Combination therapy involving anti-pl20.2 antibodies for the treatment of cancer

Abstract

The present application provides a combination therapy effective for treating and / or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases, such as pancreatic cancer and metastases thereof.

Description

[0001] Pancreatic cancer is one of the deadliest cancers. Its mortality rate is close to 100% due to its tendency to metastasize early and its high resistance to radiation and chemotherapy. With 27,000 new cases diagnosed annually in North America and 68,000 in Europe, there is an urgent need to develop new treatment strategies to reduce the mortality rate of pancreatic cancer patients.

[0002] Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)) is a member of the Claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa transmembrane protein comprising four transmembrane domains with two small extracellular loops. CLDN18.2 expression is undetectable by RT-PCR in normal tissues (except the stomach). Immunohistochemistry with CLDN18.2-specific antibodies shows the stomach as the only positive tissue. CLDN18.2 is a highly selective gastric lineage antigen uniquely expressed in transiently differentiated gastric epithelial cells. CLDN18.2 is retained during malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells. Furthermore, this pan-tumor antigen is ectopically activated at significant levels in esophageal adenocarcinoma, pancreatic cancer, and lung adenocarcinoma.

[0003] The chimeric IgG1 antibody IMAB362 against CLDN18.2 has been 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 members of the cleavage protein family, including the closely related splice variant 1 of cleavage protein 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and binds to four independent and highly efficient mechanisms of action. Upon target binding, IMAB362 mediates cell killing by inducing ADCC and CDC on the tumor cell surface through target cross-linking, as well as inducing apoptosis and directly inhibiting proliferation. Therefore, IMAB362 effectively 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 dose range discovery studies, a 28-day repeated-dose toxicity study in cynomolgus monkeys, and a 3-month repeated-dose toxicity study in mice. Repeated doses of IMAB362 IV were well tolerated in mice (maximum treatment duration of weekly administration for 3 months, maximum dose level of 400 mg / kg) and cynomolgus monkeys (up to 100 mg / kg administered up to 5 times weekly). No signs of systemic or local toxicity were induced. Specifically, gastric toxicity was not observed in any toxicity studies. IMAB362 does not induce immune activation or cytokine release. No adverse effects were recorded in male or female reproductive organs. IMAB362 does not bind to tissues without a target. Biodistribution studies in mice suggest that the absence of gastric toxicity is most likely due to the tight compartmentalization of the compartmentalized sites in healthy gastric epithelial cells, which appears to severely impair the accessibility of the IMAB362 epitope.

[0005] IMAB362 is in early clinical testing. A Phase I clinical trial has already been conducted in humans. Three patients have each received one of five dose groups (33 mg / m²). 2 100mg / m 2 300mg / m 2 600mg / m 2 1000mg / m 2 IMAB362 was administered intravenously once and observed for 28 days. IMAB362 was well tolerated, with no relevant safety observations in patients. In one patient, all measured tumor markers showed a significant reduction within 4 weeks following treatment. In an ongoing IIa clinical study, IMAB362 was administered repeatedly.

[0006] Here we present data demonstrating that chemotherapy agents can stabilize or increase CLDN18.2 expression on the surface of pancreatic cancer cells, thereby generating enhanced druggability of CLDN18.2 through anti-CLDN18.2 antibodies such as IMAB362. Synergistic effects of anti-CLDN18.2 antibodies such as IMAB362 with specific chemotherapy regimens, particularly those used for pancreatic cancer treatment, were observed. Human cancer cells pretreated with chemotherapy are more susceptible to antibody-induced target-specific killing. In mouse tumor models, tumor controls with anti-CLDN18.2 antibodies plus chemotherapy were superior to tumor controls with anti-CLDN18.2 antibodies as a single agent. Invention Overview

[0007] This invention generally provides combination therapies for the effective treatment and / or prevention of diseases associated with cells expressing CLDN18.2, including cancers such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer and their metastases, particularly gastric cancer metastases such as ovarian Krukenberg tumor, peritoneal metastases, and lymph node metastases. Pancreatic cancer and its metastases are particularly preferred.

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

[0009] The reagents for stabilizing or increasing CLDN18.2 expression can be cytotoxic agents and / or cell inhibitors. In one embodiment, the reagents for stabilizing or increasing CLDN18.2 expression include reagents that induce cell cycle arrest or induce cell accumulation at one or more phases of the cell cycle, preferably at one or more phases of the cell cycle other than G1-phase, such as S-phase, G2-phase, or a combination thereof, or a combination of S-phase or G2-phase and G1-phase. The reagents for stabilizing or increasing CLDN18.2 expression can include reagents selected from: nucleoside analogs, platinum compounds, camptothecin analogs and taxanes, their prodrugs, their salts, and combinations thereof. The nucleoside analogs can be selected from gemcitabine, 5-fluorouracil, their prodrugs, and their salts. The platinum compounds can be selected from oxaliplatin, cisplatin, their prodrugs, and their salts. The camptothecin analogs can be selected from irinotecan, topotecan, their prodrugs, and their salts. The taxanes can be selected from paclitaxel, docetaxel, their prodrugs, and their salts. The reagents used to stabilize or increase CLDN18.2 expression may include those selected from gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, their prodrugs, their salts, and combinations thereof. The reagents may also include combinations of oxaliplatin and 5-fluorouracil or their prodrugs, cisplatin and 5-fluorouracil or their prodrugs, at least one taxane and oxaliplatin, at least one taxane and cisplatin, at least one taxane and 5-fluorouracil or their prodrugs, or at least one camptothecin analog and 5-fluorouracil or their prodrugs. The reagents may also include combinations of gemcitabine and oxaliplatin, gemcitabine and cisplatin, gemcitabine and carboplatin, or oxaliplatin, 5-fluorouracil or their prodrugs, and irinotecan. Therefore, the method of the present invention may include administering a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of oxaliplatin, 5-fluorouracil or a prodrug thereof, and irinotecan. In one embodiment, the method of the present invention includes administering leucovorin, 5-fluorouracil or a prodrug thereof, irinotecan, and oxaliplatin. The reagents stabilizing or increasing CLDN18.2 expression may include reagents that induce immunogenic cell death. The reagents that induce immunogenic cell death may include oxaliplatin.

[0010] In another aspect, the present invention provides 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. In one embodiment, the cancer is selected from 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 may be ovarian Klugember's 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 present invention further includes administering an agent that stimulates γδT cells. In one embodiment, the γδT cells are Vγ9Vδ2 T cells. In one embodiment, the agent that stimulates γδT cells is a bisphosphonate (ester), such as a nitrogen-containing bisphosphonate (ester) (aminobisphosphonate (ester)). In one embodiment, the agent that stimulates γδT cells is selected from zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, opapadronic acid, alendronic acid, incardronic acid, and their salts. In one embodiment, the agent that stimulates γδT cells is administered in combination with interleukin-2.

[0012] The method of the present invention may further include the administration of at least one other chemotherapeutic agent, which may be a cytotoxic agent.

[0013] The antibody capable of binding CLDN18.2 can bind to the natural epitope of CLDN18.2 present on the surface of living cells. In one embodiment, the antibody capable of binding CLDN18.2 binds to a first extracellular loop of CLDN18.2. In one embodiment, the antibody capable of binding CLDN18.2 mediates cell killing through one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment, the antibody capable of binding CLDN18.2 is a monoclonal antibody, a chimeric antibody, or a humanized antibody or a fragment of an antibody. In one embodiment, the antibody mediates cell killing when it binds to cellular CLDN18.2, particularly to cells expressing CLDN18.2 on their cell surface, wherein the cells are preferably cancer cells, such as cancer cells as described herein. In one embodiment, the antibody capable of binding CLDN18.2 is an antibody selected from: (i) antibodies produced by clones deposited with the following accession numbers and / or antibodies obtainable from clones deposited with the following accession numbers: DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810, (ii) is an antibody in a chimeric or humanized form of the antibody described in (i), (iii) is an antibody having the specificity of the antibody described in (i), and (iv) comprises the antigen-binding portion or antigen-binding site of the antibody described in (i), particularly the variable region of the antibody described in (i), and preferably an antibody having the specificity of the antibody described in (i). In one embodiment, the antibody binds to a therapeutic agent, such as a toxin, a radioisotope, a drug, or a cytotoxic agent.

[0014] In one embodiment, the method of the present invention includes using up to 1000 mg / m³ 2 The antibody capable of binding CLDN18.2 is administered at a dose specified in the present invention. In one embodiment, the method of the present invention comprises administering the antibody at 300 mg / m². 2 Up to 600mg / m 2 The dosage of the antibody that can repeatedly bind to CLDN18.2 was administered.

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

[0016] In one embodiment, the cancer described herein is CLDN18.2 positive. In one embodiment, the cancer cells of the cancer described herein are CLDN18.2 positive. In one embodiment, the cancer cells of the cancer 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 combinations thereof, such as a combination of primary pancreatic cancer and metastatic cancer. In one embodiment, the method of the present invention is used to simultaneously treat primary cancer and metastatic cancer, such as primary pancreatic cancer and metastatic pancreatic cancer. In one embodiment, the metastatic cancer includes metastases to lymph nodes, ovaries, liver, or lungs, or combinations thereof. In one embodiment, the pancreatic cancer includes cancer of the pancreatic duct. In one embodiment, the pancreatic cancer includes adenocarcinoma or carcinoma, or combinations thereof. In one embodiment, the pancreatic cancer includes ductal adenocarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma, or acinar cell carcinoma, or combinations thereof. In one embodiment, the pancreatic cancer is partially or completely untreatable with gemcitabine treatment, such as gemcitabine monotherapy. In one embodiment, prevention of pancreatic cancer includes prevention of recurrence of pancreatic cancer.

[0018] In one embodiment, according to the invention, the patient to be treated has undergone surgery for pancreatic cancer. In another embodiment, the patient has precancerous pancreatic lesions, particularly precancerous pancreatic lesions including those initiating malignant histological changes in the pancreatic ducts. In these embodiments, the method of the invention is preferably intended to prevent the formation of malignant pancreatic cancer.

[0019] In another aspect, the present invention provides a pharmaceutical preparation for treating or preventing pancreatic cancer, comprising (i) an antibody capable of binding to CLDN18.2; and (ii) a reagent stabilizing or increasing CLDN18.2 expression. The pharmaceutical preparation of the present invention may also comprise a reagent stimulating γδT cells. The antibody capable of binding to CLDN18.2 and the reagent stabilizing or increasing CLDN18.2 expression, and optionally, the reagent stimulating γδT cells, may be present in the pharmaceutical preparation in a mixed or separate manner. The pharmaceutical preparation may be present in the form of a kit comprising a first container and a second container, and optionally, a container comprising a reagent stimulating γδT cells, the first container comprising the antibody capable of binding to CLDN18.2, and the second container comprising the reagent stabilizing or increasing CLDN18.2 expression. The pharmaceutical preparation may also include printed instructions for using the preparation for treating or preventing pancreatic cancer, particularly in the methods of the present invention. Different embodiments of the pharmaceutical preparation, particularly the antibody capable of binding to CLDN18.2, the reagent stabilizing or increasing CLDN18.2 expression, and the reagent stimulating γδT cells, are as described above with respect to the methods of the present invention.

[0020] In a particular aspect, the present invention provides a pharmaceutical formulation comprising (i) an antibody capable of binding to CLDN18.2, and (ii) gemcitabine. The pharmaceutical formulation of the present invention may also comprise an agent for stimulating γδT cells. The antibody capable of binding to CLDN18.2 and gemcitabine, and optionally, the agent for stimulating γδT cells, may be present in the pharmaceutical formulation in a mixed or separate manner. The pharmaceutical formulation may be used to treat or prevent cancers such as pancreatic cancer. The pharmaceutical formulation may be present in the form of a kit comprising a first container and a second container, and optionally, a container comprising the agent for stimulating γδT cells, the first container comprising the antibody capable of binding to CLDN18.2, and the second container comprising gemcitabine. The pharmaceutical formulation may also include printed instructions for use in treating or preventing cancers such as pancreatic cancer, particularly in the methods of the present invention. Different embodiments of the pharmaceutical formulation, particularly the antibody capable of binding to CLDN18.2, the agent stabilizing or increasing CLDN18.2 expression, and the agent for stimulating γδT cells, are as described above with respect to the methods of the present invention.

[0021] The present invention also provides reagents such as antibodies capable of binding CLDN18.2 and / or reagents described herein that stabilize or increase CLDN18.2 expression for use in the methods described herein. For example, the present invention also provides antibodies capable of binding CLDN18.2 that are co-administered with reagents such as gemcitabine that stabilize or increase CLDN18.2 expression, and optionally, reagents that stimulate γδT cells.

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

[0023] Figure 1 A lentiviral vector for transducing the human CLDN18.2 pancreatic cancer cell line was cloned downstream of the EF1α promoter. The expression cassette was integrated between long terminal repeats (5' and 3'-LTRs), enabling viral mRNA packaging and reverse transcription. RSV: Rous sarcoma virus, enabling Tat-independent viral mRNA production. Amp: Ampicillin resistance gene. PGKp: Mefenamic acid promoter. WPRE: Marmot posttranscriptional regulatory element; enhances transgene expression. LTR: Long terminal repeat, enabling viral packaging. SV40A terminates transcription and polyadenylates mRNA. pUC: Bacterial vector backbone. Bla: Ampicillin promoter.

[0024] Figure 2 Analysis of pancreatic cell metastasis in mouse lungs. A schematic diagram of mouse lung anatomy after intravenous injection of pancreatic cancer cells.

[0025] Figure 3 CLDN18.2 expression in normal and cancerous pancreatic tissues. Normal pancreatic tissue (A) and pancreatic cancer tissue (B) fixed in formalin and embedded in paraffin (FFPE) were stained with monoclonal mouse 35-22A antibody (0.2 μg / ml). Hematoxylin counterstaining (2:00 min). Magnification 200x.

[0026] Figure 4 CLDN18.2 expression in normal and precancerous pancreatic tissues. Different precancerous structures: (A) normal and PanIN1; (B) PanIN2; (C) 43-14A staining of PanIN3. Magnification 200x.

[0027] Figure 5 Preliminary Study - Correlation between CLDN18.2 signal intensity and the number of positive tumor cells in primary pancreatic tumors analyzed. Each point represents a case of primary pancreatic cancer analyzed by staining FFPE sections with monoclonal mouse 35-22A antibody (0.2 μg / ml). Dashed lines indicate the 10% value.

[0028] Figure 6 Preliminary study - Expression of CLDN18.2 in primary and metastatic pancreatic tumor tissues. Mouse monoclonal 35-22A antibody was used to stain FFPE tissue sections (3 μm) from (A) primary adenocarcinoma tumors and (B) lymph node metastases. Hematoxylin (Mayers) counterstaining was performed.

[0029] Figure 7 Main study: The correlation between the CLDN18.2 signal intensity of primary pancreatic tumors and the number of positive tumor cells was analyzed. Each point represents a case of primary pancreatic ductal adenocarcinoma (solid circle) or primary neuroendocrine tumor (hollow circle) analyzed by staining FFPE sections with monoclonal mouse 43-14A antibody (0.2 μg / ml).

[0030] Figure 8 The correlation between the CLDN18.2 signal intensity and the amount of positive tumor cells in pancreatic metastases was analyzed. Each point represents a case of pancreatic lymph node (solid circle) or liver (hollow circle) metastasis analyzed by staining FFPE sections with monoclonal mouse 43-14A antibody (0.2 μg / ml). Dashed lines indicate the 10% value.

[0031] Figure 9Expression of CLDN18.2 in primary and metastatic pancreatic tumor tissues. Primary adenocarcinoma (A, C, E) tumors and lymph node metastases (B, D, F) were stained with mouse monoclonal 43-14A antibody in FFPE tissue sections (3 μm). The sections were counterstained with Mayers hematoxylin.

[0032] Figure 10 Graphical analysis of CLDN18.2 expression in paired primary pancreatic tumors and metastatic lymph nodes.

[0033] Figure 11 Expression of .CLDN18.2 in paired primary and metastatic pancreatic tumors. FFPE tissue sections (3 μm) of (A) primary adenocarcinoma, (B) liver metastases, and (C) lymph node metastases were stained using mouse monoclonal 43-14A antibody. Sections were counterstained with Mayers hematoxylin. Magnification: 200x.

[0034] Figure 12 CLDN18.2 mRNA levels in pancreatic cancer cell lines. (A) Q-PCR expression analysis of different pancreatic CA cell lines, lentivirally transduced (LVT) cell lines (grey bars), gastric cancer cell line KATO-III (positive control), and breast cancer cell line SKBR-3 (negative control). CLDN18.2 transcripts were amplified using gene-specific primers. Relative expression levels above 1x10⁻⁶ will be displayed. 5 The endogenous cell line was scored as CLDN18.2 positive (shaded column). NTC: H2O control sample. Error bars: mean + SD. (BD) Passage-dependent CLDN18.2 expression analysis in Patu8988S (B), Panc05.04 (C), and the LVT cell line shown (D). Passage number is shown below each column.

