms of action. After target binding, IMAB362 mediates cell death through ADCC, CDC, and induction of apoptosis induced by target cross-linking on the tumor cell surface, as well as direct inhibition of proliferation. Accordingly, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo.

[0004] The toxicity and PK / TK profile of IMAB362 have been thoroughly investigated in mice and cynomolgus monkeys, including a dose-ranging study, a 28-day repeat-dose toxicity study in cynomolgus monkeys, and a 3-month repeat-dose toxicity study in mice. Repeated administration of IMAB362 iv is well tolerated in both mice (maximum treatment duration of 3 months, once weekly, at a maximum dose level of 400 mg / kg) and cynomolgus monkeys (up to 5 weeks, once weekly, at a maximum dose of 100 mg / kg). No signs of systemic or local toxicity are induced. In particular, gastric toxicity has not been observed in any toxicity study. IMAB362 does not induce immune activation or cytokine release. No adverse effects on male or female reproductive organs were recorded. IMAB362 does not bind to target-deprived tissues. Biodistribution in mice indicates that the absence of gastric toxicity is likely due to compartmentalization of tight junctions at the luminal site of healthy gastric epithelium, which 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) of 3 patients each were included. 2, 100 mg / m 2 , 300 mg / m 2 , 600 mg / m 2 , 1000 mg / 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. IMAB362 is being administered in multiple doses 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. Synergistic effects were observed between certain chemotherapy regimens, particularly those used to treat pancreatic cancer, 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 was superior to that achieved by anti-CLDN18.2 antibodies as a single agent. [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 metastasis, e.g., Krukenberg tumor, peritoneal metastasis and lymph node metastasis.

[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 CLDN18.2 expression can be administered before, simultaneously with, or after the administration of the antibody capable of binding to CLDN18.2, or a combination thereof.

[0009] Agents that stabilize or increase CLDN18.2 expression can be cytotoxic and / or cytostatic drugs. In one embodiment, agents that stabilize or increase CLDN18.2 expression include agents that induce 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, such as S phase, G2 phase, a combination thereof, or a combination of S phase or G2 phase and G1 phase. Agents that stabilize or increase CLDN18.2 expression can include agents selected from the group consisting of nucleoside analogs, platinum compounds, camptothecin analogs, and taxanes, their prodrugs, salts thereof, and combinations thereof. Nucleoside analogs can be selected from the group consisting of gemcitabine, 5-fluorouracil, their prodrugs, and salts thereof. Platinum compounds can be selected from the group consisting of oxaliplatin, cisplatin, their prodrugs, and salts thereof. The camptothecin analog may be selected from the group consisting of irinotecan, topotecan, prodrugs thereof, and salts thereof. The taxane may be selected from the group consisting of paclitaxel, docetaxel, prodrugs thereof, and salts thereof. The agent that stabilizes or increases the expression of CLDN18.2 may include an agent selected from the group consisting of gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, prodrugs thereof, salts thereof, and combinations thereof. Agents that stabilize or increase the 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 CLDN18.2 expression 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 a prodrug thereof, and irinotecan. Thus, the methods 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 a prodrug thereof, and irinotecan. In one embodiment, the methods of the present invention include administering folinic acid, 5-fluorouracil or a prodrug thereof, irinotecan, and oxaliplatin. Agents that stabilize or increase CLDN18.2 expression 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] An antibody capable of binding to CLDN18.2 can bind to a natural epitope of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, an antibody capable of binding to CLDN18.2 mediates cell 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, an antibody capable of binding to CLDN18.2 is a monoclonal, chimeric, or humanized antibody, or an antibody fragment. In one embodiment, the antibody mediates cell killing when bound to CLDN18.2, particularly CLDN18.2 expressed by a cell on its cell surface, wherein the cell is preferably a cancer cell, e.g., 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 number DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810; (ii) an antibody that is a chimeric or humanized form of an antibody included in (i); (iii) an antibody that has the specificity of an antibody included in (i); and (iv) an antibody that contains an antigen-binding portion or antigen-binding site, in particular the variable region, of an antibody included in (i) and preferably has the specificity of an antibody included in (i). In one embodiment, the antibody is linked to a therapeutic agent, such as a toxin, radioisotope, drug, or cytotoxic agent.

[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 repeated administration 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 primary pancreatic 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 primary pancreatic cancer and metastatic pancreatic cancer. In one embodiment, metastatic cancer includes metastasis to lymph nodes, ovaries, liver, or lungs, 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, particularly precancerous pancreatic lesions including early malignant histological changes in the 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 pharmaceutical preparation for treating or preventing pancreatic cancer, the pharmaceutical preparation comprising (i) an antibody capable of binding to CLDN18.2 and (ii) an agent that stabilizes or increases CLDN18.2 expression. The pharmaceutical 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 CLDN18.2 expression, and optionally the agent that stimulates γδ T cells, may be present in the pharmaceutical preparation as a mixture or separately from each other. The pharmaceutical preparation may be 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 CLDN18.2 expression, and optionally a container containing an agent that stimulates γδ T cells. The pharmaceutical preparation may further comprise printed instructions for using the preparation for treating or preventing pancreatic cancer, particularly for using the preparation in the methods 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 in relation to the methods of the present invention.

[0020] In a specific aspect, the present invention provides a pharmaceutical preparation containing (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine. The pharmaceutical preparation of the present invention may further contain 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 pharmaceutical preparation as a mixture or separately from each other. The pharmaceutical preparation may be intended for treating or preventing cancer, such as pancreatic cancer. The pharmaceutical preparation may be 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 pharmaceutical preparation may further comprise printed instructions for using the preparation for treating or preventing cancer, such as pancreatic cancer, particularly for using the preparation in the methods of the present invention. Various embodiments of the pharmaceutical preparation and, in particular, the antibody capable of binding to CLDN18.2, the agent that stabilizes or increases CLDN18.2 expression, and the agent that stimulates γδ T cells are as described above with respect to the methods of the present invention.

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

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

[0023] [Figure 1]Lentiviral vector used to transduce 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), which allow packaging and reverse transcription of viral mRNA. RSV: Rous sarcoma virus, allows Tat-independent production of viral mRNA. Amp: ampicillin resistance gene. PGKp: blasticidin promoter. WPRE: woodchuck posttranscriptional regulatory element, enhances transgene expression. LTR: long terminal repeats allow viral packaging. SV40A allows transcription termination and mRNA polyadenylation. pUC: bacterial vector backbone. Bla: ampicillin promoter. [Figure 2] Metastasis analysis of pancreatic cells in mouse lungs. Dissection of mouse lung after iv injection of mice with pancreatic cancer cells. [Figure 3] CLDN18.2 expression in normal and cancerous pancreatic tissues. Staining of formalin-fixed, paraffin-embedded (FFPE) normal pancreatic tissue (A) and pancreatic adenocarcinoma tissue (B) with monoclonal mouse 35-22A antibody (0.2 μg / ml). Hematoxylin counterstain (2:00 min). Magnification 200x. [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. [Figure 5] Pilot study—Correlation between CLDN18.2 signal intensity and the 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. FFPE tissue sections (3 μm) of (A) adenocarcinoma primary tumors and (B) lymph node metastases were stained with mouse monoclonal 35-22A antibody. Counterstaining was performed with hematoxylin (Mayers). [Figure 7] Main study: Correlation between CLDN18.2 signal intensity and the 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 the 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. FFPE tissue sections (3 μm) were stained with mouse monoclonal 43-14A antibody for (A, C, E) 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] CLDN18.2 expression in matched pancreatic primary tumor and metastatic 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, lentivirally transduced (LVT) cell lines (gray 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 greater than 1 × 10 were scored as CLDN18.2 positive (hatched bars). NTC: HO control sample. 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 a 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 seconds (Pierce SuperSignal West Dura) and 20 seconds (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 was compared between untransduced 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 glass coverslips. Antibody: 35-22A (20x magnification, exposure times 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 the 35-22A antibody after fixation and permeabilization. Alexa488- or Alexa555-conjugated 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. [Figure 16ABCDEF] Binding of IMAB362 to the cell surface of CLDN18.2-positive pancreatic CA cell lines (pharmacodynamics). [Figure 16GHIJKL] Binding (pharmacodynamics) of IMAB362 to the cell surface of CLDN18.2-positive pancreatic cancer cell lines. IF analysis of CLDN18.2-expressing pancreatic cancer cell lines (A, B, D, E), lentiviral-transduced pancreatic cell lines (G-L), 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 cell fixation and permeabilization (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. CLDN18.2 expression 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 using 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 an 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 i.v. at 1 x 10 or 2 x 10 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 percent 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 the tail vein were sacrificed at various time points (A-D = day 70, E-H = day 86), and lung tissue was isolated and stained with a 1:1000 dilution of MHC-I (EPR1394Y) antibody (A, B, E, F) or 0.2 μg / ml of 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 in gemcitabine-treated pancreatic tumor cells. Apoptosis induced by cross-linking of CLDN18.2 on BxPC3-CLDN18 after 48 hours. BxPC3-CLDN18 cells were cultured in medium or medium plus 100 ng / ml gemcitabine. The proportion of apoptotic cells in mononuclear cells shifted. A similar shift was obtained by incubation of tumor cells with camptothecin. [Figure 22ABCD] Efficacy of IMAB362-induced ADCC activity on pancreatic cancer cells. [Figure 22EFGH] Efficacy of IMAB362-induced ADCC activity towards pancreatic cancer cells. (A) ADCC performed on CLDN18.2-positive pancreatic cancer cell lines using PBMCs from various donors. (B-F) ADCC performed on LVT pancreatic cell lines ectopically expressing CLDN18.2 and the corresponding parental cells. (G) Dot plot. [Figure 23AB] Efficacy of IMAB362-induced CDC activity on pancreatic cancer cells. [Figure 23CDE] Efficacy of IMAB362-induced CDC activity on pancreatic cancer cells. (A) CDC performed in four independent experiments with a pool of healthy human serum as a complement source, IMAB362, and CLDN18.2-positive pancreatic CDOK1-p740 control cells. (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 the IMAB362 concentration that produced half-maximal lysis (EC50) for pancreatic cancer cell lines. (E) Maximum killing obtained with IMAB362 for 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 with 200 μg of IMAB362 or control was initiated 3 days after tumor cell injection. Treatment continued twice weekly, alternating between ip and iv injections, until the 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. [Figure 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 with 200 μg of IMAB362 or control was initiated 3 days after tumor cell injection. Treatment continued twice weekly, alternating between ip and iv injections, until the 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 became ulcerated. [Figure 26]Effect of IMAB362 treatment on the growth of Suit2-LVT pancreatic metastases. 2 x 10 Suit2-LVT tumor cells were injected intravenously into the tail vein of 12 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment began 3 days after tumor cell injection with 200 μg of IMAB362, 200 μg of 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 using an anti-human MHC class I antibody. *p<0.05 (Kruskal-Wallis test). Error bars: mean ± SD. [Figure 27AB] Q-PCR and IHC analysis of Patu8988S lung metastases. Each mouse was injected with 2x106 Patu8988S cells. Animals were sacrificed after 65 days. Open circle: mice sacrificed after 63 days. [Figure 27CD] Q-PCR and IHC analysis of Patu8988S lung metastases. Each mouse was injected with 2 x 106 Patu8988S cells. Animals were sacrificed after 65 days. Open circles: mice sacrificed after 63 days. (A) Mice were treated twice weekly with 200 μg of IMAB362 or saline control. The amount of human DNA (ng) detected by Q-PCR was 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 in the IMAB362 group (open triangle). Significance is indicated by including or excluding the outlier. (D / E) Same experiment as (C). Here, the surface of the 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 exhibit very different sensitivities to gemcitabine. Cell lines were exposed to various concentrations of gemcitabine for 4 days, and inhibition of growth was analyzed by viability assay. [Figure 29] Dose-response curves for oxaliplatin. Pancreatic cancer cell lines exhibit very different sensitivities to oxaliplatin. Cell lines were exposed to various concentrations of oxaliplatin for 4 days, and inhibition of growth was analyzed by viability assay. [Figure 30] Effect of chemotherapy treatment on CLDN18.2 expression (RNA). RNA from untreated DANG cells (A) pretreated with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) for 2 days, or Patu8988S cells (B) pretreated with Gem (10 ng / ml) or GemOx (Gem 10 ng / ml + Ox 100 ng / ml) for 3 days. 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 level of the housekeeping gene HPRT. [Figure 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 demonstrate equal protein loading. [Figure 32] FACS analysis of CLDN18.2 cell surface expression. CLDN18 expression (black histogram) of Patu8988S cultured in medium (left) and Gem-treated (right) is shown overlaid compared to isotype control. Patu8988S are treated with gemcitabine (10 ng / ml) for 3 days. [Figure 33]Cell cycle analysis of DANG cells treated or not with either gemcitabine (Gem; 2 ng / ml) or gemcitabine plus 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. [Figure 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. 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 hours 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). [Figure 36] Effect of chemotherapy on gastric cancer cells. Cytostatic compounds cause cell cycle arrest at various phases of the cell cycle: S / G2 (irinotecan) or G2 (docetaxel). The area of ​​each bar is 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. [Figure 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 for two independent assays. MiaPaCa2-LVT cells were cultured for 70 hours in medium, Gem (10 ng / ml), or GemOx (10 ng / ml Gem + 100 ng / ml Ox). [Figure 40] Effect of chemotherapy on IMAB362-induced CDC. [Figure 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 flanks of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Treatment with chemotherapy (50 mg / kg gemcitabine ip, 50 mg / kg gemcitabine + 5 mg / kg oxaliplatin ip, respectively) began 3 days after tumor cell injection and continued weekly for 6 weeks. 24 hours after chemotherapy injection, 800 μg of IMAB362 or control was administered intravenously via the tail vein. IMAB362 treatment continued until the mice were sacrificed. (A) Growth curves of subcutaneous BxPC3-LVT xenograft tumors. Sc tumor size was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or when tumors became ulcerated. [Figure 42] Enhanced antitumor efficacy by combining IMAB362 with gemcitabine regimens. BxPC3-LVT xenograft tumors were inoculated by subcutaneous injection of 8.5e6 BxPC3-LVT cells into the flanks of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Chemotherapy treatment (100 mg / kg gemcitabine ip or 100 mg / kg gemcitabine + 5 mg / kg oxaliplatin ip) began 3 days after tumor cell injection and continued weekly for 6 weeks. 24 hours after chemotherapy injection, 200 μg (1 / 2 dose) or 400 μg (total dose) of IMAB362 was administered intravenously via the tail vein. IMAB362 treatment continued twice weekly, alternating between ip and iv injections, until mice were sacrificed. (A) Growth curve of subcutaneous BxPC3-LVT xenograft tumors. The size of the sc tumors was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival curve. Mice were sacrificed when the tumors reached a volume of 1400 mm3 or became ulcerated. [Figure 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 flanks of 10 female Hsd:athymic nude-Foxn1nu mice per treatment group. Chemotherapy treatment (50 mg / kg gemcitabine ip) began 4 days after tumor cell injection and continued weekly for 6 weeks. 24 hours after chemotherapy injection, 200 μg of IMAB362 or control was administered intravenously via the tail vein. IMAB362 treatment continued twice weekly, alternating between ip and iv injections, until mice were sacrificed. (A) Growth of subcutaneous xenograft tumors. Tumor size was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumors reached a volume of 1400 mm3 or when tumors became ulcerated. [Figure 44] Effect 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 flanks of female Hsd:athymic nude-Foxn1nu mice. Nine days after subcutaneous tumor inoculation, tumor-bearing mice were regrouped into uniform treatment groups with eight animals per group, and treatment began. Mice were treated with 150 mg / kg gemcitabine ip twice weekly for 4 weeks. 24 hours after gemcitabine injection, 200 μg of IMAB362 or control was applied intravenously via the tail vein. Treatment with 200 μg of IMAB362 continued twice weekly, alternating between ip and iv injections, until the mice were sacrificed. (A) Subcutaneous tumor size was measured twice weekly (mean + SEM; **=p<0.01). (B) Kaplan-Meier survival curve. Mice were sacrificed when tumors reached a volume of 1400 mm3 or became ulcerated (log-rank (Mantel-Cox) test; **=p<0.01). [Figure 45AB] Effect of IMAB362 in combination with gemcitabine on lung metastases in the Patu8988S xenograft model. [Figure 45CD]Effect of IMAB362 in combination with gemcitabine on lung metastasis in the Patu8988S xenograft model. 2 x 10 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 began with a maintenance treatment of 200 μg of IMAB362 twice weekly (iv / ip) in combination with 100 mg / kg gemcitabine ip twice weekly for 4 weeks. A control group was treated with 200 μg of isotype control antibody in combination with 100 mg / kg gemcitabine twice weekly. Animals were sacrificed 70 days after implantation. (A) Quantitative PCR analysis of human DNA in lung samples from mice treated with IMAB362 and isotype antibody (mean of triplicate reactions for each sample). Significant difference compared to 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 using anti-human MHC-I antibody (clone EPR1394Y) (mean ± SEM; P = 0.0003, Mann-Whitney test). (C and D) Examples of immunohistological 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 INVENTION

[0024] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, which may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

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

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

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

[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 present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0030] The term "CLDN18" refers to claudin 18 and encompasses any variants, including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

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

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

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

[0038] According to the present invention, CLDN18.2 is expressed in cells when the expression level is at least 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10,000-fold higher than the expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in cells when the expression level is higher than the detection limit and / or when the expression level is high enough to allow binding by a CLDN18.2-specific antibody added to the cells. Preferably, CLDN18.2 expressed in cells is expressed or exposed on the surface of the cells.

[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 that express CLDN18.2.

[0040] According to the present invention, a "disease associated with cells expressing CLDN18.2" or similar expression means that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. Increased refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is observed only in the diseased tissue, while expression in the corresponding healthy tissue is suppressed. For example, CLDN18.2 is expressed in pancreatic cancer tissue, but its expression is undetectable in non-cancerous pancreatic tissue. 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" refer to abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow even after the stimulus that initiated the new growth has ceased. Preferably, "cancer diseases" are characterized by cells that express CLDN18.2, and the cancer cells express CLDN18.2. The CLDN18.2-expressing cells are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0042] According to the present invention, a "carcinoma" is a malignant tumor derived 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 lining body cavities and organs. Epithelium is embryologically derived from ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated forms may not. By staining cells from a biopsy, a pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Due to the ubiquity of glands in the body, adenocarcinoma can arise in many tissues. Although not every gland secretes the same substances, as long as the cells have an exocrine function, they are considered glandular, and therefore their malignant forms are named adenocarcinoma. Malignant adenocarcinomas can invade other tissues and often metastasize if given enough time.

[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. Organized into functional units along the ductal network, acinar cells synthesize and secrete enzymes into the ductal lumen in response to cues from the stomach and duodenum. Cardioatrial cells reside within 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 originating from transformed cells that arise in the tissues that form the pancreas. It is the fourth leading cause of cancer-related death in the United States and the eighth leading cause worldwide. Early-stage pancreatic cancer often causes no symptoms, and later-stage symptoms are usually nonspecific and variable. As a result, 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 approximately 20%, while the median survival times for locally advanced and metastatic disease, which together account for more than 80% of individuals, are approximately 10 months and 6 months, respectively.