[0035] Figure 13CLDN18.2 protein levels in cell lysates of pancreatic cancer cell lines. Proteins were isolated on 12.5% ​​SDS-PAGE. Western blot analysis was performed using a CLDN18 antibody (Zymed-MID) detecting the C-terminus of CLDN18.1 and CLDN18.2, and a loading control antibody detecting β-actin. Exposure times were 140 seconds (Pierce SuperSignal West Dura) and 20 seconds (Pierce SuperSignal West Pico), respectively. (A) CLDN18 was detected in pancreatic cell line lysates, a positive control (HEK293-p740), and a negative control cell lysate (SKBR-3). (B) CLDN18.2 expression was compared between uninduced parental cell lysates and lentivirally transduced (LVT) cell line lysates. Patu8988S and SKBR-3 were added as positive and negative controls, respectively.

[0036] Figure 14 Detection and localization of CLDN18 expression in pancreatic cancer cell lines. Pancreatic cancer cell lines grown on coverslips were stained. Antibody: 35-22A (20x magnification, exposure time shown below each image). DAPI was used to stain cell 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).

[0037] Figure 15 Detection and localization of CLDN18 expression in CLDN18.2-transduced pancreatic cancer cell lines. CLDN18 expression was detected in lentivirally transduced (LVT) pancreatic cancer cell lines using antibody 35-22A after fixation and permeabilization. Alexa488 or Alexa555, labeled as 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.

[0038] Figure 16IMAB362 binds to the cell surface of CLDN18.2-positive pancreatic CA cell lines (pharmacokinetics). IF analysis was performed on CLDN18.2-expressing pancreatic cancer cell lines (A, B, D, E), lentivirally transduced pancreatic cell lines (GL), and KATO-III gastric cancer control cells (C, F). Cells were stained with IMAB362 under natural conditions (DE) and compared with staining with 35-22A after fixation and permeabilization (AC). DAPI was used to stain cell nuclei. Exposure time is shown in each image. G: BxPC3-LVT; H: CAPAN1-LVT; I: DANG-LVT; J: MiaPaCa2-LVT; K: Patu8902-LVT; L: Suit2-LVT.

[0039] Figure 17 CLDN18.2 expression in xenograft tumors of different 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. Tissue staining with Zymed-MID antibody. Magnification: 10x (A,C,E,G,J,L) and 20x (B,D,F,H,K,M).

[0040] Figure 18 Engraftment examination of Suit-2 and MiaPaCa2 pancreatic cancer cell lines. Cells were injected into the tail vein of nude mice. Animals were sacrificed at 45 days (A), 52 days (B), and 59 days (C) after Suit-2 (AC) administration, or at 59 days (D), 66 days (E), and 73 days (F) after MiaPaCa2 (DF) administration. Lung tissues were prepared and stained with MHC class I antibody (anti-human MHC I, clone EPR1394Y) to detect human cells in mouse tissues.

[0041] Figure 19 Metastasis and implantation analysis of .Patu8988S. Using 1x10 6 Or 2x10 6 Patu8988S cells were injected iv into Nu / Nu mice and the lungs (A) and liver (B) of the mice were isolated at different time points shown below the x-axis. To calculate the percentage of human DNA present in each tissue preparation, a standard curve was prepared by mixing human DNA and mouse DNA and preparing a 7x 5-fold dilution that produced 100% (1) to 0.0064% (7) of human DNA.

[0042] Figure 20IHC analysis of Patu8988S cells transferred into mouse lung tissue. Mice injected with Patu8988S cells via tail vein were sacrificed at different time points (AD = 70 days, EH = 86 days), lung tissue was isolated, and stained with MHC-I (EPR1394Y) antibody (A, B, E, F) diluted 1:1000 or with anti-McCl3 18 (Zymed-Mid) (C, D, G, H) at 0.2 μg / ml. Magnification: A, C, E, G = 10x, B, D, F, H = 20x.

[0043] Figure 21 IMAB362-mediated apoptosis in gemcitabine-treated pancreatic tumor cells. Apoptosis was induced after 48 hours by crosslinking CLDN18.2 onto BxPC3–CLDN18. BxPC3–CLDN18 was cultured in medium or medium + 100 ng / ml gemcitabine. Apoptotic cytokines of monocytes underwent translocation. Similar translocation was obtained by incubating tumor cells with camptothecin.

[0044] Figure 22 The efficacy of .IMAB362-induced ADCC activity against pancreatic cancer cells. (A) ADCC performed on CLDN18.2-positive pancreatic cancer cell lines using PBMCs from different donors. (BF) ADCC performed on LVT pancreatic cell lines ectopically expressing CLDN18.2 and corresponding parental cells. (G) Dot plot.

[0045] Figure 23 Efficacy of IMAB362-induced CDC activity against pancreatic cancer cells. (A) CDC in four independent experiments using healthy human serum as complement source, IMAB362, and CLDN18.2-positive pancreatic CDOK1-p740 control cells. (B) CDC in CLDN18.2-positive (Patu8988S, DANG, Panc05.04) and CLDN18.2-negative (CAPAN1, Suit2, BxPC3, YAPC) pancreatic cell lines. (C) CDC in ectopically expressed LVT cell lines. (D) Dot plot showing the concentration of IMAB362 that induces the half-maximal lysis rate (EC50) in pancreatic cancer cell lines. (E) Maximum killing rate of pancreatic cancer cell lines obtained with IMAB362.

[0046] Figure 24Effect of IMAB362 treatment on subcutaneous MiaPaCa2-LVT xenografts. Each treatment group was inoculated with MiaPaCa2-LVT xenograft tumors by subcutaneous injection of 1e7 MiaPaCa2-LVT cells into the flanks of 15 female Hsd: thymic athymic naked-Foxn1nu mice. Treatment began on day 3 post-injection with 200 μg IMAB362 or as a control. Treatment continued bi-weekly with alternating intraperitoneal (ip) and intravenous (iv) injections until animal sacrifice. (A) Effect of IMAB362 treatment on tumor growth. Subcutaneous tumor size (mean + SEM) was measured twice weekly. (B) Kaplan-Meier survival plot. When the tumor volume reached 1400 mm²... 3 Mice were euthanized when the tumors became ulcerative.

[0047] Figure 25 Subcutaneous BxPC3-LVT xenograft treatment with IMAB362. Each treatment group was inoculated with BxPC3-LVT xenograft tumors by subcutaneous injection of 1e7 BxPC3-LVT cells into the flanks of 15 female Hsd: thymic athymic naked-Foxn1nu mice. Treatment began on day 3 post-injection with 200 μg IMAB362 or as a control. Treatment continued bi-weekly with alternating intraperitoneal (ip) and intravenous (iv) injections until animal sacrifice. (A) Effect of IMAB362 treatment on tumor growth. Subcutaneous tumor size was measured twice weekly (mean + SEM, *p<0.05). (B) Kaplan-Meier survival plot. When the tumor volume reached 1400 mm²... 3 Mice were euthanized when the tumors became ulcerative.

[0048] Figure 26 The effect of IMAB362 treatment on pancreatic metastatic growth of Suit2-LVT. (2x10) 6 Suit2-LVT tumor cells were intravenously injected into the tail vein of 12 female Hsd: thymic athymic naked-Foxn1nu mice / treatment group. On day 3 post-tumor cell injection, treatment began with 200 μg IMAB362, 200 μg allotype control, or an equal volume of PBS. Animals were sacrificed on day 42 post-transplantation. (A) qPCR analysis (mean of 2–4 reactions / samples) determined the percentage of human DNA present in mouse lung samples. (B) The percentage of human cells covering the surface of mouse lungs was determined by planimetry. Human cells were immunohistochemically stained in tissue sections with anti-human MHC class I antibody. *p<0.05 (Kruskal-Wallis test). Error bar: mean ± SD.

[0049] Figure 27Q-PCR and IHC analysis of lung metastases in .Patu8988S mice. Each mouse was injected with 2x10 6 Patu8988S cells. Animals were sacrificed after 65 days. Hollow circles: Mice were sacrificed after 63 days. (A) Mice were treated with 200 μg IMAB362 or saline control every half week. The amount of human DNA (ng) was detected by Q-PCR and calculated by Ct value. (B) The Q-PCR experiment as described in A was repeated. The percentage of human DNA present in mouse DNA was calculated by Ct value. (C) Mice were treated with IMAB362 and an allotype control antibody (rituximab). The percentage of human DNA present in mouse lungs was calculated by Ct value. For the IMAB362 group, one outlier (hollow triangle) was detected. Significance was shown by including or excluding the outlier. (D / E) is the same experiment as C. The surface of transfer 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 bar ± SD

[0050] Figure 28 Dose-response curves of gemcitabine. Pancreatic cancer cell lines showed very different sensitivities to gemcitabine. Cell lines were exposed to different concentrations of gemcitabine for 4 days, and the inhibition of proliferation was analyzed by viability assays.

[0051] Figure 29 Dose-response curves of oxaliplatin. Pancreatic cancer cell lines showed very different sensitivities to oxaliplatin. Cell lines were exposed to different concentrations of oxaliplatin for 4 days, and the inhibition of proliferation was analyzed by viability assay.

[0052] Figure 30 Effects of chemotherapeutic treatment on CLDN18.2 expression (RNA). RNA was from untreated DANG cells (2 days) pretreated with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + 0x 10 ng / ml) (A), or from Patu8988S cells pretreated with Gem (10 ng / ml) or GemOx (Gem 10 ng / ml + 0x 100 ng / ml) for 3 days (B). RNA was converted to cDNA and CLDN18.2 transcript levels were analyzed by quantitative real-time PCR. Results are shown in relative units compared to the transcript levels of the housekeeping gene HPRT.

[0053] Figure 31The effect of chemotherapy on CLDN18.2 protein levels in pancreatic cancer cells. Proteins from total cell lysates of untreated (med), Gem (1 ng / ml), or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) DANG or Patu8988S cells were analyzed to detect CLDN18.2 expression using Zymed C-terminal polyclonal antiserum. Actin was used to visualize proteins loaded in equal volumes.

[0054] Figure 32 FACS analysis of CLDN18 expression on cell surface. Solid histograms of CLDN18 expression in cultured (left) and Gem-treated (right) Patu8988S cells are shown in an overlay plot compared to Isotyp Co. Patu8988S cells were treated with gemcitabine (10 ng / ml) for 3 days.

[0055] Figure 33 Cell cycle analysis of DANG cells treated with gemcitabine (Gem; 2 ng / ml) or gemcitabine + oxaliplatin (GemOx; 1 ng / ml + 10 ng / ml) for two days or untreated. (A) Gemcitabine treatment resulted in cell cycle arrest in S-phase cells. Each column was divided to show the percentage of cells in G0 / G1, S, and G2 phases. (B) Western blot analysis showed upregulation of CLDN18 after Gem treatment.

[0056] Figure 34 Effects of gemcitabine on cell cycle (A) and CLDN18.2 expression (B, C) in Patu8988S cells. Patu8988S cells were untreated and treated with gemcitabine (10 ng / ml) for 2 days. (A) Each column region was divided to show the percentage of cells in G0 / G1, S, and G2 phases. CLDN18.2 density (x-axis) was plotted against cell number (y-axis). (B) CLDN18.2 expression was blotted between untreated (dashed line) and gemcitabine-treated (solid line). (C) CLDN18.2 expression in gemcitabine-treated Patu8988S cells in G0 / G1 phase (dashed line) was compared with that in cells in S phase (solid line).

[0057] Figure 35 Effects of chemotherapy on gastric cancer cells. Culture of Kato III cells for 96 hours induced cell cycle arrest at the G0 / G1 phase (a) and downregulation of CLDN18.2 (c). Cell-inhibiting compounds that caused cell cycle arrest at different stages of cell cycle arrest stabilized CLDN18.2 expression (c).

[0058] Figure 36 The effects of chemotherapy on gastric cancer cells. Cell-inhibiting compounds cause cell cycle arrest at different stages of the cell cycle (S / G2- phase (irinotecan) or G2- phase (docetaxel)). Each column region is divided to show the percentage of cells in the G0 / G1, S, and G2 phases.

[0059] Figure 37 Dose-response curves of IMAB362-mediated ADCC following DANG chemotherapy. (A) Dose-response curve of a representative donor 40 h after pretreatment with Gem or GemOx on DANG pancreatic cancer cells. (B) EC50 values ​​(mean) of IMAB362-mediated ADCC. P-value: unpaired t-test.

[0060] Figure 38 Effects of chemotherapy on gastric cancer cells. a: Cells treated with irinotecan, docetaxel, or cisplatin showed lower levels of viable cells compared to target cells cultured in medium. b: CLDN18.2 expression was increased in cells treated with irinotecan, docetaxel, or cisplatin compared to cells cultured in medium. c / d: Cell treatment with irinotecan, docetaxel, or cisplatin increased the efficacy of IMAB362-induced ADCC.

[0061] Figure 39 Effects of chemotherapy agents on CDC mediated by IMAB362 in MiaPaCa2-LVT cells. The figure shows two independently measured dose-response curves. MiaPaCa2-LVT cells were cultured for 70 h in medium, Gem (10 ng / ml), or GemOx (10 ng / ml Gem + 100 ng / ml Ox).

[0062] Figure 40 The effect of chemotherapy on IMAB362-induced CDC.

[0063] Figure 41Effects of IMAB362 in combination with Gem or GemOx on BxPC3-LVT xenografts. In each treatment group, BxPC3-LVT xenograft tumors were inoculated by subcutaneous injection of 8.5e6 BxPC3-LVT cells into the flank of 10 female Hsd: thymic naked-Foxn1nu mice. On day 3 post-injection, chemotherapy (50 mg / kg gemcitabine ip, 50 mg / kg gemcitabine plus 5 mg / kg oxaliplatin ip, respectively) was initiated and continued weekly for six weeks. 24 hours after chemotherapy, 800 μg IMAB362 or control was administered intravenously via the tail vein. IMAB362 treatment continued weekly until mouse sacrifice. (A) Growth curves of subcutaneous BxPC3-LVT xenografts. Subcutaneous tumor size (mean + SEM) was measured twice weekly. (B) Kaplan-Meier survival plot. When the tumor volume reached 1400 mm²... 3 Mice were euthanized when the tumors became ulcerative.

[0064] Figure 42 Enhanced antitumor efficacy was achieved by combining gemcitabine with IMAB362. Each treatment group was inoculated with BxPC3-LVT xenograft tumors by subcutaneous injection of 8.5e6 BxPC3-LVT cells into the flanks of 10 female Hsd:athymic naked-Foxn1nu mice. On day 3 post-injection, chemotherapy (100 mg / kg gemcitabine ip, or 100 mg / kg gemcitabine plus 5 mg / kg oxaliplatin ip) was initiated and continued weekly for six weeks. 24 hours after chemotherapy, 200 μg (half dose) or 400 μg (full dose) of IMAB362 was administered intravenously into the tail vein. IMAB362 treatment continued bi-weekly with alternating ip and iv injections until mouse sacrifice. (A) Growth curves of subcutaneous BxPC3-LVT xenografts. Subcutaneous tumor size (mean + SEM) was measured twice weekly. (B) Kaplan-Meier survival plot. When the tumor volume reaches 1400 mm 3 Mice were euthanized when the tumors became ulcerative.

[0065] Figure 43Effects of IMAB362 in combination with gemcitabine on MiaPaCa2-LVT xenografts. Each treatment group was inoculated with MiaPaCa2-LVT xenograft tumors by subcutaneous injection of 5e6 MiaPaCa2-LVT cells into the flank of 10 female Hsd:athymic naked-Foxn1nu mice. Treatment was initiated on day 4 post-injection with chemotherapy (50 mg / kg gemcitabine ip) and continued weekly for six weeks. 24 hours after chemotherapy, 200 μg IMAB362 or control was administered intravenously via the tail vein. IMAB362 treatment was continued bi-weekly with alternating ip and iv injections until mouse sacrifice. (A) Growth of the subcutaneous xenograft. Tumor size (mean + SEM) was measured twice weekly. (B) Kaplan-Meier survival plot. When the tumor volume reached 1400 mm²... 3 Mice were euthanized when the tumors became ulcerative.

[0066] Figure 44 The effect of IMAB362 treatment in combination with gemcitabine on established MiaPaCa2-LVT xenograft tumors. This was achieved by subcutaneous injection of 1e7 MiaPaCa2-LVT cells into female Hsd: thymic naked-Foxn1 cells. nu MiaPaCa2-LVT xenograft tumors were inoculated into the flanks of mice. Nine days after subcutaneous tumor inoculation, tumor-bearing mice were rearranged into homogeneous treatment groups of eight animals each and treatment began. Mice were treated with 150 mg / kg gemcitabine intraperitoneally (IP) every two weeks for four weeks. Twenty-four hours after gemcitabine injection, 200 μg IMAB362 or a control was administered intravenously into the tail vein. Treatment with 200 μg IMAB362 continued every two weeks with alternating IP and IV injections until sacrifice. (A) Subcutaneous tumor size was measured twice weekly (mean + SEM; ** = p < 0.01). (B) Kaplan-Meier survival curves. When the tumor volume reached 1400 mm²... 3 Mice were euthanized when the tumor became ulcerative (Mantel-Cox test; ** = p < 0.01).

[0067] Figure 45 The effect of IMAB362 in combination with gemcitabine on lung metastases in the Patu8988S xenograft model. (2x10) 6 Patu8988S tumor cells were intravenously injected into 12 female Hsd: athymic naked-Foxn1 mice in various treatment groups. nuIn the tail vein of mice. Two weeks after intravenous injection of tumor cells, mice were treated with 200 μg IMAB362 (iv / i, p.) every half week in combination with 100 mg / kg gemcitabine every half week for 4 weeks. The control group was treated with 200 μg of the allotype control antibody every half week in combination with 100 mg / kg gemcitabine. Animals were sacrificed on day 70 post-transplantation. (A) Quantitative PCR analysis of human DNA in lung samples from mice treated with IMAB362 and the allotype antibody (mean of 3 reactions / samples). Significant difference was found compared with the allotype control (P = 0.0035, Mann-Whitney test). (B) Determination of the percentage of stained human cells covering the surface of mouse lungs by computer-based analysis. Immunohistochemical staining of paraffin-embedded lung tissue with anti-human MHC-I antibody (clone EPR1394Y) (mean ± SEM; P = 0.0003, Mann-Whitney test). C and D: Examples of immunohistochemical staining of Patu8988s lung metastases in mice treated with anti-MHC-I antibody against IMAB362 + gemcitabine (C) or allotype antibody + gemcitabine (D). Invention Details

[0068] While the invention is described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0069] The elements of the invention will be described below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The different described embodiments and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described in this application should be considered as disclosed in the specification of this application.

[0070] Preferably, the terms used herein are defined as such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. Leuenberger, B. Nagel, and H. As described in Eds., Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

[0071] Unless otherwise indicated, the present invention will be practiced using conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as explained in the literature in the field (cf., Molecular Cloning: A Laboratory Manual, 2nd edition, J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0072] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to include the specified member, integer or group of members, integer or step, but not exclude any other member, integer or group of members, integer or step, although in some embodiments such other member, integer or group of members, integer or step may be excluded, i.e., the subject matter consists of the specified member, integer or group of members, integer or step. The terms “a,” “an,” and “the,” and similar indicators used in the context of describing the invention (especially in the context of the following claims) shall be interpreted to cover both the singular and plural, unless otherwise indicated herein or obviously contradicted by the context. The enumeration of ranges of numerical values ​​herein is intended only as a shorthand method for separately referring to each individual numerical value belonging to the range. Unless otherwise indicated herein, each individual numerical value is incorporated into the specification as if it were separately enumerated herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or obviously contradicted by the context. The use of any and all instances or exemplary language (such as "such as") provided herein is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention as otherwise claimed. The language in the specification should not be construed as indicating any unclaimed essential elements for carrying out the invention.

[0073] Several documents are referenced throughout the text of this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether mentioned above or below, is incorporated herein by reference in its entirety. Nothing herein shall be construed as allowing the invention to rely on prior art without authorization.

[0074] The term “CLDN18” refers to micrin 18 and includes any variants, including micrin 18 splice variant 1 (micrin 18.1 (CLDN18.1)) and micrin 18 splice variant 2 (micrin 18.2 (CLDN18.2)).

[0075] The term "CLDN18.2" preferably refers to human CLDN18.2, and in particular to proteins comprising, preferably, an amino acid sequence according to SEQ ID NO:1 or a variant thereof.

[0076] The term "CLDN18.1" preferably refers to human CLDN18.1, and in particular to proteins comprising, preferably, an amino acid sequence according to SEQ ID NO:2 or a variant of said amino acid sequence.

[0077] According to the present invention, the term "variant" specifically refers to mutants, splicing variants, conformations, isotypes, allele variants, species variants, and species homologs, especially those that occur naturally. Allele variants involve alterations to the normal sequence of a gene, the significance of which is often unclear. Whole-genome sequencing often identifies numerous allele variants of a given gene. A species homolog is a nucleic acid or amino acid sequence from a species that has a different origin than the species from which the given nucleic acid or amino acid sequence originates. The term "variant" should encompass any post-translational modification variants and conformational variants.

[0078] According to the present invention, the term "CLDN18.2 positive cancer" means cancer involving cancer cells expressing CLDN18.2, preferably expressing CLDN18.2 on the surface of said cancer cells.

[0079] "Cell surface" is used in its usual sense in the field, and therefore includes the outer part of the cell that is easily accessible to and bound by proteins and other molecules. For example, transmembrane proteins having one or more extracellular portions are considered to be expressed on the cell surface.

[0080] If CLDN18.2 is located on the surface of the cell and is readily accessible to and bound by a CLDN18.2-specific antibody added to the cell, then CLDN18.2 is expressed on the cell surface.

[0081] According to the invention, CLDN18.2 is substantially not expressed in cells if the expression level is low compared to its expression level in gastric cells or gastric tissue. Preferably, the expression level is 10% lower than the expression level in gastric cells or gastric tissue, more preferably 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% lower, or even lower. Preferably, CLDN18.2 is substantially not expressed in cells if the expression level exceeds the expression level in non-cancerous tissues other than the stomach by no more than 2-fold, preferably 1.5-fold, and preferably no more than the expression level in said non-cancerous tissues. Preferably, CLDN18.2 is substantially not expressed in cells if the expression level is below the detection limit and / or if the expression level is too low to allow binding of the CLDN18.2-specific antibody added to cells.

[0082] According to the present invention, CLDN18.2 is expressed in cells if the expression level exceeds, preferably by more than 2 times, 10 times, 100 times, 1000 times, or 10000 times, the expression level in non-cancerous tissues other than the stomach. Preferably, CLDN18.2 is expressed in cells if the expression level is above the detection limit and / or if the expression level is high enough to allow binding of a CLDN18.2-specific antibody added to the cells. Preferably, the CLDN18.2 expressed in cells is expressed on or exposed on the surface of the cells.

[0083] According to the present invention, the term "disease" refers to any pathological state, which includes cancer, particularly those forms of cancer described herein. Any reference herein to cancer or a specific form of cancer also includes its metastasis. In a preferred embodiment, the disease to be treated according to the present application involves cells expressing CLDN18.2.

[0084] "Diseases associated with cells expressing CLDN18.2" or similar expressions mean, according to the invention, 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 its state in a healthy tissue or organ. An increase is defined as 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 found only in diseased tissues, while expression is suppressed in the corresponding healthy tissues. For example, CLDN18.2 is expressed in pancreatic cancer tissues, but its expression is undetectable in non-cancerous pancreatic tissues. According to the invention, diseases associated with cells expressing CLDN18.2 include cancerous diseases. Furthermore, according to the invention, cancerous diseases are preferably those in which cancer cells express CLDN18.2.

[0085] As used herein, "cancer disease" or "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cell" means an abnormal cell that grows through rapid, uncontrolled cell proliferation and continues to grow after the stimulus that initiated new growth has ceased. Preferably, "cancer disease" is characterized by cells expressing CLDN18.2, and cancer cells expressing CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, and more preferably cancer cells of the cancers described herein.

[0086] According to the present invention, "cancer" refers to a malignant tumor originating from epithelial cells.

[0087] Adenocarcinoma is a cancer originating from glandular tissue. This tissue is also part of a larger category of tissue called epithelial tissue. Epithelial tissue includes the skin, glands, and a variety of other tissues lining the body's cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. Cells that do not need to be glandular to have secretory properties are classified as adenocarcinoma. This form of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated adenocarcinomas may not. By staining cells from a biopsy, pathologists determine whether a tumor is adenocarcinoma or some other type of cancer. Due to the ubiquitous nature of glands in the body, adenocarcinoma can occur in many tissues. Although individual glands may not secrete the same substances, as long as they have exocrine function, they are considered glandular, and thus their malignant form is named adenocarcinoma. Malignant adenocarcinomas invade other tissues and frequently metastasize (given sufficient time to do so).

[0088] The pancreas (an endoderm-derived organ) is a key 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 digestive enzymes and deliver them to the gastrointestinal tract. In response to signals from the stomach and duodenum, acinar cells, arranged in functional units along the ductal network, synthesize enzymes and secrete them into the ductal lumen. Within the acinar units, near the ducts, are acinar cells. The endocrine pancreas consists of four specialized endocrine cell types that aggregate into clusters called islets of Langerhans, regulating metabolism and glucose homeostasis by secreting hormones into the bloodstream.

[0089] Pancreatic cancer is a malignant growth derived from transforming cells that originate 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 of cancer-related death worldwide. Early-stage pancreatic cancer often presents no symptoms, while late-stage symptoms are typically nonspecific and variable. Therefore, pancreatic cancer is often not diagnosed until it reaches an advanced stage. Pancreatic cancer has a poor prognosis: the relative survival rates for year 1 and year 5 are 25% and 6%, respectively, for all combined stages. For locally advanced disease, year 5 survival is approximately 20%, while the median survival is approximately 10 months for locally advanced disease and 6 months for metastatic disease, which collectively represents over 80% of individuals.

[0090] Pancreatic cancer includes adenocarcinoma (tumors that show glandular structures) that arise from the exocrine components of the pancreas, and neuroendocrine carcinoma that arises from islet cells.

[0091] The most common form of pancreatic cancer, specifically ductal adenocarcinoma, is typically characterized by moderately to poorly differentiated glandular structures on microscopic examination. Pancreatic ductal adenocarcinoma (PDAC) usually occurs in the upper part of the pancreas, infiltrating surrounding tissues, including lymph nodes, spleen, and peritoneal cavity, and metastasizing to the liver and lungs. PDAC primarily displays duct-like structures and glandular patterns with varying degrees of cellular atypia and differentiation. Less common PDAC subtypes include glial, adenosquamous, or sarcomatoid structures. Regional variations in histological structure, tumor grade, and degree of differentiation are frequently observed within individual tumors. Even the smallest primary lesions often show perineural and lymphovascular invasion, indicating a tendency for early distant spread.

[0092] The second most common type of exocrine pancreatic cancer is mucinous adenocarcinoma. Mucinous adenocarcinoma produces large amounts of mucin, which produces a cystic appearance in imaging studies.

[0093] Pancreatic neuroendocrine tumors form in the hormone-producing cells of the pancreas (islet cells). Acinar cell vegetations arise from the acinar cells of the pancreas.

[0094] According to the present invention, the term "cancer" also includes cancer metastases of the primary tumor, such as primary pancreatic cancer. Therefore, if reference is made, pancreatic cancer, it also includes metastases of pancreatic cancer, such as metastases to the lungs, liver, and / or lymph nodes.

[0095] "Metastasis" refers to the spread of cancer cells from their initial site to another part of the body. The formation of metastasis is a highly complex process and depends on malignant cells detaching from the primary tumor, invading the extracellular matrix, penetrating the endothelial basement membrane to enter body cavities and blood vessels, and then infiltrating target organs via blood transport. Finally, the growth of the new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components can be retained and form metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis," which involves metastasis far from the primary tumor and the local lymph node system. In one embodiment, the term "metastasis" according to the invention refers to lymph node metastasis. A specific form of metastasis treatable using the therapy of the invention is metastasis originating from pancreatic cancer as the primary site. In a preferred embodiment, such pancreatic cancer metastasis is metastasis to the lymph nodes, to the lungs, and / or to the liver.

[0096] Klugember's tumor of the ovary is an uncommon metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. It is a metastatic signet ring cell adenocarcinoma of the ovary. The stomach is the primary site for the majority of cases (70%) of ovarian Klugember's tumors. Colon cancer, appendiceal cancer, and breast cancer (primarily invasive lobular carcinoma) are the next most common primary sites. Rare cases of ovarian Klugember's tumors originating from gallbladder cancer, bile duct cancer, pancreatic cancer, small bowel cancer, ampullary cancer, cervical cancer, and bladder / ureteral cancer have been reported.

[0097] Difficult-to-treat cancers are malignant tumors for which specific treatments are ineffective, and which initially do not respond to treatment or become unresponsive over time.

[0098] "Treatment" means the application of a compound or composition or combination of compounds or compositions to a subject in order to prevent or eliminate disease, including reducing the size or number of tumors in the subject; blocking or slowing the disease in the subject; inhibiting or slowing the formation of new disease in the subject; reducing the frequency or severity of symptoms and / or recurrences in subjects who currently have or have previously had disease; and / or prolonging, i.e., increasing the lifespan of the subject.

[0099] In particular, the term "treatment of a disease" includes the cure of a disease or its symptoms, the shortening of its duration, the reduction, prevention, slowing or inhibiting its progression or worsening, or the prevention or delay of its onset.

[0100] According to the invention, the term "patient" means a subject of treatment, particularly a sick subject, including humans, non-human primates, or other animals, especially mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a particular preferred embodiment, the patient is a human.

[0101] The term "agent that stabilizes or increases CLDN18.2 expression" refers to a reagent or combination of reagents that, when applied to cells, produces increased levels of CLDN18.2 RNA and / or protein in the cells, preferably increased levels of CLDN18.2 protein on the cell surface, compared to when the reagent or combination of reagents is not applied to the cells. Preferably, the cells are cancer cells, particularly cancer cells expressing CLDN18.2, and therefore targets for CLDN18.2 binding antibodies, such as cells of cancer types (particularly pancreatic cancer) described herein. The term "agent that stabilizes or increases CLDN18.2 expression" specifically refers to a reagent or combination of reagents that, when applied to cells, produces a higher density of CLDN18.2 on the cell surface, compared to when the reagent or combination of reagents is not applied to the cells. "Stabilizing CLDN18.2 expression" specifically includes situations where the reagent or combination of reagents prevents or reduces the decrease in CLDN18.2 expression, such as when CLDN18.2 expression would decrease without the reagent or combination of reagents, while providing the reagent or combination of reagents prevents or reduces the decrease in CLDN18.2 expression. "Increasing CLDN18.2 expression" specifically includes situations where the reagent or combination of reagents increases CLDN18.2 expression, such as when CLDN18.2 expression would decrease, remain substantially constant, or increase without the reagent or combination of reagents, while providing the reagent or combination of reagents increases CLDN18.2 expression to a higher level compared to the situation where CLDN18.2 expression would decrease, remain substantially constant, or increase without the reagent or combination of reagents.

[0102] According to the present invention, the term "agent that stabilizes or increases CLDN18.2 expression" includes chemotherapeutic agents or combinations of chemotherapeutic agents, such as cell inhibitors. Chemotherapeutic agents can affect cells in one of the following ways: (1) by damaging the DNA of cells so that they no longer replicate, (2) by inhibiting the synthesis of new DNA strands so that cells cannot replicate, and (3) by stopping the mitotic process of cells so that cells cannot divide into two cells.

[0103] According to the present invention, the term "reagent that stabilizes or increases CLDN18.2 expression" preferably refers to a reagent or combination of reagents, such as a cell-inhibiting compound or combination of cell-inhibiting compounds, which, when applied to cells (particularly cancer cells), cause cell arrest or accumulation at one or more phases of the cell cycle, preferably at one or more phases of the cell cycle other than G1- and G0- (preferably other than G1-), preferably at one or more G2- or S-phases of the cell cycle, such as G1 / G2-, S / G2-, G2-, or S-phases of the cell cycle. The term "cell arrest or accumulation at one or more phases of the cell cycle" means an increase in the percentage of cells at one or more phases of the cell cycle. Each cell undergoes a cycle comprising four phases for self-replication. The first phase, called G1, is when the cell prepares to replicate its chromosome. The second phase, called S, occurs during DNA synthesis and replication. The next phase is G2, during which RNA and proteins replicate. The final phase is M, which is the phase of actual cell division. In this final phase, replicated DNA and RNA separate and move to their respective ends of the cell, which effectively divides into two identical functional cells. Chemotherapy agents that disrupt DNA typically cause cells to accumulate in the G1 and / or G2 phases. Chemotherapy agents that block cell growth by interfering with DNA synthesis typically cause cells to accumulate in the S-phase; these chemotherapeutic agents include antimetabolites. Examples of such drugs are gemcitabine, 6-mercaptopurine, and 5-fluorouracil.