[0046] Pancreatic cancer includes adenocarcinomas (tumors that exhibit glandular structures) 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 structures on microscopic examination. Pancreatic ductal adenocarcinoma (PDAC) generally arises in the head of the pancreas and is associated with invasion of surrounding tissues, including lymphatics, the spleen, and the peritoneal cavity, as well as metastasis to the liver and lung. PDAC exhibits a predominantly glandular pattern with ductal-like structures and varying degrees of cellular atypia and differentiation. Less common subtypes of PDAC include colloid, adenosquamous, or sarcomatoid histology. Regional variations 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 encompasses cancer metastases of primary tumors, such as primary pancreatic cancer. Thus, for example, when referring to pancreatic cancer, this also encompasses 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 their original site to another part of the body. The formation of metastasis is a highly complex process that relies on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and vessels, and then infiltration of the target organ after transport by blood. Finally, the growth of new tumors at the target site relies on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis," which refers to metastasis far from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the therapeutic methods of the present invention is metastasis arising from pancreatic cancer as the primary site. In a preferred embodiment, such pancreatic cancer metastasis is lymph node metastasis, lung metastasis, and / or liver metastasis.

[0052] Krukenberg tumor is a rare metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. Krukenberg tumor is a metastatic signet-ring cell adenocarcinoma of the ovary. The stomach is the primary site in most Krukenberg tumor cases (70%). Cancers of the colon, appendix, and breast (primarily invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor arising 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, and 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 worsening of, or preventing or delaying the onset of a disease or its symptoms.

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

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

[0058] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" includes a chemotherapeutic agent or combination of chemotherapeutic agents, such as cytostatic agents. Chemotherapeutic agents can affect cells in one of the following ways: (1) by damaging the cell's DNA so that the cell can no longer replicate, (2) by inhibiting the synthesis of new DNA strands so that cell replication is not possible, or (3) by stopping the cell's mitotic process so that the cell cannot divide into two cells.

[0059] According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" preferably relates to an agent or combination of agents, such as a cytostatic compound or combination of cytostatic compounds, that, when provided to cells, particularly cancer cells, causes the cells to arrest or accumulate in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the G1 and G0 phases, preferably other than the G1 phase, preferably in the G2 or S phase of the cell cycle, such as one or more of the G1 / G2, S / G2, G2 or S phases of the cell cycle. The term "cells arresting or accumulating in one or more phases of the cell cycle" means that the percentage of cells in said one or more phases of the cell cycle increases. Each cell goes through a cycle containing four phases to replicate itself. The first phase, called G1, is the stage in which the cell prepares to replicate its chromosomes. The second phase, called S, is the phase in which DNA synthesis occurs and DNA is replicated. The next phase is G2, where RNA and proteins replicate. The final phase is M, which is the actual cell division phase. In this final phase, the replicated DNA and RNA separate and move to separate ends of the cell, and the cell actually divides into two identical, functional cells. Chemotherapeutic agents that are DNA-damaging agents usually result in the accumulation of cells in G1 and / or G2 phases. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, usually result in the accumulation of cells in S phase. Examples of these agents are 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 analogs, such as gemcitabine, 5-fluorouracil or its prodrugs, platinum compounds, such as oxaliplatin and cisplatin, taxanes, such as paclitaxel and docetaxel, and camptothecin analogs, such as irinotecan and topotecan, as well as drug combinations, such as drug combinations comprising one or more of gemcitabine, oxaliplatin, and 5-fluorouracil, for example, 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 of said agent, such as a conjugate of said agent. Examples are conjugates of said agent with a carrier substance, such as protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salt of said agent is pharmaceutically 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 can enter a lethal stress pathway that results in the release of a spatiotemporally defined combination of signals that are decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:798-804). In such situations, cancer cells initiate the release of signals that are sensed by innate immune effectors such as dendritic cells and trigger cognate immune responses, including CD8+ T cell and IFN-γ signaling, so that tumor cell death can elicit an effective anticancer immune response. These signals include preapoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) at the cell surface, preapoptotic secretion of ATP, and postapoptotic release of the nuclear protein HMGB1. Taken together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Although anthracyclines, oxaliplatin, and gamma irradiation can induce all of the signals that define ICD, cisplatin, for example, lacks the ability to induce CRT translocation from the ER to the surface of dying cells, a step that requires ER stress, and requires complementation with the ER stress inducer thapsigargin.

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

[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 that is a nucleoside analogue of

[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 / m once weekly for 7 out of 8 weeks. 2 and then administered once a week for three out of four weeks.

[0069] The term "nucleoside analogue" 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) refers to a compound of the following formula: [ka] It is a compound that is a pyrimidine analogue of

[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. Orally administered capecitabine has the following formula: [ka] It 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 containing platinum in its structure, such as a platinum complex, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

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

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

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

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

[0078] Taxanes are a class of diterpene compounds originally derived from natural sources such as yew plants, although some have been synthetically synthesized. The primary mechanism of action of the taxane class of drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0079] According to the present invention, the term "docetaxel" means a compound of the formula: [ka] It refers to a compound having the 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-trityl 4-acetate 2-benzoate 13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropanoate}.

[0081] According to the present invention, the term "paclitaxel" means a compound of the formula: [ka] It refers to a compound having the 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 having the following structure: [ka] It refers to a compound containing

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

[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-dioxo1H-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 that are also antibiotics and are commonly used in cancer chemotherapy. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

[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 enzymes, preventing the relaxation of supercoiled DNA and thus blocking DNA transcription and replication; 3. Generate iron-mediated free oxygen radicals that damage DNA and cell membranes.

[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 analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs thereof.

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

[0094] Particularly preferred anthracyclines according to the present invention are those of the following formula: [ka] [In the formula, R1 is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and OMe, R3 is selected from the group consisting of H and OH, and R4 is selected from the group consisting of H and OH. is a compound of

[0095] In one embodiment, R1 is H, R2 is OMe, R3 is H, and R4 is OH. In another embodiment, R1 is OH, R2 is OMe, R3 is H, and R4 is OH. In another embodiment, R1 is OH, R2 is OMe, R3 is OH, and R4 is H. In another embodiment, R1 is H, R2 is H, R3 is H, and R4 is OH.

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

[0097] According to the present invention, the agent that stabilizes or increases the expression of CLDN18.2 may be a chemotherapeutic agent, particularly a chemotherapeutic agent established in cancer treatment, or may be part of a drug combination, for example, a drug combination established for use in cancer treatment.Such a drug combination may be a drug combination used in chemotherapy, or may be a drug combination used in 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 / m² 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 can 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] It 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 possess a unique T cell receptor (TCR) on their surface. The majority of T cells possess a TCR consisting of two glycoprotein chains, termed the α-TCR chain and the β-TCR chain. In contrast, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is typically much rarer than αβ T cells. Human γδ T cells play an important role in stress surveillance responses, such as infection and autoimmunity. It has been suggested that transformation-induced changes in tumors also generate a stress surveillance response mediated by γδ T cells, enhancing antitumor immunity. Importantly, after antigen engagement, activated γδ T cells at the lesion site deliver cytokines (e.g., INFγ, TNFα) and / or chemokines that mediate the recruitment of other effector cells, exhibiting immediate effector functions such as cytotoxicity (via death receptor and cytolytic granule pathways) and ADCC.

[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 dramatically expanded during many acute infections, including tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria, where they can outnumber all other lymphocytes within days, suggesting that they play an early and essential role in sensing "danger" from invading pathogens.

[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. While IPP production in normal cells is insufficient for γδ T cell activation, dysregulation of the mevalonate pathway in tumor cells leads to IPP accumulation and γδ T cell activation. IPP can also be therapeutically increased by aminobisphosphonates, which inhibit farnesyl pyrophosphate synthase (FPPS), an enzyme in the mevalonate pathway. Zoledronic acid (ZA, zoledronate, Zometa™, Novartis), among others, is a representative such aminobisphosphonate and has already been administered clinically to patients for the treatment of osteoporosis and metastatic bone disease. Following in vitro treatment of PBMCs, ZA is specifically taken up by monocytes. IPP accumulates in monocytes and differentiates into antigen-presenting cells, which stimulate the development of γδ T cells. In this situation, the addition of interleukin 2 (IL-2) as a growth and survival factor for activated γδ T cells is preferable. 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 that stimulates γδ T cells" relates to a compound that stimulates the development of γδ T cells, in particular Vγ9Vδ2 T cells, in vitro and / or in vivo, in particular by inducing γδ T cell activation and proliferation. Preferably, the term relates to a compound that increases isopentenyl pyrophosphate (IPP) production in mammalian cells in vitro and / or in vivo, preferably by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS).

[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 acid, e.g., pamidronate; (1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acids, risedronate, for example; (1-hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, minodronic acid; [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid; [4-amino-1-hydroxy-1-(hydroxy-oxide-phosphoryl)-butyl]phosphonic acid, alendronic acid, e.g., alendronate; [(cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid; (1-hydroxyethane-1,1-diyl)bis(phosphonic acid), etidronic acid, e.g., etidronate; and {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic acid.

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

[0108] In one particularly preferred embodiment, the γδ T cell stimulating agent according to the invention is administered in combination with IL-2, which has 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 infection and in distinguishing self from foreign (non-self) substances. IL-2 mediates its effects by binding to the IL-2 receptor, which is expressed by lymphocytes.