[0104] According to the present invention, the term "agent that stabilizes or increases CLDN18.2 expression" includes nucleoside analogs such as gemcitabine, 5-fluorouracil, or their prodrugs; platinum compounds such as oxaliplatin and cisplatin; taxanes such as paclitaxel and docetaxel; and camptothecin analogs such as irinotecan and topotecan; and combinations of drugs, such as combinations of drugs comprising one or more gemcitabine, oxaliplatin, and 5-fluorouracil, such as combinations of drugs comprising gemcitabine and oxaliplatin, gemcitabine and 5-fluorouracil, oxaliplatin and 5-fluorouracil, or other drug combinations described herein. According to the present invention, references to agents that stabilize or increase CLDN18.2 expression, such as references to nucleoside analogs, platinum compounds, camptothecin analogs, or taxanes, for example, references to gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, or paclitaxel, include any prodrug, such as an ester, salt, or derivative, as a conjugate of the reagent. Examples include conjugates of the reagent and a carrier substance, such as protein-bound paclitaxel, or albumin-bound paclitaxel. Preferably, the salt of the reagent is pharmaceutically acceptable.

[0105] In a preferred embodiment, "a reagent that stabilizes or increases CLDN18.2 expression" is or includes "a reagent that induces immunogenic cell death".

[0106] Under certain conditions, cancer cells can enter a lethal stress pathway to activate a tumor-specific immune response, which is linked to the emission of a spatiotemporally defined combination of signals decoded by the immune system (Zitvogel L et al. (2010) Cell 140:798–804). In such a protocol, cancer cells are triggered to emit signals sensed by innate immune effectors such as dendritic cells, thereby triggering a similar immune response involving CD8+ T cells and IFN-γ signaling, allowing tumor cell death to elicit a productive anticancer immune response. These signals include pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nucleoprotein HMGB1. In summary, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and gamma radiation can induce all the signals defining ICD, while, for example, cisplatin, which is insufficient to induce CRT translocation from the ER to the cell surface of death—a process requiring ER stress—needs to be supplemented by carotenoids (ER stress inducers).

[0107] According to the present invention, the term "agent for inducing immunogenic cell death" refers to a reagent or combination of reagents that, when administered to cells, particularly cancer cells, induces cells to enter a lethal stress pathway, ultimately resulting in a tumor-specific immune response. Specifically, when administered to cells, the agent for inducing immunogenic cell death induces the cells to emit a spatiotemporally defined combination of signals, including, in particular, pre-apoptotic exposure to the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nucleoprotein HMGB1.

[0108] According to the present invention, the term "agent that induces immunogenic cell death" includes anthracyclines and oxaliplatin.

[0109] The term "nucleoside analogue" refers to structural analogues of nucleosides, that is, a class that includes purine analogues and pyrimidine analogues.

[0110] The term "gemcitabine" refers to compounds containing the following nucleoside analogues:

[0111]

[0112] Specifically, the term refers to the compound 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythropentanose)pyrimidin-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]-1,2-dihydropyrimidin-2-one.

[0113] According to the present invention, gemcitabine is preferably administered intravenously. Preferably, gemcitabine is administered at a dose of 0.5 to 2 g / m². 2 Preferably 0.8 to 1.5 g / m 2 More preferably 1 to 1.2 g / m 2 Dosage range based on body surface area. For example, gemcitabine can be administered once weekly for 7 out of 8 weeks at a dose of 1000 mg / m², followed by once weekly for 3 out of 4 weeks.

[0114] The term "nucleoside analogue" includes fluoropyrimidine derivatives, such as fluorouracil and its prodrugs. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the trademarks Adrucil, Carac, Efudix, Efudex, and Fluoroplex) refers to compounds that are pyrimidine analogues of the following formula:

[0115]

[0116] Specifically, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0117] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is converted into a prodrug of 5-FU in tissues. Capecitabine, which can be administered orally, has the following formula:

[0118]

[0119] Specifically, the term refers to the compound [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

[0120] According to the present invention, the term "platinum compound" refers to a compound containing platinum in its structure, such as platinum complexes, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

[0121] The term "cisplatin" refers to the compound cis-dichlorodiammineplatinum (CDDP):

[0122]

[0123] The term "carboplatin" refers to the compound cis-(1,1-cyclobutanedihydroxy acid)diaminoplatinum(II):

[0124]

[0125] The term "oxaliplatin" refers to a platinum-based compound that coordinates with a diaminocyclohexane carrier ligand of the following formula:

[0126]

[0127] Specifically, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](oxalic acid-O,O')platinum(II). Oxaliplatin for injection is also marketed under the trade name Eloxatine.

[0128] Taxanes are a class of diterpenoid compounds, originally derived from natural sources such as plants in the genus *Taxus*, but some have been synthesized artificially. The main mechanism of action of taxane drugs is to disrupt microtubule function, thereby inhibiting cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0129] According to the present invention, the term "docetaxel" refers to a compound having the following formula:

[0130]

[0131] Specifically, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytaxane-11-ene-2α,4,13α-triyl-4-acetate 2-benzoate 13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropionate}.

[0132] According to the present invention, the term "paclitaxel" refers to a compound having the following formula:

[0133]

[0134] Specifically, 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-phenylpropionyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytaxyl-11-ene-2-ylbenzoate.

[0135] According to the present invention, the term "camptothecin analogue" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analogue" refers to a compound comprising the following structure:

[0136]

[0137] According to the present invention, preferred camptothecin analogues are inhibitors of DNase topoisomerase I (topo I). According to the present invention, preferred camptothecin analogues are irinotecan and topotecan.

[0138] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. In chemical terms, it is a semi-synthetic analog of the natural alkaloid camptothecin, having the following formula:

[0139]

[0140] Specifically, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H-pyrano[3',4':6,7]-indolazino[1,2-b]quinoline-9-yl-[1,4'-dipiperidine]-1'-carboxylic acid ester.

[0141] Topotecan is a topoisomerase inhibitor with the following formula:

[0142]

[0143] Specifically, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0144] Anthracyclines are a class of drugs commonly used in cancer chemotherapy; these drugs are also antibiotics. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetraphenyl-5,12-quinone structure, and typically require glycosylation at specific sites.

[0145] Anthracycline drugs preferably exert one or more of the following mechanisms of action: 1. By intercalating between base pairs in the DNA / RNA chain, they inhibit DNA and RNA synthesis, thereby preventing the replication of rapidly growing cancer cells. 2. They inhibit topoisomerase II, thereby preventing the relaxation of supercoiled DNA and thus blocking DNA transcription and replication. 3. They generate ion-mediated free oxygen radicals that damage DNA and cell membranes.

[0146] According to the present invention, the term "anthracycline drug" preferably refers to a reagent, more preferably to an anticancer agent that induces apoptosis, and more preferably to an anticancer agent that induces apoptosis by inhibiting DNA rebinding in topoisomerase II.

[0147] Preferably, according to the present invention, the term "anthracycline drugs" generally refers to a class of compounds having the following ring structures,

[0148]

[0149] This includes its analogues and derivatives, pharmaceutical salts, hydrates, esters, conjugates, and prodrugs.

[0150] Examples of anthracycline drugs and anthracycline drug analogues include, but are not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, rhodomycin, pirarubicin, pentorubicin, N-trifluoroacetyl-daunorubicin-14-valerate, aclacinomycin, morpholino-daunorubicin (morpholino-DOX), cyanomorpholino-daunorubicin (cyanomorpholino-DOX), 2-pyrrolino-daunorubicin (2-PDOX), 5-iminodaunorubicin, mitoxantrone, and aclacimycin A (arirubicin). Mitoxantrone is a member of the anthracene dione class of compounds, which are anthracene analogues lacking the sugar moiety of the anthracene ring but retaining a planar polycyclic aromatic ring structure that allows insertion into DNA.

[0151] According to the present invention, anthracycline drugs are particularly preferred to be compounds of the following formula:

[0152]

[0153] in

[0154] R1 is selected from H and OH, R2 is selected from H and OMe, R3 is selected from H and OH, and R4 is selected from H and OH.

[0155] 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.

[0156] Epirubicin is explicitly defined as an anthracycline drug in the context of this invention. Epirubicin is an anthracycline drug having the following formula:

[0157]

[0158] In the United States, it is marketed under the brand name Ellence, while elsewhere it is marketed under the brand names Pharmorubicin or Epirubicin Ebewe. Specifically, the term "epirarubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxane-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetraphenyl-5,12-dione]. In some chemotherapy regimens, epirubicin is preferred over the most common anthracycline drug, doxorubicin, because it appears to cause fewer side effects.

[0159] According to the present invention, the reagent that stabilizes or increases CLDN18.2 expression can be a chemotherapeutic agent, particularly a chemotherapeutic agent established for cancer treatment, or it can be part of a pharmaceutical combination, such as a pharmaceutical combination established for cancer treatment. Such a pharmaceutical combination can be a combination used in chemotherapy, or a combination used in the FOLFIRINOX chemotherapy regimen.

[0160] The combination of drugs used in FOLFIRINOX chemotherapy includes leucovorin, fluorouracil, irinotecan (e.g., irinotecan hydrochloride), and oxaliplatin. Oxaliplatin can be administered at 85 mg / m² body surface area; irinotecan can be administered at 180 mg / m² body surface area; leucovorin calcium can be administered at 400 mg / m² body surface area; fluorouracil can be administered at 400 mg / m² body surface area in pill form, followed by 5-fluorouracil at 2400 mg / m² body surface area in a continuous infusion, preferably over 46 hours, preferably every 2 weeks.

[0161] The term "folinic acid / leucovorin" refers to a compound that can be used in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Therefore, if this document refers to the administration of 5-fluorouracil or its prodrug, in one embodiment, said administration may include co-administration with folinic acid. Follic acid has the following formula:

[0162]

[0163] Specifically, 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}glutaric acid.

[0164] γδT cells (gmma delta T cells) represent a small subset of T cells with a unique T cell receptor (TCR) on their surface. Most T cells have a TCR composed of two glycoprotein chains called the α-TCR chain and the β-TCR chain. In contrast, in γδT cells, the TCR consists of one γ-chain and one δ-chain. This group of T cells is generally far less common than αβT cells. Human γδT cells play important roles in stress-surveillance responses such as infectious diseases and autoimmunity. Transformation-induced changes in tumors have also been shown to induce stress-surveillance responses mediated by γδT cells and enhance anti-tumor immunity. Importantly, following antigen binding, γδT cells activated at the site of injury provide cytokines (such as INFγ, TNFα) and / or chemokines that mediate the recruitment of other effector cells and exhibit direct effector functions such as cytotoxicity (via death receptors and cytolytic granule pathways) and ADCC.

[0165] Most γδ T cells in peripheral blood express the Vγ9Vδ2 T cell receptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates and are thought to play an early and important role in sensing “danger” caused by invading pathogens, as they proliferate rapidly in many acute infections and can outnumber all other lymphocytes within days, such as tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria.

[0166] γδT cells respond to small non-peptide phosphorylated antigens (phosphate antigens), such as pyrophosphate synthesized in bacteria and isopentenyl pyrophosphate (IPP) produced in mammalian cells via the mevalonate pathway. However, IPP production in normal cells is insufficient to activate γδT cells, while dysregulation of the mevalonate pathway in tumor cells leads to IPP accumulation and activation of γδT cells. IPP can also be therapeutically enhanced by aminobisphosphonates, which inhibit the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). Zoledronic acid (ZA, zoledronic acid, Zoomita) is one such inhibitor. TM Novartis indicates that such aminobisphosphonates have been clinically administered to patients for the treatment of osteoporosis and metastatic bone disease. Following in vitro treatment of PBMCs, ZA is specifically absorbed by monocytes. IPP accumulates in monocytes, which differentiate into antigen-presenting cells that stimulate the formation of γδT cells. In this setting, interleukin-2 (IL-2) is preferably added as a growth and survival factor for activated γδT cells. Finally, certain alkylated amines (however only at millimolecular concentrations) have been described for the in vitro activation of Vγ9Vδ2T cells.

[0167] According to the present invention, the term "reagent for stimulating γδT cells" refers to compounds that stimulate the formation of γδT cells (especially Vγ9Vδ2 T cells) in vitro and / or in vivo, particularly compounds that stimulate the formation of γδT cells in vitro and / or in vivo by inducing the activation and expansion of γδT cells. Preferably, the term refers to compounds that increase the production of isopentenyl pyrophosphate (IPP) by mammalian cells in vitro and / or in vivo, and more preferably compounds that increase the production of isopentenyl pyrophosphate (IPP) by mammalian cells in vitro and / or in vivo by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS).

[0168] A specific group of compounds that stimulate γδT cells is a bisphosphonate, especially nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

[0169] For example, suitable bisphosphonates for use in this invention may include one or more of the following compounds, including their analogues and derivatives, pharmaceutical salts, hydrates, esters, conjugates, and prodrugs:

[0170] [1-Hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, such as zoledronic acid salts;

[0171] (Dichlorophosphoryl-methyl)phosphonic acids, such as chlorophosphonates;

[0172] {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronic acid, such as ibandronate;

[0173] (3-Amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic acid, such as pamidronate;

[0174] (1-Hydroxy-1-phosphoryl-2-pyridin-3-yl-ethyl)phosphonic acid, risedronic acid, such as risedronate;

[0175] (1-Hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphatidylethyl)phosphonic acid, minophosphonic acid;

[0176] [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), opaldonic acid;

[0177] [4-Amino-1-hydroxy-1-(hydroxy-oxidized-phosphoryl)-butyl]phosphonic acid, alendronic acid, such as alendronate;

[0178] [(cycloheptaylamino)methylene]bis(phosphonic acid), incarphosphonic acid;

[0179] (1-Hydroxyethyl-1,1-diyl)bis(phosphonic acid), etidronic acid, such as etidronate; and

[0180] {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), teludromic acid.

[0181] According to the present invention, zoledronic acid (INN) or zoledronic acid salts (sold by Novarti under the trade names Zometa, Zomera, Aclasta, and Reclast) are particularly preferred bisphosphonates. Zometa is used to prevent fractures in patients with cancers such as multiple myeloma and prostate cancer, and for the treatment of osteoporosis. It can also be used to treat malignant hypercalcemia and may help treat pain from bone metastases.

[0182] In a particularly preferred embodiment, the reagent for stimulating γδT cells according to the invention is administered in combination with IL-2. Such a combination has been shown to be particularly effective in mediating the expansion and activation of γ9δ2T cells.

[0183] Interleukin-2 (IL-2) is a type of cytokine signaling molecule in the immune system. The protein attracts lymphocytes, and this is part of the body's natural response to microbial infection, distinguishing between exogenous (non-self) and self-infected components. IL-2 mediates its action by binding to IL-2 receptors expressed by lymphocytes.

[0184] According to the present invention, the IL-2 used can be any IL-2 that supports or enables the stimulation of γδT cells and can be derived from any species, preferably human. The IL-2 can be isolated, recombinant, or synthetic IL-2, and can be naturally occurring or modified IL-2.

[0185] The term "antigen" refers to reagents such as proteins or peptides that contain epitopes that are directed and / or to be directed in response to an immune response. In a preferred embodiment, the antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a cancer cell component that may be derived from the cytoplasm, cell surface, and cell nucleus, particularly those antigens that are preferably produced in large quantities intracellularly or those that serve as surface antigens on cancer cells.

[0186] In the context of this invention, the term "tumor-associated antigen" preferably refers to a protein that is specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage, and is expressed or aberrantly expressed in one or more tumors or cancerous tissues. In the context of this invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed in normal tissues or are expressed only sparingly in normal tissues.

[0187] The term "epitope" refers to an antigenic determinant in a molecule, that is, a portion of the molecule that is recognized by the immune system (e.g., by antibodies). For example, an epitope is a three-dimensional site separated on an antigen that is recognized by the immune system. Epitopes typically consist of chemically active surface groups or sugar side chains of a molecule (such as an amino acid) and usually have specific three-dimensional structural features and specific charge features. The difference between configurational epitopes and non-configurational epitopes is that, in the presence of denaturing solvents, binding to the former is lost while binding to the latter is not lost. Epitopes of proteins (such as CLDN18.2) preferably comprise continuous or discontinuous portions of the protein, and are preferably 5 to 100, more preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acid lengths. For example, epitopes can preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths.

[0188] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any molecule containing its antigen-binding moiety. The term "antibody" includes monoclonal antibodies and fragments or derivatives of antibodies, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies (such as scFv), and antigen-binding antibody fragments such as Fab and Fab' fragments, and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.

[0189] The antibodies described herein may be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies described herein may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutations).

[0190] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of human immunoglobulins. The antigen-binding site may include an intact variable domain fused to a constant domain or a complementarity-determining region (CDR) grafted only to an appropriate frame region within the variable domain. The antigen-binding site may be wild-type or modified by substitution of one or more amino acids, such as modifications to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., humanized mouse antibodies, which contain all six CDRs derived from mouse antibodies). Other forms have one or more CDRs that are altered relative to the original antibody.