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

[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 cancer cells that can originate from the cytoplasm, cell surface and cell nucleus, in particular an antigen that is produced intracellularly or as a surface antigen on cancer cells, preferably in large amounts.

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

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

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

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

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

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

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

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

[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, as well as analogs or homologs thereof. Suitable therapeutic agents for forming antibody conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), and steroids (e.g., steroids ... Examples of therapeutic agents include, but are not limited to, cyclosporine (rubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0121] Antibodies can also be conjugated 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 possessing a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon gamma; or biological response modifiers, such as lymphokines, interleukin 1 ("IL-1"), interleukin 2 ("IL-2"), interleukin 6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other growth factors.

[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 Prospect of The Therapeutic Use Of Radiolabeled Antibodies" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); See "In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[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%, and even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a particular germline sequence displays no more than 10 amino acid differences, more preferably no more than 5, or even more preferably no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0125] As used herein, the term "heteroantibody" refers to two or more antibodies, derivatives thereof, or antigen-binding regions thereof linked together, at least two of which have different specificities, including 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 may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal, such as a mouse, fused to an immortalized cell.

[0127] The antibodies described herein may be recombinant antibodies. The term "recombinant antibody," as used herein, encompasses all antibodies made, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a hybridoma made therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies made, expressed, created, or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences.

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

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

[0130] The term "transfectoma" as used herein includes recombinant eukaryotic host cells that express 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 that 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, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind to antigens other than CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may, however, have cross-reactivity with other related antigens, such as related antigens from other species (e.g., CLDN18.2 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, an "isolated" monoclonal antibody combination relates to antibodies with different specificities that are combined in a well-defined composition or mixture.

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

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

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

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

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

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

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

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

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

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

[0149] In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 is produced by a hybridoma deposited at DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) with the following name and accession number: a.182-D1106-055, accession number DSM ACC2737, deposited October 19, 2005 b.182-D1106-056, accession number DSM ACC2738, deposited October 19, 2005 c.182-D1106-057, accession number DSM ACC2739, deposited October 19, 2005 d.182-D1106-058, accession number DSM ACC2740, deposited October 19, 2005 e.182-D1106-059, accession number DSM ACC2741, deposited October 19, 2005 f.182-D1106-062, accession number DSM ACC2742, deposited October 19, 2005 g.182-D1106-067, accession number DSM ACC2743, deposited October 19, 2005 h.182-D758-035, accession number DSM ACC2745, deposited November 17, 2005 i.182-D758-036, accession number DSM ACC2746, deposited November 17, 2005 j.182-D758-040, accession number DSM ACC2747, deposited November 17, 2005 k.182-D1106-061, accession number DSM ACC2748, deposited November 17, 2005 l.182-D1106-279, accession number DSM ACC2808, deposited October 26, 2006 m.182-D1106-294, accession number DSM ACC2809, deposited October 26, 2006 n.182-D1106-362, accession number DSM ACC2810, deposited October 26, 2006.

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

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

[0152] In preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a heavy chain constant region (CH) having an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof. In further preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a light chain constant region (CL) having an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In particularly preferred embodiments, antibodies, particularly chimeric forms of antibodies according to the invention, include antibodies comprising a CH having an amino acid sequence derived from a human CH, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof, and a CL having an amino acid sequence derived from a human CL, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.

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

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

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

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

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

[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 RNA and protein / peptide. This term also includes partial expression of a nucleic acid. Furthermore, expression can be performed transiently or stably.

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

[0170] Those skilled in the art will recognize that the sequences of the CDRs, hypervariable regions, and variable regions, in particular, can be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions can be identical or highly homologous to regions of the antibodies identified herein. By "highly homologous," it is contemplated that one to five, preferably one to four, e.g., one to three, or one or two substitutions can be made within the CDRs. In addition, the hypervariable and variable regions can be modified to exhibit substantial homology with regions of the antibodies specifically disclosed herein.

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

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

[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] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the modification occurs at a position in the amino acid sequence that is not conserved among homologous proteins or peptides and / or the amino acid is replaced with another amino acid with similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

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

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

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

[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 containing one or more transgenes, preferably heavy and / or light chain transgenes, or transchromosomes (integrated or not integrated into the animal's native genomic DNA), and preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with a CLDN18.2 antigen and / or cells expressing CLDN18.2. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as in the case of transgenic mice such as HCo7 or HCol2 mice, e.g., HuMAb mice, or the human heavy chain transgene can be maintained extrachromosomally, as in the case of transchromosomal (e.g., KM) mice described in WO 02 / 43478. Such transgenic and transchromosomal mice are capable of producing multiple isotypes (eg, IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN18.2 by undergoing VDJ recombination and isotype switching.

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

[0182] Terms such as "increase" or "enhance" relate to an increase or enhancement of preferably at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

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

[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 through a complementary mechanism of action, which may involve an anti-tumor immune response that may be impaired due to the cytotoxic side effects of the chemotherapeutic agent on T lymphocytes. However, the antibodies described herein can also act simply by binding to CLDN18.2 on the cell surface, thus, for example, blocking cell proliferation.

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

[0187] Complement-dependent cytotoxicity CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is initiated by the C-terminal end of the participating antibody molecules, such as IgG molecules. H This results in the exposure of multiple C1q binding sites in close proximity on the two domains (C1q is one of three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-avidity interaction, which initiates a cascade of events involving a series of other complement proteins, leading to the proteolytic release of the effector cell chemotactic / activators C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

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

[0189] The preferred animal system for producing hybridomas secreting monoclonal antibodies is the murine system. The production of hybridomas in mice is a very well-established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

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

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

[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, an enriched preparation of recombinantly expressed antigen or its fragment, and / or cells expressing the antigen, as described above. Alternatively, mice can be immunized with DNA encoding the antigen or its fragment. If immunization with a purified or enriched preparation of antigen does not produce antibodies, mice can also be immunized with cells, e.g., a cell line, expressing the antigen to stimulate an immune response.

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

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

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

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

[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 Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with toxins or radioisotopes. Unlabeled murine antibodies are highly immunogenic in humans when administered repeatedly, resulting in reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of murine antibodies in humans can be reduced or completely avoided by chimerizing or humanizing the respective antibodies. Chimeric antibodies are antibodies whose different portions are derived from different animal species, such as those with variable regions derived from a murine antibody and human immunoglobulin constant regions. Antibody chimerization is achieved by linking the heavy and light chain variable regions of a murine antibody with human heavy and light chain constant regions (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by linking a human kappa light chain constant region to a murine light chain variable region. In an equally preferred embodiment, chimeric antibodies can be produced by linking a human lambda light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD, and IgM.

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

[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 cytometry analysis.

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

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

[0205] Flow cytometry can be used to determine the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection, as well as negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated for 30 minutes at 4°C. After washing, APC- or Alexa647-labeled anti-IgG antibodies can be bound to the antigen-bound monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry on a FACS instrument using side light scatter characteristics to gate on single live cells. Cotransfection methods can be used to distinguish antigen-specific monoclonal antibodies from nonspecific binders in a single measurement. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. Because the majority of transfected cells express both transgenes, antigen-specific monoclonal antibodies will selectively bind to cells expressing the fluorescent marker, while nonspecific antibodies will bind to non-transfected cells in equal proportions. A selective assay using fluorescence microscopy can be used in addition to or instead of a flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

[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, as well as negative controls lacking antigen expression, are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against the antigen for 30 minutes at 25°C. After washing, cells can be reacted with Alexa555-conjugated anti-mouse IgG secondary antibodies (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

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

[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 (PMNs), NK cells, monocytes, mononuclear cells, or other effector cells from healthy donors can be purified by Ficoll Hypaque density gradient centrifugation followed by lysis of contaminating red blood cells. Washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum and purified to express CLDN18.2. 51 Cr-labeled target cells can be mixed with various ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence-enhancing ligand (BATDA). Highly fluorescent chelates of europium with the enhancing ligand, released from dead cells, can be measured by fluorometer. Another alternative technique utilizes transfection of target cells with luciferase. Added lucifer yellow can then be oxidized only by live cells. Purified anti-CLDN18.2 IgG can then be added at various concentrations. An irrelevant human IgG can be used as a negative control. The assay can be performed at 37°C for 4 to 20 hours, depending on the effector cell type used. The elution of the culture supernatant 51 Samples can be assayed for cell lysis by measuring Cr release or the presence of 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 various known techniques. For example, serum for complement can be obtained from blood using methods known to those skilled in the art. Various methods can be used to measure the CDC activity of mAbs. For example, 51 Cr release can be measured, or a propidium iodide (PI) exclusion assay can be used to assess membrane permeabilization. Briefly, target cells were washed and 5×10 5 1 / ml can be incubated with various concentrations of mAb for 10-30 minutes at room temperature or 37°C. Serum or plasma can then be added to a final concentration of 20% (v / v), and the cells can be incubated for 20-30 minutes at 37°C. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be analyzed immediately by flow cytometry using a FACSArray.

[0212] In an alternative assay, induction of CDC can be measured in adherent cells. In one embodiment of this assay, cells are plated at 3x10 in tissue culture flat-bottom microtiter plates 24 hours prior to the assay. 4 Cells are seeded at a density of 1000 μg / well. The next day, the growth medium is removed, and the cells are incubated with the antibody in triplicate. Control cells are incubated with growth medium or growth medium containing 0.2% saponin for measurements of background lysis and maximum lysis, respectively. After 20 minutes of incubation at room temperature, the supernatant is removed, and 20% (v / v) human plasma or serum in DMEM (prewarmed to 37°C) is added to the cells and incubated for an additional 20 minutes at 37°C. All cells from each sample are added to a propidium iodide solution (10 μg / ml). The supernatant is then replaced with PBS containing 2.5 μg / ml ethidium bromide, and fluorescence emission at 520 nm excitation is measured at 600 nm using a Tecan Safire. The percentage of specific lysis is calculated as follows: % specific lysis = (sample fluorescence - background fluorescence) / (maximum lysis fluorescence - background fluorescence) × 100.

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

[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 CLDN18.2-expressing cell lines, such as DAN-G, SNU-16, or KATO-III, or with cell lines that express CLDN18.2 after transfection, such as HEK293) to measure their effectiveness in controlling the growth of tumor cells that express CLDN18.2.

[0215] In vivo testing can be performed using the antibodies described herein after xenografting CLDN18.2-expressing tumor cells into immunocompromised mice or other animals. Antibodies can be administered to tumor-free mice, followed by injection of tumor cells, to measure the antibody's ability to prevent tumor formation or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice, and the therapeutic effect of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms can be measured. Antibody administration can be combined with the administration of other substances, such as cytostatics, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies, to measure synergistic effects and potential toxicity of the combination. To analyze antibody-mediated toxic side effects, animals can be inoculated with the antibody or a control agent and thoroughly examined for symptoms potentially associated with CLDN18.2 antibody treatment. Potential side effects of in vivo administration of CLDN18.2 antibodies include toxicity, particularly in CLDN18.2-expressing tissues, including the stomach. Antibodies that recognize CLDN18.2 in humans and other species, such as mice, are particularly useful for predicting potential side effects mediated by the application of monoclonal CLDN18.2 antibodies in humans.

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

[0220] Pharmaceutically unacceptable salts can be used to prepare pharmaceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[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 pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0223] Injectable formulations may contain pharmaceutically 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 encompasses one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are 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, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

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

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

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

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

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

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

[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 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 described herein.

[0234] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

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

[0238] Deparaffinization After melting and drying, the slides were deparaffinized using two xylol steps (5 min) and rehydrated using a decreasing alcohol array (at ambient temperature 20–27 °C): · 5 (± 1) minutes in a xylene bath; 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) minutes; · Remove excess liquid; · 70% ethanol for 5 (± 1) minutes; · 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. To do 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 minutes in a pressure cooker (PASCAL, Dako). The jar was then removed from the cooker and allowed to cool in the epitope retrieval solution at room temperature for 10 (±1) minutes. Slides were then washed in wash buffer (1x PBS).

[0240] After cooling, the sections were transferred to a staining jar 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 washing buffer.

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

[0242] The next day, the primary antibody solution was removed, and the sections were washed 3 x 5 min with 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 the slides were washed 3 x 5 min in fresh wash buffer.

[0243] Substrate reaction and counterstaining After removing 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] Counterstaining of the tissue was then performed by immersing the sections in a jar containing 200 ml of Mayer's hematoxylin for 2 minutes, after which the sections were placed in tap water for 5-10 minutes to stain the nuclei blue.

[0245] Dehydration and mounting Counterstaining was performed and sections were dehydrated using an ascending alcohol array: 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) xylene for 5 minutes -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 datasheets of their 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)) prepared with an ARCA cap and dissolved in HO. The RNA was stored in 22 μl aliquots at -80°C. For all pancreatic cell lines, optimal electroporation conditions were determined to yield the highest transfection rate and cell viability. For each assay, cells were detached with PBS / 5 mM EDTA and 2.5 × 10 cells were dissolved in 250 μl of X-Vivo. 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 cells. 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 either 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 quadruplicate in 1000 x 1000 cells / well and incubated for 24 hours. Luciferase activity was then measured for 90 minutes in a luminometer (Tecan Infinite200) 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 onto 10 cm dishes and grown for 2–3 days to 80% confluence. RNA was isolated using the RNeasy® Mini Kit (Qiagen) according to the provided instructions. cDNA preparation was performed using the SuperScript® III First Strand Kit (Invitrogen) according to the manufacturer's instructions. 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-PRISM 7900 Sequence Detection System 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 from pancreatic cancer cell lines, cells were seeded into 10 cm dishes and grown for 2–3 days to 80% confluence. 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: output control: level 1, duty cycle: 70% for 20–25 seconds. Protein concentration was measured spectrophotometrically (absorbance at 280 nm), and samples were stored at -80°C until use.

[0254] To detect CLDN18.2 expression by Western blotting, a separating 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, 3.2 ml of HO, 100 μl of APS, and 10 μl of TEMED) was prepared between two fixed glass plates. After polymerization, the gel was overlaid with 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, 5.8 ml of HO, 100 μl of APS, and 10 μl of TEMED), and a gel comb was placed between the glass plates. After polymerization, 75 μg of each protein sample, prepared by adding (1:20) 4× SDS sample buffer (250 mM Tris-HCl, 34% glycerol, 8.2% SDS, pH 6.8) and 7.5 μl of size marker mixture (1.5 μl Magic Mark XP Western Standard mixed with 6 μl SeaBlue Plus2 Prestained Standard), were loaded onto the gel. Gels were 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 gel onto a nitrocellulose membrane was performed at 160 mA for 90 min in 1× transfer buffer (25 mM Tris, 0.192 mM glycine, 20% MeOH). Blots were first blocked in 5% milk powder / PBS, and primary antibodies (0.25 μg / ml anti-claudin-18 (C-terminal) or 0.1 μg / ml anti-β-actin) were added in 1% milk powder / PBS. Blots were incubated overnight at 4°C, washed three times for 10 min in 1x PBS / 0.05% Tween 20, and then incubated for 1 h with labeled secondary antibodies (goat anti-rabbit IgG (FC) diluted 1:1000) in 1% milk powder / PBS at room temperature.The blots were again washed three times for 10 min each in 1× PBS / 0.05% Tween 20, and detection was performed by adding 1–3 ml of detection solution (Pico and Dura Detection System (Pierce)) for 1 min and scanning the blots in an LAS-3000 detection box (increment: 10 s, time interval: 10 s, sensitivity: high) according to GA_056_Chemolumineszenzentwickler 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 cells were adjusted to a concentration of 1 / ml. 100 μl of cells were plated onto a round-bottom 96-well plate and centrifuged again (5 min, 468 g). IMAB362 (or isotype control rituximab) was serially diluted in 50 μl of FACS buffer from 0.1 to 200 μg / ml (11 dilutions + no antibody control) and added to the cells for 30 min at 4°C. Next, 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 cycle was repeated. A secondary goat anti-human antibody (FC-specific, APC-conjugated F(ab')2 (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 again washed 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 are 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 previous in vivo testing. Selection criteria included uniform subcutaneous growth in nude mice and a therapeutic window of 20 to 100 days. Three cell lines (DANG, YAPC, and BxPC3) that already showed weak expression of CLDN18.2 mRNA and three cell lines (MiaPaCa-2, Patu8902, and Suit-2) that were known to be capable of metastasizing according to the literature were included. Two other cell lines (HPAC and CAPAN1) known to grow as uniform subcutaneous tumors in vivo were randomly selected.

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

[0259] Envelope Selection: For lentiviral transduction, the GFP-lentiviral control vector pL64B42E-(EF1α-GFP)-blasticidin was packaged into various envelope particles (VSV-G, GALV, RD114, Mokola-G, and Rabies-G). Attachment to target cancer cells is generally efficient, depending on the proteins present in the envelope and the composition of the cell membrane. For all pancreatic cancer cell lines, the VSV-G envelope showed the highest transduction efficiency (68.5–91.2%) (Table 5). Consequently, 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 transduced to a high titer (3.86 × 10 7 The virus was isolated from the culture medium at 1000x the number of particles / ml. The viral supernatant was 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.), sealed with Parafilm®, and incubated at 4°C for 3 to 16 hours. The plates were washed with 200 ml of PBS and blocked with PBS / 2% BSA for 30 minutes at room temperature. The plates were washed again and loaded with 300 μl of viral supernatant by centrifugation at 2500 rpm for 25 minutes at 15°C. The supernatant was removed, and the loading was repeated three times. The 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 cells were used per 24-well plate. 5 ~1×10 7 Cells were seeded on plates and 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 the start of ADCC to obtain 80-90% confluent cultures. Pancreatic cancer cells were transfected with luciferase RNA and cultured at 1 x 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. Additionally, NUGC-4 sub 10cH11 subE10 Luci#2 cells (8,000 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 of blood was diluted (1:2) with PBS and carefully layered on 4 × 15 ml of 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 of PBS / 2 mM EDTA, and the 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 x 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 PBMCs were then added to each well (E:T ratio = 40:1), and the plate was incubated at 37°C, 5% CO2 for 24 hours ± 1 hour.

[0264] The next day, 10 μl of 8% Triton X100 / 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 in the dark at room temperature for 80 minutes. 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 using 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 (10,000 cells / well) in 50 μl of assay medium in 96-well white assay plates and grown at 37°C, 7.5% CO2, and 95% relative humidity for 24 hours + 20 minutes before sample addition. Each 96-well assay plate contained a total of three different negative controls (heat-inactivated serum, serum with and without IMAB362, and serum with an isotype control antibody (rituximab)) and a positive control of a 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 X-100 to a second medium control well to cause total lysis. One 96-well assay plate contained a functional positive control generated by seven serial 3.16-fold dilutions of IMAB362 (10,000 to 31.8 ng / ml). This control resulted in sigmoidal dose-dependent lysis of target cells. All samples were prepared simultaneously (200 μl each) in a 96-well deep-well dilution plate. Samples were taken three times from each well by reverse pipetting to generate triplicates in the assay plate. After adding 50 μl of each test and control to the assay plate, the plate was incubated at 37°C, 7.5% CO2, and 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 of Aqua bidest, 2496 μl of HEPES (1M), 1998 μl of 1x DPBS, 4992 μl of D-luciferin stock solution (12 mg / ml)). 50 μl of luciferin substrate solution was added to each well. The plate was incubated at 37°C, 7.5% CO2, and 95% relative humidity for 45 minutes. The plate was then read using a microplate reader. Complement-dependent lysis is achieved by the following:

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 10 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 cultured at 37°C and 5% CO2. CDC assays on cells pretreated with chemotherapy agents were performed at the following IMAB362 concentrations or ch78H11 1H6 antibody concentrations as isotype control antibody: 640000, 160000, 40000, 10000, 2500, 625, 156 and 39 ng / ml.