[0191] The term "chimeric antibody" refers to antibodies in which a portion of each amino acid sequence of the heavy and light chains is homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to a corresponding sequence in another. Typically, the variable regions of the light and heavy chains mimic the variable regions of antibodies derived from one mammalian species, while the constant regions are homologous to sequences from antibodies derived from another mammalian species. A significant advantage of such a chimeric form is that the variable regions, combined with constant regions derived from currently known sources, can be conveniently derived using readily available B cells or hybridomas from non-human host organisms. While variable regions offer the advantage of ease of preparation and specificity unaffected by source, constant regions derived from humans are less likely to elicit an immune response in human subjects when the antibody is injected than constant regions derived from non-human sources. However, the definition is not limited to this specific instance.

[0192] The terms “antigen-binding portion” (or simply “binding portion”) or “antigen-binding fragment” (or simply “binding fragment”) of an antibody, or similar terms, refer to one or more fragments of an antibody that 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 within the term "antigen-binding portion" of antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) Fd fragments, consisting of VH and CH domains; (iv) Fv fragments, consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al. (1989) Nature 341:544-546), consisting of a VH domain; (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, optionally connected by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using a recombinant approach via a synthetic linker that allows them to form a single protein chain in which the VL and VH regions pair up to form a monovalent molecule (referred to 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 within the term "antigen-binding fragment" of antibody. Other examples are binding domain immunoglobulin fusion proteins 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 may be a heavy chain variable region or a light chain variable region. Domain-binding immunoglobulin fusion proteins are further disclosed in US2003 / 0118592 and US2003 / 0133939. These antibody fragments were obtained using conventional techniques known to those skilled in the art, and the fragments were screened for practicality in the same manner as intact antibodies.

[0193] The term "bispecific molecule" is intended to include any agent, such as a protein, peptide, or protein complex or peptide complex, having two different binding specificities. For example, a molecule may bind to (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell, 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 "heterogeneous specific molecule" is intended to include any agent, such as a protein, peptide, or protein complex or peptide complex, having more than two different binding specificities. For example, a molecule may bind to (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Therefore, the present invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules relating to CLDN18.2 and other targets, such as Fc receptors on effector cells. The term "bispecific antibody" also includes bispecific antibodies. Biantibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linkers used are too short to pair between the two domains on the same chain, thus forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger, P et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0194] Antibodies can be conjugated to therapeutic components or agents, such as cytotoxins, drugs (e.g., immunosuppressants), or radioisotopes. Cytotoxins or cytotoxic agents include any agent that is harmful to cells and, in particular, kills them. Examples include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraquinone, mitoxantrone, styracin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologs. Suitable therapeutic agents for forming antibody-drug conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine), and alkylating agents (e.g., nitrogen mustard, thiopental chlorambucil). The therapeutic agents include chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, photomycin and amiodarone (AMC), and antimitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In 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.

[0195] Antibodies can also be conjugated to radioactive isotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals.

[0196] The antibody conjugates of the present invention can be used to modify a given biological response, and the pharmaceutical portion should not be construed as limited to classical chemotherapeutic agents. For example, the pharmaceutical portion can be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, enzymatically active toxins or their active fragments, such as abrinogen, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”), granulocyte colony-stimulating factor (“G-CSF”), or other growth factors.

[0197] The technique of conjugating such therapeutic components to antibodies is well known; see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer". Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 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).

[0198] As used herein, if antibodies are obtained from a system by immunizing animals or by screening an immunoglobulin gene library, the antibodies are “derived” from a specific germline sequence, and the amino acid sequence of said selected antibody has at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identity with the amino acid sequence encoded by the germline immunoglobulin gene. Typically, antibodies derived from a specific germline sequence show a difference of no more than 10 amino acids from the amino acid sequence encoded by the germline immunoglobulin gene, more preferably no more than 5 amino acids, or even more preferably no more than 4, 3, 2, or 1 amino acid.

[0199] As used herein, the term "heteroantibody" refers to two or more antibodies, their derivatives, or antigen-binding regions linked together, at least two of which have different specificities. These different specificities include binding specificity to Fc receptors on effector cells and binding specificity to antigens or epitopes on target cells (such as tumor cells).

[0200] The antibodies described herein may be monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule of a single molecular composition. Monoclonal antibodies exhibit single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by fusion into an immortalized hybridoma cell, said hybridoma comprising B cells obtained from a non-human animal (such as a mouse).

[0201] The antibodies described herein may be recombinant antibodies. As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, generated, or isolated by recombinant methods, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomally derived relative to an immunoglobulin gene or a hybridoma prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfected tumors), (c) antibodies isolated from recombinant, combined antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other method involving splicing an immunoglobulin gene sequence into another DNA sequence.

[0202] The antibodies described in this article can be derived from different species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0203] The antibodies described herein include polyclonal and monoclonal antibodies, including 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 specifically an IgG1, κ, or IgG1,λ 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).

[0204] As used herein, the term “transfected tumor” includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS / O cells, HEK293 cells, HEK293T cells, plant cells, or fungi (including yeast cells).

[0205] As used herein, “heterogeneous antibody” is defined in relation to the transgenic organism that produces such an antibody. The term refers to an antibody having an amino acid sequence or a nucleic acid sequence encoding the corresponding amino acid sequence that is found in an organism not composed of transgenic organisms and is usually derived from a species other than transgenic organisms.

[0206] As used in this article, a "heterohybrid antibody" refers to an antibody containing light and heavy chains from different biological sources. For example, an antibody containing a human heavy chain associated with a mouse light chain is a heteroohybrid antibody.

[0207] This invention includes all antibodies and antibody derivatives as described herein, and for the purposes of this invention, they are covered within the term "antibody". The term "antibody derivative" refers to any modified form of antibody, such as a conjugate of the antibody with another reagent or antibody or antibody fragment.

[0208] The antibodies described herein are preferably isolated. As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody specifically binding to CLDN18.2 is substantially free of antibodies specifically binding to antigens other than CLDN18.2). However, isolated antibodies that specifically bind to epitopes, isotypes, or variants of human CLDN18.2 may be cross-reactive with other related antigens (e.g., from other species (e.g., CLDN18.2 species homologs)). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals. In one embodiment of the invention, the combination of "isolated" monoclonal antibodies involves antibodies with different specificities combined in a well-defined composition or mixture.

[0209] According to the present invention, the term "binding" preferably refers to specific binding.

[0210] According to the present invention, an antibody is capable of binding to a predetermined target if it has a significant affinity for the target and binds to the target in a standard assay. "Affinity" or "binding affinity" is typically measured by the equilibrium dissociation constant (K0). D Measurement. Preferably, the term "significant affinity" refers to the dissociation constant (K0) bound to the predetermined target. D ) is 10 -5 M or smaller, 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller.

[0211] If the antibody has no significant affinity for the target and does not bind significantly to the target in a standard assay, particularly if it binds undetectably to the target in a standard assay, then the antibody (substantially) cannot bind to the target. Preferably, the antibody binds undetectably to the target if present at a concentration of at most 2 μg / ml, preferably 10 μg / ml, more preferably 20 μg / ml, particularly 50 μg / ml or 100 μg / ml or higher. Preferably, if the antibody binds to the K of the target... D Compared to the antibody binding to the predetermined target that it can bind to, K D At least 10 times, 100 times, 10 3 - times, 10 4 - times, 10 5 - times or 10 6 If the antibody binds to a target at a concentration of - times the target's K, then the antibody does not have significant affinity for the target. For example, if the antibody binds to the K value of the target to which the antibody can bind, then the antibody does not have significant affinity for the target. D 10 -7 M, then the antibody does not bind to the target K with significant affinity. D At least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 - 2 M or 10 -1 M.

[0212] An antibody is specific to the predetermined target if it can bind to the target but not to other targets, i.e., it has no significant affinity for other targets and does not significantly bind to other targets in a standard assay. According to the invention, an antibody is specific to CLDN18.2 if it can bind to CLDN18.2 but (substantially) not to other targets. Preferably, the antibody is specific to CLDN18.2 if the affinity for such other targets and the affinity for binding to such other targets do not significantly exceed the affinity for or to CLDN18.2-unrelated proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-dense transmembrane proteins (such as MHC molecules or transferrin receptors or any other specified polypeptides). Preferably, if the antibody binds to the K of the target… D Compared to the nonspecific binding of antibodies to the target K D At least 10-fold, 100-fold, 10 3 - times, 10 4 - times, 10 5 - times or 10 6 If the antibody binds to a K-value that is not specific to its intended target, then the antibody is specific to that target. For example, if the antibody binds to the K-value of its non-specific target, then the antibody is specific to that target. D 10 -7 M then binds to K, which is a non-specific target of the antibody. D For at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 M.

[0213] Antibody-target binding can be determined experimentally 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, such as by equilibrium dialysis; by using a BIAcore 2000 instrument with a general procedure outlined by the manufacturer; by using a radioimmunoassay of a radiolabeled target antigen; or by another method known to the technician. Affinity data can be analyzed, for example, by the method of Scatchard et al. (Ann NYAcad. ScL, 51:660 (1949)). The affinity of a particular antibody-antigen interaction can be altered if measured under different conditions (e.g., salt concentration, pH). Therefore, affinity, as well as other antigen-binding parameters such as K, can be affected. D IC 50 The measurement is preferably performed using standard solutions of antibodies and antigens, as well as standard buffer solutions.

[0214] As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by genes in the heavy chain constant region.

[0215] As used in this article, "isotype switching" refers to the phenomenon where the antibody class or isotype changes from one Ig class to another Ig class.

[0216] As used herein, the term "naturally occurring" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0217] As used herein, the term "rearranged" refers to the configuration of a heavy-chain or light-chain immunoglobulin locus where, in a configuration encoding a substantially complete VH or VL domain, the V segment is located immediately adjacent to the DJ or J segment. Rearranged immunoglobulin (antibody) gene loci can be identified by comparing germline DNA; rearranged loci will have at least one recombinant heptamer / nonamer homologous element.

[0218] When the terms “unrearranged” or “phylogenetic configuration” are used in this article to refer to the V segment, it means a configuration in which the V segment has not been rearranged to be adjacent to the D or J segment.

[0219] According to the present invention, the antibody capable of binding CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, wherein the epitope is preferably located within the extracellular domain of CLDN18.2 (particularly the first extracellular domain), and preferably at amino acid sites 29 to 78 of CLDN18.2. In a particular embodiment, the antibody capable of binding CLDN18.2 is an antibody capable of binding the following epitopes: (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-ring 1, preferably SEQ ID NO: 8; (iii) an epitope located on CLDN18.2-ring 2, preferably SEQ ID NO: 10; (iv) an epitope located on CLDN18.2-ring D3, preferably SEQ ID NO: 11; (v) an epitope covering CLDN18.2-ring 1 and CLDN18.2-ring D3; or (vi) a non-glycosylated epitope located on CLDN18.2-ring D3, preferably SEQ ID NO: 9.

[0220] According to the present invention, antibodies capable of binding CLDN18.2 are preferably antibodies capable of binding CLDN18.2 but not CLDN18.1. Preferably, antibodies capable of binding CLDN18.2 are specific for CLDN18.2. Preferably, antibodies capable of binding CLDN18.2 are preferably antibodies capable of binding CLDN18.2 expressed on the cell surface. In a particularly preferred embodiment, the antibody capable of binding CLDN18.2 binds to the natural epitope of CLDN18.2 present on the surface of living cells. Preferably, the antibody capable of binding CLDN18.2 binds to one or more peptides selected from: SEQ ID NOs: 1, 3-11, 44, 46 and 48-50. Preferably, the antibody capable of binding CLDN18.2 is specific to the aforementioned proteins, peptides, or immunogenic fragments or derivatives thereof. Antibodies capable of binding CLDN18.2 can be obtained by a method comprising the following steps: immunizing an animal with a protein or peptide comprising an amino acid sequence selected from SEQ ID NO: 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 cancer types mentioned above, and, preferably, substantially does not bind to non-cancerous cells.

[0221] Preferably, the antibody capable of binding to CLDN18.2 binds to cells expressing CLDN18.2 to induce or mediate the killing of cells expressing CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, and particularly selected from tumorigenic gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancer cells, and gallbladder cancer cells. Preferably, the antibody induces or mediates cell killing by inducing one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of cell proliferation expressing CLDN18.2. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, and in a particular embodiment, the effector cells are selected from monocytes / mononuclear cells, NK cells, and PMNs. The inhibition of cell proliferation can be measured in vitro by determining cell proliferation using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) in an assay. BrdU is a synthetic nucleoside, an analogue of thymidine, that can be incorporated into newly synthesized DNA in replicating cells (during the S phase of the cell cycle), thereby replacing thymidine during DNA replication. The incorporation of this chemical agent, for example, using antibodies specific to BrdU, indicates cells actively replicating their DNA.

[0222] In a preferred embodiment, the antibody described herein can be characterized by one or more of the following properties:

[0223] a) Specificity for CLDN18.2;

[0224] b) The binding affinity with CLDN18.2 is about 100 nM or less, preferably about 5-10 nM or less, more preferably about 1-3 nM or less.

[0225] c) It can induce or mediate CDC on CLDN18.2 positive cells;

[0226] d) It can induce or mediate ADCC on CLDN18.2 positive cells;

[0227] e) It can inhibit the growth of CLDN18.2 positive cells;

[0228] f) It can induce apoptosis in CLDN18.2 positive cells.

[0229] In a particularly preferred embodiment, the antibody capable of binding CLDN18.2 is generated via a hybridoma deposited in DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) with the following name and accession number:

[0230] a.182-D1106-055, accession number DSM ACC2737, accessed on October 19, 2005.

[0231] b.182-D1106-056, accession number DSM ACC2738, accessed on October 19, 2005.

[0232] c.182-D1106-057, accession number DSM ACC2739, accessed on October 19, 2005.

[0233] d.182-D1106-058, accession number DSM ACC2740, accessed on October 19, 2005.

[0234] e.182-D1106-059, accession number DSM ACC2741, accessed on October 19, 2005.

[0235] f.182-D1106-062, accession number DSM ACC2742, accessed on October 19, 2005.

[0236] g.182-D1106-067, accession number DSM ACC2743, accessed on October 19, 2005.

[0237] h.182-D758-035, accession number DSM ACC2745, accessed on November 17, 2005.

[0238] i.182-D758-036, accession number DSM ACC2746, accessed on November 17, 2005.

[0239] j.182-D758-040, accession number DSM ACC2747, accessed on November 17, 2005.

[0240] k.182-D1106-061, accession number DSM ACC2748, accessed on November 17, 2005.

[0241] l.182-D1106-279, accession number DSM ACC2808, accessed on October 26, 2006.

[0242] m.182-D1106-294, accession number DSM ACC2809, accessed on October 26, 2006.

[0243] n.182-D1106-362, accession number DSM ACC2810, accessed on October 26, 2006.

[0244] According to the present invention, preferred antibodies are those produced by and obtained from the hybridoma described above; that is, 37G11 in the case of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in the case of 182-D1106-059, 43A11 in the case of 182-D1106-062, and 43A11 in the case of 182-D1106-062. The value is 61C2 in case 7, 26B5 in case 182-D758-035, 26D12 in case 182-D758-036, 28D10 in case 182-D758-040, 42E12 in case 182-D1106-061, 125E1 in case 182-D1106-279, 163E12 in case 182-D1106-294, and 175D10 in case 182-D1106-362; as well as its chimeric and anthropomorphic forms.

[0245] The table below shows preferred chimeric antibodies and their sequences.

[0246]

[0247]

[0248] In a preferred embodiment, the antibody, particularly the chimeric form of the antibody according to the invention, comprises an antibody containing a heavy chain constant region (CH), said heavy chain constant region (CH) comprising an amino acid sequence derived from the human heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO:13 or a fragment thereof. In a further preferred embodiment, the antibody, particularly the chimeric form of the antibody according to the invention, comprises an antibody containing a light chain constant region (CL), said light chain constant region (CL) comprising an amino acid sequence derived from the human light chain constant region, such as the amino acid sequence shown in SEQ ID NO:12 or a fragment thereof. In a particularly preferred embodiment, the antibody, particularly the chimeric form of the antibody according to the invention, comprises an antibody containing CH and a CL, said CH comprising an amino acid sequence derived from the human CH, such as the amino acid sequence shown in SEQ ID NO:13 or a fragment thereof, said CL comprising an amino acid sequence derived from the human CL, such as the amino acid sequence shown in SEQ ID NO:12 or a fragment thereof.

[0249] In one embodiment, the antibody capable of binding CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising κ, mouse variable light chain, human κ light chain constant region allotype Km(3), mouse heavy chain variable region, human IgG1 constant region, and allotype G1m(3).

[0250] In some preferred embodiments, the chimeric form of the antibody comprises an antibody containing a heavy chain and / or a light chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 16, 17, 18, 19 and fragments thereof, and the light chain comprises an amino acid sequence selected from SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28 and fragments thereof.

[0251] In some preferred embodiments, the chimeric form of the antibody comprises an antibody containing a combination of heavy and light chains selected from the following possible (i) to (ix):

[0252] (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:14 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:21 or a fragment thereof.