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

[0270] Cells were seeded in 96-well plates and after 4-6 hours gemcitabine or oxaliplatin was added at the following concentrations: 1000, 500, 250, 100, and 20 ng / ml. The proliferation assay was incubated at 37°C and 5% CO2 for 4 days. 50 μl of XTT complete reagent (50 parts XTT + 1 part coupling reagent) was added and incubated at 37°C. Absorbance (cells + supernatant) measurements were performed on a Tecan Safire after 3 and 4 hours. Growth inhibition was calculated relative to the median, which was set at 100%. EC values ​​for gemcitabine and oxaliplatin were calculated. 50 Values ​​were calculated with the GraphPad Prism program.

[0271] 12. Culturing 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 + 1 ng / ml gemcitabine or 1 ng / ml gemcitabine + 10 ng / ml oxaliplatin. 1-1.4E+07 Patu8988S were seeded and cultured without or with 10 ng / ml gemcitabine, or with or without 10 ng / ml gemcitabine combined with 100 ng / ml oxaliplatin.

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

[0273] 13. Cell cycle analysis Cells were plated in 6-well plates, and after 5-6 hours, chemotherapy drugs were added for 24, 48, or 3 days. Cells floating in the medium were combined with the adherent cell layer and trypsinized. The cells were then washed. Cell cycle analysis was initiated directly or cell surface staining was performed as previously described. Cells were resuspended in 1 ml of PBS and added to 3 ml of 4% PFA. Cells were fixed for 15 minutes at room temperature, then pelleted and washed. For RNase treatment, cells were resuspended in 200 μl of RNase (10,000 U / ml) plus 0.05% Triton X-100 and incubated at 37°C for 30 minutes. 1 ml of PBS was added, the sample was centrifuged, and resuspended in 200 μl of PBS / PJ 50 μg / ml. After at least 30 minutes, the sample was ready for flow cytometry analysis. Cell cycle distribution was determined using FlowJo software to analyze 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 in the dark for 15 minutes at room temperature. Cells were immediately analyzed by flow cytometry. Viable 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 positive for 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 (TdT) and FITC-dUTP for 90 minutes at 37°C to label DNA fragments. Cells were washed and incubated in RNase A / propidium iodide in the dark for 30 minutes at room temperature to stain total DNA, followed by flow cytometry analysis. 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 of 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. Chemotherapeutic agents were administered 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 0.05 mm or became ulcerated. Tumor samples were either cryopreserved 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 x 10 cells were injected into 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 point of metastatic engraftment and growth.

[0279] Transplant treatment was performed on 10-12 Hsd:Athymic Nude-Foxn1 mice per treatment group. nu These mice were then injected 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 clear physiological signs of metastatic disease (weight loss, weakness, shortness of breath). All organs of the mice were macroscopically analyzed for metastases. Only for Patu8988S and Suit-2 cells, the lungs and lung / liver showed macroscopic metastases, respectively. These organs were cut into four equal pieces, and two (lung: right upper lobe and left lower lobe) were preserved for genomic DNA isolation. The other two pieces were formalin-fixed and preserved 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. Because this method is biased by the fact that metastases do not spread uniformly within the lung and sometimes one lobe is more heavily affected than the other, two different regions of the lung were mixed in one DNA preparation (Figure 2).

[0283] Q-PCR reactions were performed using the primer pair No. 5861 5'-GGGATAATTTCAGCTGACTAAACAG-3' and No. 5862 5'-TTCCGTTTAGTTAGGTGCAGTTATC-3', which specifically amplify α-satellite DNA present in human chromosome 17 but absent in mouse DNA. To generate a standard curve and as a positive control, Patu8988S DNA was mixed with mouse DNA and five-fold dilutions were prepared to yield 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 translocated DNA present in mouse lung tissue (linear regression). Q-PCR reactions were performed in a final volume of 50 μl consisting of 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 1.8 μl H 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 using two mouse monoclonal antibody reagents (Figure 3).

[0285] Preliminary pilot experiments were performed using the prototype antibody 35-22A on tissue microarrays (TMAs). The major drawbacks of TMAs 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 resulted in 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, and 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, and oriented perpendicular to the basement membrane. There is a histologic overlap between non-neoplastic flat hyperplastic lesions and flat neoplastic lesions without atypia.

[0289] PanIN-1B subtype lesions have papillary, micropapillary, or basally pseudostratified structures 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 atypical (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, intraluminal budding of small clusters of epithelial cells, and luminal necrosis suggest the diagnosis of PanIN-3. The lesions are characterized by loss of nuclear polarity, dystrophic goblet cells (with nuclei oriented toward the lumen and mucinous cytoplasm oriented toward the basement membrane), occasionally aberrant mitoses, nuclear irregularities, 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 using 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 in 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 conducted to evaluate CLDN18.2 expression in primary pancreatic cancer. For the pilot study, several TMAs from 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 uneven counterstaining with hematoxylin suggested suboptimal tissue processing of 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 were stained without background staining 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, Figure 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 (Figure 7, Table 11). Again, a correlation was observed between CLDN18.2 expression levels and the proportion of positive tumor cells. The majority of analyzed cases (62%) had grade 3 tumors (Table 12). [Table 11] [Table 12]

[0300] Pancreatic cancer is diagnosed at an advanced stage in the majority of patients, whose tumors have already metastasized to lymph nodes and other organs, particularly the liver. In the main study, 79 FFPE tissue samples of pancreatic cancer lymph node and liver metastases were analyzed in an immunohistochemistry 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 an additional weaker cytoplasmic signal in some cases (Figure 9). Consistent with the results of primary tumor analysis, a correlation was observed between CLDN18.2 expression levels and the proportion 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 is 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 metastases were positive for CLDN18.2. In one case, both tissues were negative, and in another case, the primary tumor was CLDN18.2 positive but the metastasis was negative.

[0305] In 21 (80.7%) of the 26 positive doublets, the signal intensity was the same in the primary and metastatic tumor cells. In 5 cases, the 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 were 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 equivalent in terms of CLDN18.2 positivity score (Figure 11). In three cases, some tumor cells were CLDN18.2 positive in the primary lesion, but the metastatic lesion 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 for pancreatic cancer models Cell line source The primary objective of this preclinical evaluation study was to analyze the inhibitory effects 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 activity, a set of 26 commercially available pancreatic cancer cell lines was screened for CLDN18.2 expression and extensively characterized. 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 that binds to exon 1 of CLDN18.2 and a reverse primer that binds to exon 3 of CLDN18.2. 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 × 10 human pancreatic cancer cell lines DANG, Panc03.27, Panc05.04, Patu8988S, and YAPC.5 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, 1 x 10 8 Only in HAPC-LVT and Suit2-LVT cells was the expression level detected at a relative CLDN18.2 expression level of 1 × 10 5 It was higher.

[0313] We investigated whether CLDN18.2 expression is stable during in vitro culture. Patu8988S, Panc05.04 cells, and the lentiviral-transduced cell lines Suit2-LVT, MiaPaCa2-LVT, and Patu8902-LVT were passaged up to 15 times and analyzed for CLDN18.2 transcripts (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, we used early passages whenever possible for in vitro experiments, and confirmed CLDN18.2 expression in tumor xenografts 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 transcript analysis, CLDN18.2 expression was analyzed at the protein level by Western blotting and IF. For Western blot analysis, cell lysates from 26 pancreatic cancer cell lines were examined by Western blotting (WB) using a CLDN18-specific antibody, anti-claudin-18 (C-terminus). Lysates from SKBR-3 cells were again used as a negative control, and lysates from 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 a smaller size by Western blotting. All other cell lines were negative (Figure 13).

[0315] Cellular expression of CLDN18 in pancreatic cancer cells To obtain corroborating 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, showing 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 (e.g., AsPC1, DANG, HUP-T3, HUP-T4, and Panc01), which are likely staining artifacts. DANG, Panc03.27, and BxPC3 cells, which were identified as featuring low CLDN18.2 at the RNA and / or protein levels, 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 over 80% of all cells.