[0253] (ii) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:20 or a fragment thereof.

[0254] (iii) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:16 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:22 or a fragment thereof.

[0255] (iv) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:18 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:25 or a fragment thereof.

[0256] (v) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:17 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:24 or a fragment thereof.

[0257] (vi) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:23 or a fragment thereof.

[0258] (vii) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:26 or a fragment thereof.

[0259] (viii) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO:27 or a fragment thereof.

[0260] (ix) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 or a fragment thereof and the light chain comprises the amino acid sequence shown in SEQ ID NO:28 or a fragment thereof.

[0261] As used above, "fragment" or "fragment of amino acid sequence" refers to a portion of an antibody sequence, specifically a sequence of antibody sequences shortened at the N- and / or C-terminus, which, when replacing the antibody sequence in the antibody, preserves the binding of the antibody to CLDN18.2 and preferably preserves the antibody's function as described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage. Preferably, the fragment of amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues from the amino acid sequence. Fragments of amino acid sequences selected from SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 preferably involve a sequence in which 17, 18, 19, 20, 21, 22, or 23 amino acids have been removed from the N-terminus.

[0262] In a preferred embodiment, the antibody capable of binding CLDN18.2 includes a heavy chain variable region (VH) comprising an amino acid sequence and fragments thereof selected from SEQ ID NO:29, 30, 31, 32, 33, 34.

[0263] In a preferred embodiment, the antibody capable of binding CLDN18.2 includes a light chain variable region (VL) comprising an amino acid sequence and fragments thereof selected from SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43.

[0264] In some preferred embodiments, the antibody capable of binding CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possible (i) to (ix):

[0265] (i) The VH contains the amino acid sequence shown in SEQ ID NO:29 or a fragment thereof, and the VL contains the amino acid sequence shown in SEQ ID NO:36 or a fragment thereof.

[0266] (ii) The VH comprises the amino acid sequence shown in SEQ ID NO:30 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:35 or a fragment thereof.

[0267] (iii) The VH comprises the amino acid sequence shown in SEQ ID NO:31 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:37 or a fragment thereof.

[0268] (iv) The VH comprises the amino acid sequence shown in SEQ ID NO:33 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:40 or a fragment thereof.

[0269] (v) The VH comprises the amino acid sequence shown in SEQ ID NO:32 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:39 or a fragment thereof.

[0270] (vi) The VH comprises the amino acid sequence shown in SEQ ID NO:34 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:38 or a fragment thereof.

[0271] (vii) The VH comprises the amino acid sequence shown in SEQ ID NO:34 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:41 or a fragment thereof.

[0272] (viii) The VH comprises the amino acid sequence shown in SEQ ID NO:34 or a fragment thereof, and the VL comprises the amino acid sequence shown in SEQ ID NO:42 or a fragment thereof.

[0273] (ix) The VH contains the amino acid sequence shown in SEQ ID NO:34 or a fragment thereof and the VL contains the amino acid sequence shown in SEQ ID NO:43 or a fragment thereof.

[0274] In a preferred embodiment, the antibody capable of binding CLDN18.2 comprises a VH containing a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from embodiments (i) to (vi) below:

[0275] (i) CDR1: sites 45-52 of SEQ ID NO:14, CDR2: sites 70-77 of SEQ ID NO:14, CDR3: sites 116-125 of SEQ ID NO:14,

[0276] (ii) CDR1: sites 45-52 of SEQ ID NO:15, CDR2: sites 70-77 of SEQ ID NO:15, CDR3: sites 116-126 of SEQ ID NO:15.

[0277] (iii) CDR1: sites 45-52 of SEQ ID NO:16, CDR2: sites 70-77 of SEQ ID NO:16, CDR3: sites 116-124 of SEQ ID NO:16.

[0278] (iv) CDR1: sites 45-52 of SEQ ID NO:17, CDR2: sites 70-77 of SEQ ID NO:17, CDR3: sites 116-126 of SEQ ID NO:17.

[0279] (v) CDR1: sites 44-51 of SEQ ID NO:18, CDR2: sites 69-76 of SEQ ID NO:18, CDR3: sites 115-125 of SEQ ID NO:18, and

[0280] (vi)CDR1: sites 45-53 of SEQ ID NO:19, CDR2: sites 71-78 of SEQ ID NO:19, CDR3: sites 117-128 of SEQ ID NO:19.

[0281] In a preferred embodiment, the antibody capable of binding CLDN18.2 comprises a VL containing a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from embodiments (i) to (ix) below:

[0282] (i) CDR1: Positions 47-58 of SEQ ID NO:20, CDR2: Positions 76-78 of SEQ ID NO:20, CDR3: Positions 115-123 of SEQ ID NO:20

[0283] (ii) CDR1: sites 49-53 of SEQ ID NO:21, CDR2: sites 71-73 of SEQ ID NO:21, CDR3: sites 110-118 of SEQ ID NO:21,

[0284] (iii) CDR1: sites 47-52 of SEQ ID NO:22, CDR2: sites 70-72 of SEQ ID NO:22, CDR3: sites 109-117 of SEQ ID NO:22.

[0285] (iv) CDR1: sites 47-58 of SEQ ID NO:23, CDR2: sites 76-78 of SEQ ID NO:23, CDR3: sites 115-123 of SEQ ID NO:23.

[0286] (v) CDR1: sites 47-58 of SEQ ID NO:24, CDR2: sites 76-78 of SEQ ID NO:24, CDR3: sites 115-123 of SEQ ID NO:24.

[0287] (vi) CDR1: sites 47-58 of SEQ ID NO:25, CDR2: sites 76-78 of SEQ ID NO:25, CDR3: sites 115-122 of SEQ ID NO:25.

[0288] (vii) CDR1: sites 47-58 of SEQ ID NO:26, CDR2: sites 76-78 of SEQ ID NO:26, CDR3: sites 115-123 of SEQ ID NO:26

[0289] (viii) CDR1: sites 47-58 of SEQ ID NO:27, CDR2: sites 76-78 of SEQ ID NO:27, CDR3: sites 115-123 of SEQ ID NO:27, and

[0290] (ix)CDR1: sites 47-52 of SEQ ID NO:28, CDR2: sites 70-72 of SEQ ID NO:28, CDR3: sites 109-117 of SEQ ID NO:28.

[0291] In a preferred embodiment, the antibody capable of binding CLDN18.2 comprises a combination of VH and VL, each containing a set of complementarity-determining regions CDR1, CDR2, and CDR3 selected from embodiments (i) to (ix) below:

[0292] (i) VH: CDR1: sites 45-52 of SEQ ID NO:14, CDR2: sites 70-77 of SEQ ID NO:14, CDR3: sites 116-125 of SEQ ID NO:14; VL: CDR1: sites 49-53 of SEQ ID NO:21, CDR2: sites 71-73 of SEQ ID NO:21, CDR3: sites 110-118 of SEQ ID NO:21.

[0293] (ii) VH: CDR1: sites 45-52 of SEQ ID NO:15, CDR2: sites 70-77 of SEQ ID NO:15, CDR3: sites 116-126 of SEQ ID NO:15; VL: CDR1: sites 47-58 of SEQ ID NO:20, CDR2: sites 76-78 of SEQ ID NO:20, CDR3: sites 115-123 of SEQ ID NO:20.

[0294] (iii) VH: CDR1: sites 45-52 of SEQ ID NO:16, CDR2: sites 70-77 of SEQ ID NO:16, CDR3: sites 116-124 of SEQ ID NO:16; VL: CDR1: sites 47-52 of SEQ ID NO:22, CDR2: sites 70-72 of SEQ ID NO:22, CDR3: sites 109-117 of SEQ ID NO:22.

[0295] (iv) VH: CDR1: sites 44-51 of SEQ ID NO:18, CDR2: sites 69-76 of SEQ ID NO:18, CDR3: sites 115-125 of SEQ ID NO:18; VL: CDR1: sites 47-58 of SEQ ID NO:25, CDR2: sites 76-78 of SEQ ID NO:25, CDR3: sites 115-122 of SEQ ID NO:25.

[0296] (v) VH: CDR1: sites 45-52 of SEQ ID NO:17, CDR2: sites 70-77 of SEQ ID NO:17, CDR3: sites 116-126 of SEQ ID NO:17, VL: CDR1: sites 47-58 of SEQ ID NO:24, CDR2: sites 76-78 of SEQ ID NO:24, CDR3: sites 115-123 of SEQ ID NO:24.

[0297] (vi) VH: CDR1: sites 45-53 of SEQ ID NO:19, CDR2: sites 71-78 of SEQ ID NO:19, CDR3: sites 117-128 of SEQ ID NO:19; VL: CDR1: sites 47-58 of SEQ ID NO:23, CDR2: sites 76-78 of SEQ ID NO:23, CDR3: sites 115-123 of SEQ ID NO:23.

[0298] (vii) VH: CDR1: sites 45-53 of SEQ ID NO:19, CDR2: sites 71-78 of SEQ ID NO:19, CDR3: sites 117-128 of SEQ ID NO:19; VL: CDR1: sites 47-58 of SEQ ID NO:26, CDR2: sites 76-78 of SEQ ID NO:26, CDR3: sites 115-123 of SEQ ID NO:26.

[0299] (viii) VH: CDR1: sites 45-53 of SEQ ID NO:19, CDR2: sites 71-78 of SEQ ID NO:19, CDR3: sites 117-128 of SEQ ID NO:19; VL: CDR1: sites 47-58 of SEQ ID NO:27, CDR2: sites 76-78 of SEQ ID NO:27, CDR3: sites 115-123 of SEQ ID NO:27, and

[0300] (ix)VH:CDR1: sites 45-53 of SEQ ID NO:19,CDR2: sites 71-78 of SEQ ID NO:19,CDR3: sites 117-128 of SEQ ID NO:19,VL:CDR1: sites 47-52 of SEQ ID NO:28,CDR2: sites 70-72 of SEQ ID NO:28,CDR3: sites 109-117 of SEQ ID NO:28.

[0301] In a further preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises one or more complementarity-determining regions (CDRs) 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 as described herein), preferably at least the CDR3 variable region, and preferably comprises one or more complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL) as described herein, preferably at least the CDR3 variable region. In one embodiment, the one or more 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 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 as described herein), and preferably comprises complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) as described herein.

[0302] In one embodiment, an antibody comprising one or more CDRs as described herein, or a combination of a set of CDRs or groups of CDRs, comprises the CDRs along with the frame regions in which they are interposed. Preferably, the portion further comprises at least about 50% of any one or both of the first and fourth frame regions, the 50% being 50% of the C-terminus of the first frame region and 50% of the N-terminus of the fourth frame region. Antibody construction using recombinant DNA technology can result in the introduction of a variable region encoded by a linker at the N- or C-terminus of a residue, the linker being introduced to facilitate cloning or other manipulative steps, including the introduction of a linker to ligate the variable region of the invention to other protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the production of biantibodies), or protein tags.

[0303] In one implementation, an antibody comprising one or more CDRs as described herein, or a combination of a set of CDRs or groups of CDRs, is included in the human antibody framework.

[0304] The antibodies mentioned herein that contain a specific chain, region, or sequence relative to their heavy chain preferably refer to cases where all heavy chains of said antibody contain said specific chain, region, or sequence. This applies accordingly to the light chains of antibodies.

[0305] As used herein, the term "nucleic acid" is intended to include both DNA and RNA. Nucleic acids can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0306] 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. It also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable.

[0307] The teachings given herein with respect to specific amino acid sequences (such as those shown in the sequence listing) should be understood to also relate to variants of said specific sequences, thereby producing sequences that are functionally equivalent to said specific sequences, such as amino acid sequences exhibiting properties equivalent to or similar to those of said specific amino acid sequences. An important property is the preservation of antibody binding to its target or the maintenance of the antibody's effector function. Preferably, when a specific sequence in the antibody is replaced with a sequence that is a variant of the specific sequence, said sequence preserves the antibody's binding to CLDN18.2, and preferably preserves the antibody's function as described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage.

[0308] Those skilled in the art will understand that, in particular, the sequences of the CDR, hypervariable regions, and variable regions can be modified without losing their ability to bind to CLDN18.2. For example, the CDR region may be identical or highly homologous to the antibody region specified herein. "Highly homologous" is contemplated as allowing for 1 to 5, preferably 1 to 4, such as 1 to 3, or 1 or 2 substitutions in the CDR. Furthermore, the hypervariable and variable regions may be modified such that they exhibit substantially homology to the antibody regions explicitly disclosed herein.

[0309] For the purposes of this invention, "variants" of amino acid sequences include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants, including those with deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncated variants.

[0310] 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 into a specific site in the amino acid sequence, but random insertions may also be made, with appropriate screening for the resulting product.

[0311] Amino acid addition variants include amino- and / or carboxyl-terminal fusions of one or more amino acids (such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids).

[0312] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can occur at any position in a protein.

[0313] Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue into its position. Modifications at non-conserved amino acid sequence sites between homologous proteins or peptides and / or substitution of amino acids with other amino acids having similar properties should be considered based on preference. Preferably, the amino acid changes in protein variants are conserved amino acid changes, i.e., substitutions with similar charged or uncharged amino acids. Conserved amino acid changes involve substitutions of one family of amino acids whose side chains are associated. Naturally occurring amino acids are generally classified into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

[0314] Preferably, the similarity, preferably identity, between a given amino acid sequence and a variant thereof 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%. Preferably, the similarity or identity of amino acid regions is considered, said amino acid regions being 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, it is preferable to consider the similarity or identity of at least about 20 amino acids, at least about 40 amino acids, at least about 60 amino acids, at least about 80 amino acids, at least about 100 amino acids, at least about 120 amino acids, at least about 140 amino acids, at least about 160 amino acids, at least about 180 amino acids, or about 200 amino acids, and preferably the similarity or identity of consecutive amino acids. In a preferred embodiment, the similarity or identity of the entire length of the reference amino acid sequence is considered. The alignment used to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0315] "Sequence similarity" indicates the percentage of amino acids that are the same or represent conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0316] The term "percentage identity" is intended to represent the percentage of amino acid residues obtained after optimal alignment that are identical between the two sequences to be compared. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are routinely performed by comparing these sequences after optimal alignment, either by segments or through a "comparison window" to identify and compare locally similar regions. Besides manual methods, optimal alignments of sequences for comparison can be generated using local homology algorithms by Smith and Waterman (1981, AdsApp.Math.2, 482), Neddleman and Wunsch (1970, J.Mol.Biol.48, 443), and Pearson and Lipman (1988, Proc.Natl.Acad.Sci.USA85, 2444), or by using computer programs based on these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST NAND TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0317] Percentage identity is calculated by determining the number of identical sites between the two sequences being compared, dividing that number by the number of sites being compared, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0318] The term "transgenic animal" refers to an animal having a genome containing one or more transgenes, preferably heavy chain transgenes and / or light chain transgenes, or transchromosomal transgenes (integrated or not integrated into the animal's natural genomic DNA), and preferably capable of expressing said transgenes. For example, a transgenic mouse may have human light chain transgenes and human heavy chain transgenes or human heavy chain transchromosomal transgenes, such that when immunized with CLDN18.2 antigen and / or cells expressing CLDN18.2, the mouse produces human anti-CLDN18.2 antibodies. Human heavy chain transgenes can be integrated into the mouse's chromosomal DNA, as in transgenic mice such as HuMAb mice, such as HCo7 or HCl2 mice, or human heavy chain transgenes can be kept extrachromosomally, as in transchromosomal (e.g., KM) mice as described in WO 02 / 43478. Through VDJ recombination and isotype conversion, such transgenic and transchromosomal mice are capable of producing multiple isotypes (e.g., IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN18.2.

[0319] As used herein, “reduce,” “lower,” or “inhibit” means an overall decrease in a level (such as an expression level or a cell proliferation level) or is capable of causing an overall decrease in a level (such as an expression level or a cell proliferation level), preferably 5% or higher, 10% or higher, 20% or higher, more preferably 50% or higher, and most preferably 75% or higher.

[0320] Terms such as “increase” or “enhance” preferably refer to an increase or enhancement of 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.

[0321] Mechanism of action of mAb

[0322] While the following provides considerations regarding the potential mechanisms of therapeutic efficacy of the antibodies of the present invention, they should not be construed as limiting the invention in any way.

[0323] The antibodies described herein preferably interact with components of the immune system, preferably via ADCC or CDC. They can also be used to target payloads (e.g., radioactive isotopes, drugs, or toxins) to directly kill tumor cells or in synergy with conventional chemotherapy agents to attack tumors through complementary mechanisms of action, which may include anti-tumor immune responses that may have been impaired due to the cytotoxic side effects of chemotherapy agents on T lymphocytes. However, the antibodies described herein can also function solely by binding to CLDN18.2 on the cell surface, thereby blocking cell proliferation.