[0316] Confirmation of CLDN18.2 expression in pancreatic cancer cells To confirm the expression of CLDN18.2 and assess the amount of this target on the cell surface, the endogenous cell lines Panc05.04 and Patu8988S, as well as the LVT cell line, 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, with a lower percentage of positive cells 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 were performed on various pancreatic cancer cell lines to identify appropriate subcutaneous xenograft models for testing the in vivo efficacy of IMAB362. Among all cell lines tested, the 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 modeling, demonstrating high engraftment rates and uniform tumor growth. Additionally, the Patu8988S and DANG cell lines, which endogenously express CLDN18.2, were selected for subcutaneous xenograft testing of IMAB362 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 appropriate metastatic models To investigate the effect of IMAB362 on metastasis formation, a metastatic cancer model was established in nude mice. Pancreatic cancer cell lines were analyzed for their ability to metastasize after intravenous administration. 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 cells was not feasible because most mice died almost immediately. No macroscopic metastases were detected in the lungs or livers of the five surviving mice challenged with CAPAN-LVT cells after 72 days. In contrast, Suit-2 and MiaPaCa2 cell injections were well tolerated. Lung tissue from these mice was analyzed by IHC at various time points after injection. In mice challenged with MiaPaCa-2 cells, no metastases were detected in the lungs until 73 days after injection; therefore, 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 at two different cell numbers per mouse (1 × 10 6 , 2 × 10 6 ) was injected intravenously. 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 alpha-satellite DNA on 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 x 10 6 or 2×10 6 After 70 days, iv application of cells allowed the detection of 5.8% and 3.7% human DNA, 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 metastases, lung tissue was immunohistochemically stained using an anti-human MHC class I antibody and an anti-claudin 18 (center) antibody to detect human cells in mouse tissue. 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 an anti-claudin 18 (center) antibody, demonstrating clear expression of the IMAB362 target protein in these cells. Therefore, in addition to the Suit2-LVT model, this endogenous metastasis model was chosen for the study of IMAB362 treatment.

[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 leads to 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 example, in some CD20-positive lymphoma cell lines, induction of apoptosis by rituximab is only observed under cross-linking conditions. Such cross-linking can 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 to 42 hours in human gastric cancer cells NUGC-4 and KATO-III, as measured by TUNEL assay. The degree of apoptosis correlates with the antibody dose and the level of target expression on the cancer cells. Treatment with gemcitabine leads to cell cycle arrest and subsequent apoptotic cell death of tumor cells. 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 at 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) through granzymes and perforin secreted by effector cells when their Fcγ receptors bind to antibodies. The impact of this mechanism of action was previously demonstrated on luciferase-positive and CLDN18.2-positive gastric cancer cells (such as NUGC-4 and KATO-III) by 24-hour incubation with IMAB362 in the presence of human peripheral blood mononuclear cells (PBMCs) (effector-to-target ratio = 40:1). Application of up to 200 μg / ml of IMAB362 resulted in 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 various cell lines at an E:T ratio of 40:1. PBMCs from 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). CLDN18.2 surface expression was not detectable by FACS for Panc05.04 and DANG, but 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 .gamma.-glucan are efficiently lysed.

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

[0328] The IMAB362 concentrations that caused half-maximal killing of target cells as well as maximum killing are shown in Figures 22G-H. While the LVT pancreatic cancer cell line was killed at a high rate after the addition of small amounts of antibody, for DANG and Panc05, the highest antibody concentrations were required to reach approximately 50% maximum killing. For Panc05.04, the results obtained for subclone 15D3 (a CLDN18.2-positive clone selected by limiting dilution of Panc05.04 and FACS) are included in the figures, showing ADCC lysis rates comparable to those of the LVT cell line. Unfortunately, CLDN18.2 expression in this clone was rapidly silenced in vitro after cell passaging, 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. Additionally, LVT cell lines and parental lines were tested in 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), which 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). The cell lines DANG, BxPC3, YAPC, Patu8988S, Panc05.04, CAPAN1, and Suit2 were not lysed by the addition of IMAB362 and a 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 (e.g., 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 affects 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 line, 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 EC200-EC200-EC200 range of 0.3-2.6 μg / ml. 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 examine the effects of IMAB362 in vivo. Using a pancreatic xenograft model with high CLDN18.2 expression, IMAB362 treatment demonstrated a significant antitumor effect. This was examined by treating mice bearing BxPC3-LVT or MiaPaCa-2-LVT xenografts subcutaneously in the left flank. Treatment began 3 days after tumor inoculation with 200 μg of IMAB362 injected twice weekly. IMAB362-treated mice showed significantly inhibited tumor growth compared with mice treated with saline control. In addition, tumor growth suppression in IMAB362-treated mice resulted in an extended median survival time (Figures 24 and 25). The effect of IMAB362 correlated with the duration of treatment. Initiating IMAB362 treatment at an early time point had an enhanced effect on tumor growth inhibition compared with initiating treatment later to examine the effect on established tumors. Furthermore, the antitumor effect of IMAB362 depended 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 than that of 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 the mouse 6Suit2-LVT cells were intravenously injected and treated with 200 μg of IMAB362, an isotype control antibody (IMAB027), or PBS, as shown in Table 20. After 35 days, the first mouse (isotype control group) died. Consequently, all mice were sacrificed on day 42, and livers were collected 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 the percentage of human DNA revealed a significant reduction (P<0.05) in Suit2-LVT metastases detected in the lungs of mice treated with IMAB362 compared with both PBS and isotype control treatments (Figure 26A). To confirm these results, tissue sections of lung samples were prepared and stained with an MHC-I antibody. The surface area of ​​positively stained cells in the lung sections was calculated using the ImageJ program. Significant inhibition (P<0.05) was observed for IMAB362 treatment compared with PBS treatment, confirming the results obtained with Q-PCR. For the isotype control antibody, however, the difference was not significant (Figure 26B). This discrepancy is likely due to differences in tissue processing: IHC processing of tissue sections provides insight into only a very small section of the lung compared with Q-PCR analysis, and genomic DNA is extracted from half of the tissue for Q-PCR analysis.

[0338] In addition to tissue processing, it is possible that the results indicate an unexpected inhibitory effect of the isotype control antibody targeting CLDN6. To explore this option, Suit2-LVT cells were analyzed for CLDN6 expression and IMAB027 binding by FACS. Addition of 200 μg / ml of IMAB362 to Suit2-LVT cells confirmed strong binding to the cells, whereas addition of 200 μg / ml of IMAB027 resulted in weak binding of the antibody to these target cells, indicating that CLDN6 is indeed weakly expressed in these cells. These results suggest that at least two factors (tissue processing and weak IMAB027 inhibition) contributed to the discrepancy observed with 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 mice. The first experiment was performed by comparing IMAB362 treatment with PBS-treated mice. One mouse in each group died immediately after cell injection. 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 by Q-PCR experiment (Figure 27). The results show that IMAB362 inhibits the growth of metastases in lung tissue.

[0340] A second experiment, involving 11 mice per group, was performed 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 by Q-PCR, and IMAB362 again reduced metastatic growth. One mouse in the IMAB362 group was identified as an outlier, and excluding this outlier resulted in a near-significant inhibition (P = 0.0588). These data were confirmed by IHC surface analysis as described for the Suit2-LVT metastasis experiment. Again, the same outlier could be identified, and after excluding this value in a t-test, IMAB362 inhibition again reached borderline significance (P = 0.0691), and the outlier was 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 analog. Like 5-fluorouracil (5-FU) and other analogs of pyrimidines, the triphosphate analog of gemcitabine replaces one of the building blocks of nucleic acid during DNA replication. This process halts tumor growth and leads 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, which 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 different sensitivities 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 Patu8988S cell proliferation. DANG and BxPC3-LVT cells are highly sensitive to gemcitabine but not to oxaliplatin. MiaPaCa-2-LVT cells are most sensitive to oxaliplatin but are less sensitive to gemcitabine treatment (Figures 28, 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 elevated CLDN18.2 mRNA and protein levels in untreated and chemotherapy-pretreated cells, as shown by RT-PCR (Figure 30) and Western blot (Figure 31) analysis. Consequently, the amount of IMAB362-targetable CLDN18.2 protein 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 resting 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 prevent all cell cycle progression in any phase of the cell cycle. For example, certain chemotherapeutic agents can block progression in either the G2 or M phase, or both the G2 and M phases (G2 / M).

[0349] Gemcitabine treatment of DANG or Patu8988S cells results in cell cycle arrest at S phase (Figures 33 and 34). Patu8988S cells cultured with Gem were analyzed. Gemcitabine treatment not only results in cell cycle arrest, but also alters CLDN18.2 expression (Figure 34B). The change in CLDN18.2 density after gemcitabine treatment is even greater when proliferating cells in S phase are compared with 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 observed in the S phase cell population.

[0350] This variation in tumor cell phenotype significantly impacts the biological efficacy of therapeutic antibodies. ADCC and CDC are dose-related, and therefore increasing 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 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. 8 × 10 5Kato III cells were cultured for 96 hours without medium changes or for 72 hours followed by 24 hours in standard medium to release the cells from cell cycle arrest in 6-well tissue culture plates at 37°C and 5% CO. Cells were harvested with EDTA / trypsin, washed, and analyzed.

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

[0353] The cell cycle stage was determined based on the measurement of the DNA content of the cells. This allows for the identification of cells in the G1, S, or G2 phase of the cell cycle. DNA replication occurs during the S phase, while cells proliferate and prepare for mitosis during the G2 phase. 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 using a BD FACS Canto II (BD Biosciences) and FlowJo (Tree Star) software.

[0354] The columns in Figures 35a and 35b show the percentage of cells in the G1, S, or G2 phase of the cell cycle, respectively. Kato III cells cultured in medium exhibit cell cycle arrest primarily in the G1 phase. Cells treated with 5-FU are primarily blocked in the S phase. Kato III cells treated with oxaliplatin exhibit enrichment of cells primarily in the G1 and G2 phases. As seen in Figure 35c, cell cycle arrest in the S or G2 phase results in the stabilization or upregulation of CLDN18.2. As soon as cells are released from either stage of the cell cycle (Figure 35b), CLDN18.2 expression on 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 resulted in dose-dependent inhibition of cell proliferation and cell cycle arrest at S / G2 phase (Figure 36). Treatment of cells with docetaxel resulted in 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) To investigate the effect of gemcitabine (Gem) or gemcitabine plus oxaliplatin (GemOx) on IMAB362-mediated ADCC, a series of experiments was performed on the pancreatic cancer cell lines Patu8988S and DANG, which constitutively express CLDN18.2. The dose-response curves for IMAB362-mediated cytolysis of pretreated cells were compared with those of the culture medium.

[0357] The dose-response curve of DANG cells pretreated with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) for 2 days is shifted upward and leftward compared to untreated target cells (Figure 37A). Treatment of tumor cells with Gem or GemOx results in upregulation of CLDN18.2 and higher susceptibility to IMAB362-mediated ADCC. We observed 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 into X-Vivo medium. Kato III cells, which endogenously express CLDN18.2 and are of gastric origin, were used as target cells in this setting. The target cells stably expressed Lucifer Yellow, a luciferase that is only oxidized by viable cells. Purified anti-CLDN18.2 antibody IMAB362 was added at various concentrations, and an unrelated 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, which is a measure of the amount of viable cells remaining after IMAB362 induces cytotoxicity. IMAB362-induced ADCC was quantified in Kato III cells pretreated with irinotecan (1000 ng / ml), docetaxel (5 ng / ml), or cisplatin (2000 ng / ml) for 3 days compared with 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 in cells pretreated with irinotecan, docetaxel, or cisplatin was increased 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] Medium-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 an increase in maximal 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 cytolysis 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] Nude mice bearing BxPC3-LVT or MiaPaCa-2-LVT tumors treated with IMAB362 showed significant tumor growth delay compared with control mice treated with saline. Chemotherapy with gemcitabine up to 100 mg / kg without additional IMAB362 treatment did not show significant therapeutic effects on BxPC3-LVT or MiaPaCa-2-LVT xenografts. In contrast, combined treatment with 50-100 mg / kg gemcitabine plus IMAB362 resulted in significantly increased tumor growth inhibition and prolonged survival of tumor-bearing mice compared with mice treated with chemotherapy alone (Figures 41, 42, and 43). These findings indicate the existence of a synergistic therapeutic effect of the combination of gemcitabine and IMAB362 immunotherapy.

[0366] When a high dose of gemcitabine, 2 × 150 mg / kg per week, was used, established MiaPaCa-2-LVT xenograft tumors showed potent tumor growth inhibition independent of IMAB362 treatment (Fig. 44A). However, mice treated with the combination of IMAB362 and gemcitabine showed highly significant survival benefits compared with 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 for 14 days in RPMI medium supplemented with 300 U / ml IL-2 with or without 1 μM zoledronic acid (ZA). The percentages of Vγ9+Vδ2+ T cells within the CD3+ lymphocyte population and 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 portion 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 ZA / IL-2 treatment of PBMC in vitro results in ADCC-mediated upregulation of the FcγIII receptor CD16 in a significant proportion of γδ T cells.

[0369] Similar to NK cells, ZA / IL-2-expanded Vy9V52 T cells are positive for CD16, the FcγRIII receptor through which cell-bound antibodies trigger ADCC. A series of experiments was performed to assess whether Vy9V52 T cells could induce potent ADCC 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, cells were harvested and added to NUGC-4 cells expressing CLDN18.2, along with increasing concentrations (0.26 ng / ml to 200 μg / ml) of IMAB362. Specific killing was measured in a luciferase assay. ADCC assays were performed on 27 donors grown in 300 U / ml IL-2 with or without ZA, using NUGC-4 as target cells. EC values ​​calculated from dose-response curves for each donor were used. 50 The values ​​and the maximum specific killing rate at a dose of 200 μg / ml IMAB362 were evaluated in scatter plots.

[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 cells are cultured without ZA, ADCC activity is reduced for most donors. In these cultures, residual ADCC activity is NK cell-dependent. By testing over 20 donors, ADCC assays demonstrated that ZA / IL-2 treatment of PBMCs significantly increased EC2 activity compared to PBMCs cultured with IL-2 alone. 50 and improves maximum specific mortality.

[0372] [Example 10] Efficacy of IMAB362 in combination with gemcitabine in a mouse metastatic 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 200 μg of IMAB362 twice weekly or with PBS (iv / ip) plus a 100 mg / kg dose of gemcitabine ip once weekly for 4 weeks. Treatment with IMAB362 or PBS was maintained until the mice were sacrificed 70 days after tumor cell injection. Analysis of xenograft tumor burden in the lungs was performed by QPCR of human DNA in lung preparations and by optical analysis of immunohistological staining with an anti-human MHC-I antibody (clone EPR1394Y). The results show that mice treated with IMAB362 plus gemcitabine had significantly lower amounts of human DNA in their lungs (Figure 45A) and that the surface of lung sections stained for human MHC-I complexes was significantly smaller than that in the lungs of mice treated with an irrelevant antibody plus gemcitabine (Figure 45B). Both methods revealed a lower tumor burden of Patu8988s xenografts in the lungs of mice treated with IMAB362 plus gemcitabine, indicating that the combination with IMAB362 significantly outperforms gemcitabine monotherapy.

Claims

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 of claim 1, wherein CLDN18.2 is expressed on the cell surface of cancer cells.

3. The method of claim 1 or 2, wherein the agent that stabilizes or increases the 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 in one or more phases of the cell cycle other than the G1 phase, more preferably in the G2 and / or S phase.

4. The method of any one of claims 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 claims 1 to 4, wherein the agent that stabilizes or increases the expression of CLDN18.2 comprises an agent selected from the group consisting of gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, a prodrug thereof, a salt thereof, and a combination thereof.

6. The method of any one of claims 1 to 5, wherein the agent that stabilizes or increases the expression of CLDN18.2 comprises an agent that induces immunogenic cell death.

7. 7. The method of claim 6, wherein the agent that induces immunogenic cell death comprises oxaliplatin.

8. 8. The method of any one of claims 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. 9. The method of any one of claims 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. 11. The method of claim 10, wherein the cancer is pancreatic cancer.

12. 12. The method of any one of claims 1 to 11, wherein the method further comprises administering an agent that stimulates γδ T cells, wherein the γδ T cells are preferably Vγ9Vδ2 T cells.

13. 13. The method of claim 12, wherein the agent that stimulates γδ T cells is a bisphosphonate.

14. 14. The method of claim 12 or 13, wherein the agent that stimulates γδ T cells is a nitrogen-containing bisphosphonate (aminobisphosphonate).

15. 15. The method of any one of claims 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. 16. The method of any one of claims 12 to 15, wherein the agent that stimulates γδ T cells is administered in combination with interleukin-2.

17. 17. The method of any one of claims 1 to 16, wherein the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.

2.

18. 18. The method of any one of claims 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 is selected from the group consisting of: (i) an antibody having 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 of any one of claims 1 to 18, wherein the antibody is selected from the group consisting of: (i) an antibody produced by and / or obtainable from the clone deposited under ACC 2810; (ii) an antibody that is a chimerized or humanized form of the antibody included in (i); (iii) an antibody that has the specificity of the antibody included in (i); and (iv) an antibody that contains an antigen-binding portion or antigen-binding site, in particular 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 the antibody having the ability to bind to CLDN18.2 at a concentration of 1000 mg / m ２ 20. The method of any one of claims 1 to 19, comprising administering at a dose of up to 100 mg / kg.

21. The method comprises administering the antibody having the ability to bind to CLDN18.2 at a concentration of 300 to 600 mg / m ２ 21. The method of any one of claims 1 to 20, comprising repeatedly administering at a dose of

22. 22. The method of any one of claims 1 to 21, wherein the cancer is CLDN18.2 positive.

23. 23. The method of any one of claims 1 to 22, wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO:

1.

24. 24. The method of any one of claims 1 to 9 and 11 to 23, wherein the pancreatic cancer comprises primary cancer, advanced cancer, or metastatic cancer, or a combination thereof, such as a combination of primary pancreatic cancer and metastatic cancer.

25. 25. The method of claim 24, wherein the metastatic cancer comprises metastasis to lymph nodes, ovaries, liver, or lungs, or a combination thereof.

26. 26. The method of any one of claims 1 to 9 and 11 to 25, wherein the pancreatic cancer comprises pancreatic ductal cancer.

27. 27. The method of any one of claims 1 to 9 and 11 to 26, wherein the pancreatic cancer comprises an adenocarcinoma or a carcinoma, or a combination thereof.

28. 28. The method of any one of claims 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. 29. The method of any one of claims 1-9 and 11-28, wherein the pancreatic cancer is partially or completely refractory to gemcitabine treatment, such as gemcitabine monotherapy.

30. 30. The method of any one of claims 1 to 9 and 11 to 29, wherein preventing pancreatic cancer comprises preventing recurrence of pancreatic cancer.

31. 31. The method of any one of claims 1 to 9 and 11 to 30, wherein the patient has undergone surgery for pancreatic cancer.

32. 32. The method of any one of claims 1 to 9 and 11 to 31, wherein the patient has a precancerous pancreatic lesion, in particular a precancerous pancreatic lesion comprising early malignant histological changes in the pancreatic duct.

33. A pharmaceutical 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. 34. The pharmaceutical preparation of claim 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. 35. The pharmaceutical preparation of claim 33 or 34, further comprising printed instructions for use of said preparation for the treatment or prevention of pancreatic cancer.

36. A pharmaceutical preparation comprising (i) an antibody capable of binding to CLDN18.2 and (ii) gemcitabine.

37. 37. The pharmaceutical preparation according to claim 36, for treating or preventing cancer, particularly pancreatic cancer.

38. 38. The pharmaceutical preparation of claim 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. 39. The pharmaceutical preparation of 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.

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