[0324] Antibody-dependent cell-mediated cytotoxicity

[0325] ADCC describes the cytotoxic capacity of effector cells (particularly lymphocytes) as described herein, which preferably require antibody-labeled target cells.

[0326] ADCC preferably occurs when an antibody binds to an antigen on tumor cells and the antibody's Fc domain binds to an Fc receptor (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism for directly inducing immediate tumor destruction to varying degrees, which leads to antigen presentation and induces a tumor-directed T-cell response. Preferably, in vivo induction of ADCC will result in both a tumor-directed T-cell response and a host-derived antibody response.

[0327] complement-dependent cytotoxicity

[0328] CDC is another cell-killing method that can be targeted by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also very effective for CDC targeting via the classical complement activation pathway. Preferably, in this cascade, the formation of the antigen-antibody complex leads to the exposure of C-cells of antibody molecules, such as IgG molecules, involved in the process. H 2. Multiple C1q binding sites closely adjacent to the domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert a previously low-affinity C1q-IgG interaction into one with high affinity, triggering a cascade involving a series of other complement protein events and leading to the proteolytic release of effector chemokines / activators C3a and C5a. Preferably, the complement cascade terminates upon the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free movement of water and solutes into and out of the cell.

[0329] Antibody production and testing

[0330] The antibodies described herein can be generated using a variety of techniques, including conventional monoclonal antibody methodologies such as the standard somatic cell hybridization technique of Kohler and Milstein (Nature 256:495, 1975). While somatic cell hybridization is preferred, other techniques for generating monoclonal antibodies may, in principle, be employed, such as oncogenic transformation of viruses or B-lymphocytes or phage display using antibody gene libraries.

[0331] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma generation in mice is a well-established procedure. Immunoassay protocols and techniques for isolating immune spleen cells for fusion are known in the field. Fusion partners (such as mouse myeloma cells) and fusion procedures are also known.

[0332] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (e.g., 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)).

[0333] In another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying portions 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.” The generation of human antibodies in such transgenic mice can be described in detail for CD20 as described in WO2004 035607.

[0334] However, another strategy for generating monoclonal antibodies involves directly isolating the antibody-encoding gene from lymphocytes that produce antibodies of defined specificities, as seen in 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 Welschof and Kraus (Recombinant antibodes for cancer therapy ISBN-0-89603-9l8-8) and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0335] To generate antibodies, as described above, mice can be immunized with a vector-conjugated peptide derived from the antigen sequence (i.e., the sequence targeting the antibody to be targeted), a recombinantly expressed antigen or fragment thereof, and / or a concentrated formulation of cells expressing the antigen. Alternatively, mice can be immunized with DNA encoding the antigen or fragment thereof. If immunization with a purified or concentrated antigen formulation does not produce antibodies, mice can also be immunized with cells expressing the antigen (such as cell lines) to promote an immune response.

[0336] Immune responses can be monitored during immunization protocols using plasma and serum samples obtained via tail vein or retroorbital blood collection. Mice with sufficient immunoglobulin titers can be used for fusion. Three days prior to sacrifice, mice can be enhanced intraperitoneally or intravenously with antigen-expressing cells, and the spleen can be removed to increase the ratio of hybridomas secreting specific antibodies.

[0337] To generate hybridomas that produce monoclonal antibodies, spleen cells and lymph node cells can be isolated from immunized mice and fused into a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for antigen-specific antibodies. Individual wells can then be screened for antibody-secreting hybridomas using ELISA. Antibodies specific to the antigen can be identified using cells expressing the antigen by immunofluorescence and FACS analysis. The antibody-secreting hybridomas can be re-inoculated and screened again; if they are still positive for monoclonal antibodies, subcloning can be performed using limiting dilutions. Stable subclones can then be cultured in vitro to generate antibodies for characterization in tissue culture medium.

[0338] Antibodies can also be generated in host cell transfected tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0339] For example, in one embodiment, a target gene (e.g., an antibody gene) may be ligated into an expression vector, such as a eukaryotic expression plasmid used in the GS gene expression system disclosed in WO87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems well known in the art. The purified plasmid containing the cloned antibody gene may be introduced into eukaryotic host cells, such as CHO cells, NS / O cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells, such as plant-derived cells, fungal cells, or yeast cells. Methods for introducing these genes may be those described in the art, such as electroporation, liposomes (lipofectine / lipofectamine), or other methods. After introducing these antibody genes into host cells, cells expressing antibodies may be identified and selected. These cells represent transfected tumors, and the expression level of the transfected tumors may then be amplified and scaled up to produce antibodies. Recombinant antibodies may be isolated and purified from these culture supernatants and / or cells.

[0340] Optionally, the cloned antibody gene can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, such as *Escherichia coli*. Furthermore, the antibody can be produced in transgenic nonhuman animals (such as sheep and rabbit milk or hen eggs) or in transgenic plants; see, for example, Verma, R., et al. (1998) J. Immunol. Meth. 216:165-181; Pollock, et al. (1999) J. Immunol. Meth. 231:147-157; and Fischer, R., et al. (1999) Biol. Chem. 380:825-839.

[0341] Chimerization

[0342] Mouse monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with toxins or radioisotopes. Unlabeled mouse antibodies are highly immunogenic in humans upon repeated application, leading to reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. If the individual antibodies are chimeric or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided. Chimeric antibodies are those whose different portions are derived from different animal species, such as those having variable regions derived from mouse antibodies and constant regions derived from human immunoglobulins. Chimerism of antibodies is achieved by linking the variable regions of the mouse heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al. in Methodsin Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-O89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the human κ-light chain constant region to the mouse light chain variable region. In another preferred embodiment, chimeric antibodies are generated by linking a human λ-light chain constant region to a mouse light chain variable region. Preferred heavy chain constant regions for generating chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0343] Humanization

[0344] Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDRs of individual antibodies are more diverse than those outside the CDRs. Because the CDR sequence is responsible for most antibody-antigen interactions, it is possible to construct expression vectors that mimic the properties of specific, naturally occurring antibodies. These expression vectors comprise CDR sequences grafted onto framework sequences from different antibodies with varying properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences are available from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences because they do not include the fully assembled variable gene, which is formed via V(D)J linkages during B cell maturation. The germline gene sequences also differ from the sequences of all components of the high-affinity secondary antibody at the uniformly distributed variable regions.

[0345] The ability of an antibody to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry).

[0346] To purify the antibody, selected hybridomas can be grown in 2-liter rotary flasks used for monoclonal antibody purification. Optionally, the antibody can be produced in a dialysis-based bioreactor. The supernatant can be filtered, and if necessary, concentrated prior to protein G-agarose or protein A-agarose affinity chromatography. The eluted IgG can be detected by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be replaced with PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80°C.

[0347] To determine whether a selected monoclonal antibody binds to a unique epitope, targeted mutagenesis or multisite targeted mutagenesis can be used.

[0348] To determine antibody isotypes, isotype ELISA with different commercial kits can be performed (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 a purified isotype control at ambient temperature for 2 hours. The wells can then be reacted with a mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probe. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at OD405-650. Alternatively, the IsoStrip mouse monoclonal antibody typing kit (Roche, catalog number 1493027) can be used as described by the manufacturer.

[0349] To demonstrate the presence of antibodies or monoclonal antibodies binding to live cells expressing antigens in the serum of immunized mice, flow cytometry can be used. Cell lines naturally expressing antigens or expressing antigens after transfection, along with negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with different concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated at 4°C for 30 min. After washing, APC- or Alexa647-labeled anti-IgG antibodies can bind to the antigen-bound monoclonal antibody as a primary antibody for staining under the same conditions. Single live cells are gated using a light and side-scattering FACS instrument, and the samples can be analyzed by flow cytometry. Co-transfection can be used to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement. Cells transiently transfected with plasmids encoding antigens and fluorescent labels can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. Because most transfected cells express two transgenes, antigen-specific monoclonal antibodies preferentially bind to cells expressing fluorescent markers, while non-specific antibodies bind to untransfected cells at a comparable rate. In addition to flow cytometry, alternative assays using fluorescence microscopy can be used, or alternative assays using fluorescence microscopy can replace flow cytometry. As mentioned above, cells can be stained and detected using a fluorescence microscope.

[0350] To demonstrate the presence of antibodies or monoclonal antibodies binding to live cells expressing antigens in the serum of immunized mice, immunofluorescence microscopy can be used. For example, under standard growth conditions, cell lines spontaneously expressing antigens or expressing antigens after transfection, and negative controls lacking antigen expression, are grown in chamber slides in DMEM / F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Cells are then fixed with methanol or paraformaldehyde, or left untreated. Cells are then reacted with a monoclonal antibody against the antigen at 25°C for 30 min. After washing, cells are reacted with an Alexa555-labeled anti-mouse IgG secondary antibody (molecular probe) under the same conditions. Cells are then examined by fluorescence microscopy.

[0351] 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 a nitrocellulose membrane, blocked, and detected with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and visualized with ECL substrate.

[0352] The reactivity of the antibody to the antigen can be further tested by immunohistochemistry in a manner well known to those skilled in the art, such as using frozen sections of non-cancerous or cancerous tissue samples fixed in paraformaldehyde or acetone, or paraffin-embedded tissue sections of non-cancerous or cancerous tissue samples fixed in paraformaldehyde, obtained from the patient during routine surgical procedures, or from xenograft tumors inoculated with cell lines expressing the antigen spontaneously or subsequently transfected. For immunostaining, the antigen-responsive antibody can then be incubated with horseradish-peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (DAKO) according to the supplier's instructions.

[0353] This assay can test the antibody's ability to mediate phagocytosis and kill cells expressing CLDN18.2. The in vitro monoclonal antibody activity assay will provide initial screening before testing in vivo models.

[0354] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0355] In short, polymorphonuclear cells (PMNs), NK cells, monocytes, or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminated red blood cells. The washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum, and then... 51Cr-labeled target cells expressing CLDN18.2 were mixed in varying ratios of effector cells to target cells. Optionally, target cells could be labeled with a fluorescently enhanced ligand (BATDA). The high-fluorescence chelate of europium containing the enhancing ligand released from dead cells could be measured using a fluorometer. Another alternative technique could utilize target cell transfection with luciferase. The added fluorescein was then oxidized only by living cells. Different concentrations of purified anti-CLDN18.2 IgG could then be added. Irrelevant human IgG could be used as a negative control. Assays could be performed at 37°C for 4 to 20 hours, depending on the type of effector cells used. Measurements were then performed... 51 The release of Cr or the presence of EuTDA chelates in culture supernatant can be used to determine cell lysis in a sample. Optionally, luminescence caused by the oxidation of fluorescein can be used as a measure of viable cells.

[0356] Anti-CLDN18.2 monoclonal antibodies can also be tested in different combinations to determine whether multiple monoclonal antibodies enhance cell lysis.

[0357] Complement-dependent cytotoxicity (CDC)

[0358] The ability of monoclonal anti-CLDN18.2 antibodies to mediate CDC can be tested using a variety of known techniques. For example, serum complement can be obtained from blood in a manner known to those skilled in the art. Different methods can be used to determine the CDC activity of the mAb. For example, it can be measured... 51 Cr release or increased membrane permeability can be assessed using propidium iodide (PI) exclusion assays. In short, target cells can be washed, and 5 x 10⁻⁶ ppm can be used. 5 / ml of mAb at different concentrations is incubated at room temperature or 37°C for 10–30 min. Serum or plasma can then be added to a final concentration of 20% (v / v) and the cells incubated at 37°C for 20–30 min. 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.

[0359] In an alternative assay, CDC induction can be determined on adherent cells. In one embodiment of this assay, 24 hours prior to the assay, the CDC is titrated in a tissue-culture flat-bottomed microtiter plate at 3 x 10⁻⁶ ppm. 4Cells were seeded at a density of 10 cells / well. The next day, the growth medium was removed, and the cells were incubated in triplicate with the antibody. Control cells were incubated with either growth medium or growth medium containing 0.2% saponin to determine background lysis and maximum lysis, respectively. After incubation at room temperature for 20 min, the supernatant was removed, and DMEM containing 20% ​​(v / v) human plasma or serum (pre-warmed to 37°C) was added to the cells, followed by incubation at 37°C for another 20 min. All cells from each sample were added to propidium iodide solution (10 μg / ml). The supernatant was then replaced with PBS containing 2.5 μg / ml ethidium bromide, and fluorescence emission after excitation at 520 nm was measured at 600 nm using Tecan Safire. The percentage of specific lysis was calculated as follows: Specific lysis % = (fluorescent sample - fluorescence background) / (maximum fluorescence lysis - fluorescence background) x 100.

[0360] Monoclonal antibody-induced apoptosis and cell proliferation inhibition

[0361] To test the ability to induce apoptosis, monoclonal anti-CLDN18.2 antibodies, for example, can be incubated with CLDN18.2-positive tumor cells (such as SNU-16, DAN-G, KATO-III, or CLDN18.2-transfected tumor cells) at 37°C for approximately 20 hours. Cells can be harvested, washed in annexin-V binding buffer (BD biosciences), and incubated in the dark for 15 min with annexin-V conjugated FITC or APC (BD biosciences). All cells from each sample can be added to PI solution (10 μg / ml in PBS) in FACS tubes and immediately evaluated by flow cytometry (as above). Alternatively, commercially available kits can be used to detect the overall inhibition of cell proliferation by the monoclonal antibody. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells in microplates. Incorporated BrdU is detected using europium-labeled monoclonal antibodies. To allow antibody detection, cells are fixed and DNA denatured using a Fix solution. Unbound antibodies are washed away, and a DELFIA inducer is added to dissociate europium ions from the labeled antibodies into solution, where they form highly fluorescent chelates with components of the DELFIA inducer. The measured fluorescence—in assays using time-resolved fluorometry—is proportional to DNA synthesis in the cells of each well.

[0362] Preclinical research

[0363] Monoclonal antibodies binding to CLDN18.2 can also be tested in in vivo models (e.g., in immunodeficient mice carrying xenograft tumors inoculated with cell lines expressing CLDN18.2 (e.g., DAN-G, SNU-16, or KATO-III) or cell lines transfected with cells expressing CLDN18.2 (e.g., HEK293)) to determine their efficacy in controlling the growth of tumor cells expressing CLDN18.2.

[0364] In in vivo studies, the antibodies described herein can be used after xenografting of 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 effect in preventing tumor formation or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice to determine the therapeutic efficacy of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibodies can be combined with other substances, such as cell-inhibiting drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibody applications, to determine the synergistic efficacy and potential toxicity of the combination. To analyze antibody-mediated toxic side effects, animals can be inoculated with antibodies or control reagents and symptoms potentially associated with CLDN18.2-antibody therapy can be thoroughly investigated. Possible side effects of in vivo application of CLDN18.2 antibodies include toxicity at tissues expressing CLDN18.2 (including the stomach). Antibodies that identify 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.

[0365] Epitope mapping by antibody recognition can be performed as detailed by Glenn E. Morris (“Epitope Mapping Protocols (Methods in Molecular Biology), ISBN-089603-375-9 and Olwyn MR Westwood, Frank C. Hay, “Epitope Mapping: A Practical Approach” Practical Approach Series, 248).

[0366] The compounds and reagents described herein can be administered in any suitable pharmaceutical composition.

[0367] Pharmaceutical compositions are typically provided in uniform dosage form and can be prepared in a manner known to them. Pharmaceutical compositions may be in the form of solutions or suspensions.

[0368] Pharmaceutical compositions may contain salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the active component of the pharmaceutical composition.

[0369] Pharmaceutically unacceptable salts can be used to prepare pharmaceutically acceptable salts, and are included within the scope of this invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, 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 from alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0370] Suitable buffering substances for use in pharmaceutical compositions include those containing acetate, citrate, borate, and phosphate.

[0371] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0372] Injectable formulations may include pharmaceutically acceptable excipients such as Ringer's lactate solution.

[0373] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, wherein an active component is combined to facilitate, enhance, or enable its application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for application to a patient.

[0374] Possible carriers for parenteral administration include sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and especially biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0375] When used herein, the term “excipient” is intended to refer to all substances that may be present in a pharmaceutical composition and are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents.

[0376] The reagents and compositions described herein can be administered via any conventional route, such as parenteral administration, including by injection or infusion. Parenteral administration is preferred, such as intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular administration.

[0377] Compositions suitable for parenteral administration typically comprise sterile aqueous or sterile non-aqueous formulations of the active compound, preferably isotonic with the recipient's blood. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. Additionally, sterile non-volatile oils are commonly used as solutions or suspension media.

[0378] The reagents and compositions described herein shall be applied in an effective amount. An "effective amount" means an amount, alone or in combination with other doses, that achieves the desired response or desired effect. In the treatment of a particular disease or patient, the desired response preferably involves inhibiting the course of the disease. This includes slowing the progression of the disease, and particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or patient may also delay or prevent the onset of said disease or patient.

[0379] The effective amount of the reagents or compositions described herein will depend on the patient to be treated, the severity of the disease, the patient's individual parameters (including age, physical condition, size, and weight), the duration of treatment, the type of concomitant therapy (if present), the specific route of administration, and similar factors. Therefore, the dosage of the reagents described herein can depend on various such parameters. In cases where the initial dose does not elicit an adequate response in the patient, a higher dose (or an effective higher dose achieved through a different, more localized route of administration) may be used.

[0380] The reagents and compositions described herein can be administered to patients (e.g., in vivo) to treat or prevent a variety of conditions, such as those described herein. Preferred patients include those with conditions that can be corrected or alleviated by administration of the reagents and compositions described herein. This includes conditions involving cells characterized by altered CLDN18.2 expression patterns.

[0381] For example, in one implementation, the antibody described herein can be used to treat patients with cancer, such as cancer characterized by the presence of cancer cells expressing CLDN18.2, as described herein.

[0382] The pharmaceutical compositions and treatment methods described in this invention can also be used for immunization or vaccination to prevent the diseases described herein.

[0383] The present invention is further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention. Example

[0384] Example 1: Materials and Methods

[0385] 1. Antibody

[0386] Table 1: Antibodies used in this article

[0387]

[0388] 2. Immunohistochemistry (IHC)

[0389] Before use, store the tissue sections (4μm thick) at 2-8℃.

[0390] Before dewaxing, the sections are kept in a drying oven at 58-60°C for 1 hour to melt the paraffin and remove moisture quantitatively, thereby improving tissue adhesion to the slide (“drying”).

[0391] Dewaxing

[0392] After melting and drying, the slides are dewaxed using two xylene steps (5 minutes each) and then rehydrated using a gradient of decreasing ethanol (at an ambient temperature of 20-27°C).

[0393] • Keep in a xylene bath for 5 (±1) minutes;

[0394] • Repeat this step once in a fresh bath;

[0395] • Excess liquid;

[0396] • Keep in anhydrous ethanol for 5 (±1) minutes;

[0397] • Repeat this step once with a fresh bath;

[0398] • Remove excess liquid;

[0399] • Keep in 96% ethanol for 5 (±1) minutes;

[0400] • Repeat this step once with a fresh bath;

[0401] • Remove excess liquid;

[0402] • Keep in 80% ethanol for 5 (±1) minutes;

[0403] • Remove excess liquid;

[0404] • Keep in 70% ethanol for 5 (±1) minutes;

[0405] • Remove excess liquid;

[0406] • Keep in distilled or deionized water for 5 minutes;

[0407] Epitope repair and quenching

[0408] After removing the paraffin, the target epitopes were repaired using a heat-induced epitope repair method. Therefore, slides were placed in stained wide-mouth bottles containing 200 ml of repair buffer (10 mM citrate buffer; 0.05% Tween-20; pH 6) and incubated at 120 °C in an autoclave (PASCAL, Dako) for 10 min. The bottles were then removed from the autoclave and cooled in epitope repair solution at room temperature for 10 (±1) min. The slides were then rinsed in washing buffer (1x PBS).

[0409] After cooling, the slides were transferred into a staining bottle containing 200 ml of quenching solution (1x PBS containing 0.3% peroxidase) and incubated at room temperature for 15 min, followed by 2 x 5 min washing steps in fresh washing buffer.

[0410] Blocking and antibody incubation

[0411] Remove excess wash buffer, cover the slide with 200 μl of blocking buffer (1x PBS containing 10% goat serum), and incubate at RT for 30 minutes. Remove the blocking buffer and replace it with 200 μl of diluted antibody solution (diluted in blocking buffer). Incubate the slide with the primary antibody overnight at 2–8 °C.

[0412] Table 2: Dilution of primary antibodies used for histological analysis – Stock and final concentrations of antibodies used in histological assays

[0413]

[0414] On the second day, remove the primary antibody solution and wash the slides 3 x 5 min with wash buffer. Then, remove excess wash buffer and add 200 μl of ready-to-use secondary antibody solution (Power Vision HRP goat-α-mouse; Immunologic; NL). Incubate the slides at RT for 30 min. Remove excess liquid and wash the slides 3 x 5 min with fresh wash buffer.

[0415] Substrate reaction and counterstaining

[0416] After removing excess wash buffer, cover the slides with approximately 50–150 μL of freshly prepared substrate-chromogenic solution (VectorRed; Vector Labs) for 2 min. Remove excess substrate and incubate the slides with deionized water in a wide-mouth flask for 1–5 min.

[0417] Subsequently, tissue counterstaining was performed by immersing the sections in a wide-mouthed bottle containing 200 ml of Mayer's hematoxylin for 2 minutes. Then, the sections were placed in tap water for 5-10 minutes to allow the cell nuclei to turn blue.

[0418] Dehydration and sealing

[0419] After counterstaining, the sections were dehydrated using a gradient of increasing ethanol concentrations:

[0420] • Soak in 70% ethanol (for approximately 5-10 seconds).

[0421] • Soak in 80% ethanol (for approximately 5-10 seconds)

[0422] • Soak in 96% ethanol (for approximately 5-10 seconds).

[0423] • Soak in 96% ethanol (for approximately 5-10 seconds).

[0424] • Soak in anhydrous ethanol (for about 5-10 seconds).

[0425] • Keep in xylene for 5 minutes

[0426] • Keep in xylene for 5 minutes

[0427] To fix the sample, an anhydrous sealing agent (X-TRA-Kit, Medite) was used. The slide was directly sealed from the last wide-mouth bottle containing xylene and air-dried at RT.

[0428] Table 3: Tissue microarrays used for histological analysis

[0429]

[0430] 3. Cell Culture

[0431] Based on the original data table and the following standard tissue culture methods, all pancreatic cancer cell lines and additional control cell lines used in the experiments presented in this paper were cultured in the culture medium. The conditions are summarized in Table 4. For all newly acquired cell lines, mycoplasma contamination of the cells was tested, and a master cell bank was prepared.

[0432] Table 4. Cell culture conditions for human pancreatic cancer and control cell lines

[0433] 1:LVT refers to a cell line stably transduced with lentivirus to express CLDN18.2.

[0434] 2: Inoculation density for culturing cells in the mentioned flasks or petri dishes for 2 or 3 days.

[0435] 3:adM: Reculturing cells derived from subcutaneous tumors

[0436]

[0437]

[0438]

[0439] 4. Luciferase transfection of pancreatic cell lines

[0440] For ADCC assays, pancreatic cancer cell lines were transiently transfected with luciferase RNA. The luciferase RNA (pST1-luc2mut-2hBgUTR-A121-EciI vector (pST1-109)) was generated using an ARCA cap and dissolved in H2O. RNA was aliquoted in 22 μl portions and stored at -80°C. Optimal electroporation conditions were determined for all pancreatic cell lines to yield the highest transfection rates and cell viability. For each assay, cells were isolated with PBS / 5 mM EDTA and dissolved in 250 μl of 2.5 x 10⁻⁶ X-Vivo solution. 6 Mix each cell with 10 μg of RNA in an ice-cold cuvette. Immediately electroporate the cells (GenePulserXcell, Biorad) and resuspend them in pre-warmed assay medium to adjust the cell size to 5 x 10⁻⁶ cells / mL. 5 Cells / ml. Electroporation conditions for all cell lines:

[0441] EP1: 250V, 475μF

[0442] EP2: 200V, 300μF

[0443] EP3: 150V, 300μF

[0444] EP4: 200V, 400μF

[0445] EP5: 250V, 950μF

[0446] Control: 0V, 0μF

[0447] Following electroporation, cell viability was determined directly using CASY or by staining cells with trypan blue and identifying the percentage of dead cells in the Neubauer chamber. Cells were seeded in quadruplicates in white 96-well plates (2.5 x 10⁶ cells / well). 4 Cells / well were incubated for 24 h. Subsequently, after adding the luciferase mixture and incubating for 90 min, luciferase activity was measured using a photometer (Tecan Infinite 200). Transfection was considered successful if the obtained RLU value was >1.000, and ADCC could then be measured.

[0448] 5. Quantitative Real-Time PCR (Q-PCR)

[0449] To isolate RNA from pancreatic cancer cell lines, cells were seeded in 10 cm culture dishes and grown for 2-3 days until 80% confluence. According to... Isolate RNA according to the instructions provided with the Mini Kit (Qiagen). Perform cDNA preparation using the manufacturer's instructions provided with the III First Strand kit (Invitrogen). Store RNA and cDNA samples at -80°C.

[0450] Quantitative analysis of CLDN18.2 transcripts was performed by amplifying oligo(dT)-guided cDNA using PCR primers #5054s (5'-AGAGAGCTCTGGCTTCACCGAGTG-3') and #5060as (5'-CCAGAAGTTAGTCACCAGCATGTTGG-3') in 40 cycles of PCR reactions to distinguish between the CLDN18.1 and CLDN18.2 isoforms. Reactions were prepared using SYBR Green (QuantiTect SYBR Green PCR Kit, Qiagen), with SYBR Green inserted into double-stranded DNA. Reactions and measurements were performed using an ABI-PRISM7900 Sequence Detection System instrument and software (AppliedBiosystems).

[0451] The relative expression level of the CLDN18 transcript relative to the housekeeping gene HPRT was calculated using the ΔΔCT method.

[0452] 6. Western blot analysis

[0453] To isolate proteins from pancreatic cancer cell lines, cells were seeded in 10 cm culture dishes and grown for 2–3 days until 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 induce genomic DNA division, protein samples were sonicated under the following conditions: output control: level 1, cycle time: 70% hold for 20–25 seconds. Protein concentration was measured using a spectrophotometer (absorbance at 280 nm), and samples were stored at -80°C before use.

[0454] To detect CLDN18.2 expression in Western blots, a 12.5% ​​polyacrylamide gel for separation was prepared between two fixed glass plates (for two small gels: 4.1 ml 29:1 acrylamide / bisacrylamide, 100 μl 10% SDS, 2.5 ml Tris pH 8.8, 3.2 ml H2O, 100 μl APS, 10 μl TEMED). After polymerization, the gel was covered with a stacking gel (1.5 ml 29:1 acrylamide / bisacrylamide, 100 μl 10% SDS, 2.5 ml Tris pH 6.8, 5.8 ml H2O, 100 μl APS, 10 μl TEMED) and a gel comb was placed between the glass plates. After polymerization, 75 μg of each protein sample was loaded onto the gel. The protein samples were prepared by adding (1:20) 4x 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). The gels were electrophoresed at 80 V in 1x SDS electrophoresis buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS) for 30 min and at 180 V in 1x SDS electrophoresis buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS) for 60 min. Semi-dry gel blots on nitrocellulose membranes were incubated at 160 mA in 1x transfer buffer (25 mM Tris, 0.192 mM glycine, 20% MeOH) for 90 min. The blots were first blocked in 5% milk powder / PBS, and primary antibody (0.25 μg / ml anti-densein 18 (C-terminus) or 0.1 μg / ml anti-β-actin) was added to 1% milk powder / PBS. The blots were incubated overnight at 4°C, washed three times for 10 min each in 1x PBS / 0.05% Tween 20, and then incubated with labeled secondary antibody in 1% milk powder / PBS (1:1000 diluted goat-anti-rabbit IgG (FC)) at room temperature for 1 h. The blot was washed three times again in 1x PBS / 0.05% Tween 20. Detection was performed by adding 1-3 ml of detection solution (Pico and Dura Detection System (Pierce)) and holding for 1 min. The blot was then scanned in a LAS-3000 detection chamber (increment: 10 s, interval: 10 s, sensitivity: high) according to GA_056_Chemolumineszenzentwickler LAS3000.

[0455] 7. Flow Cytometry (FACS)

[0456] Cells were harvested from exponentially growing cultures with 70-85% confluence using PBS / 5mM EDTA or trypsin / EDTA. Cells were counted, centrifuged for 5 min (468 g), and the pellet was resuspended in FACS buffer (PBS containing 2% FCS and 0.1% sodium azide), adjusting the concentration to 2 x 10⁻⁶. 6 / ml. Seed 100 μl of cells into a round-bottom 96-well plate and centrifuge again (5 min, 468 g). Serially dilute IMAB362 (or isotype control rituximab) in 50 μl of FACS-buffer from 0.1–200 μg / ml (11 dilution gradients + antibody-free control) and add to the cells at 4°C for 30 min. Then, add 200 μl of FACS-buffer to each well and centrifuge the plate (5 min, 468 g). Remove the supernatant and wash repeatedly. Dilute goat anti-human secondary antibody (FC-specific, APC-conjugated F(ab′)2(Dianova)) in FACS-buffer (1:100) and add 30 μl to each well. Incubate the plate at 4°C for 30 min. After incubation, the plate was washed twice with 200 μl of FACS buffer and the precipitate was finally resuspended in 100 μl of FACS buffer for measurement of FACSArray Bioanalyzer (BD) according to the GA_018_BD FACS Array Bioanalyzer.

[0457] 8. Lentiviral transduction

[0458] Lentiviral vector construction: Lentivirals are RNA viruses that stably integrate into both dividing and non-dividing cells using human genomic DNA. The vector pLenti6.4 (Invitrogen) was used as the backbone. It contains the clethodim gene for selecting positively transduced cells. CLDN18.2, fused to the EF1α promoter, was cloned into the recombinant region of the vector generating pL64B42E (EF1α-hCLaudin18.2)-clethodim. Figure 1 ).

[0459] Cell line selection: Cell lines were selected based on literature data or previous in vivo testing data. Selection criteria included uniform subcutaneous growth in nude mice and a therapeutic window of 20–100 days. Three cell lines (DANG, YAPC, and BxPC3) showing weak expression of CLDN18.2 mRNA were integrated, along with three cell lines (MiaPaCa-2, Patu8902, and Suit-2) capable of transfer according to literature. Two other cell lines (HPAC and CAPAN1, known to grow uniformly in subcutaneous tumors in vivo) were randomly selected.

[0460] Determination of levofloxacin selection criteria: For all cell lines, the required levofloxacin concentration for cell selection after lentiviral transduction was determined before transduction. Pancreatic cancer cells were seeded at high density in 6-well plates, and 80-90% confluence was achieved after 24 hours. Levofloxacin (stock solution: 10 mg / ml, Invitrogen) was added to the wells in incremental concentrations ranging from 0.5-12 μg / ml (5 dilution gradients + levofloxacin-free control). The culture medium was changed every 3-4 days, and the cells were analyzed under a microscope before removing the medium. The number of dead cells and the status of viable cells were recorded. Cells were cultured for 14 days. The lowest levofloxacin concentration that produced 100% apoptotic cells after 14 days was preferred for selecting lentiviral transduced cells. Table 4 shows the required levofloxacin concentration for each established LVT cell line.

[0461] Envelope selection: For lentiviral transduction, the GFP-lentiviral control vector pL64B42E-(EF1a-GFP)-blast was packaged into different envelope particles (VSV-G, GALV, RD114, Mokola-G, and Rabies-G). Adhesion to target cancer cells was more or less effective 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). Therefore, the CLDN18.2 expression vector pL64B42E(EF1α-hCLaudin18.2)-methionine was packaged into a VSV-G envelope. Producer cells were infected and discharged from the culture medium at a high titer (3.86 x 10⁻⁶). 7 Virus was isolated using particles per ml. The viral supernatant was stored at -80°C.

[0462] Table 5: Pancreatic cancer cell lines overexpressing CLDN18.2 generated via lentiviral transduction

[0463]

[0464] 1 The efficiency obtained by packaging the vector into VSV-G enveloped particles was measured 2 days after infection.

[0465] Lentiviral transduction of pancreatic cancer cell lines: For infection of pancreatic cancer target cell lines, use 200 μl... (20 μg / ml, Takara Inc.) was used to coat 24-well plates. Sealed plates were incubated at 4°C for 3–16 h. The plates were washed with 200 ml of PBS and blocked with PBS / 2% BSA at RT for 30 min. The plates were washed again and centrifuged at 2500 rpm for 25 min at 15°C, loading 300 μl of viral supernatant. The supernatant was removed, and the loading was repeated three times. The plates were washed once more with PBS, and low-passaged target cells were seeded into each well. For all pancreatic cancer cell lines, 5 x 10⁴ cells were seeded per 24 wells. 5 -1x10 7 Cells were collected. The plates were incubated at 37°C for 2 days. Subsequently, cells were isolated and transduction efficiency was determined by FACS using a FITC-labeled IMAB362 antibody. Cells were expanded and master cell libraries were prepared for each cell line.

[0466] 9. ADCC Measurement

[0467] Pancreatic cancer target cells were seeded in flasks two days in advance to obtain 80-90% confluence cultures on the day ADCC began. Pancreatic cancer cells were transfected with luciferase RNA and inoculated at 1x10⁻⁶ cells / mL in 50 μL of assay medium (medium containing 20 mM HEPES as described in Table 4). 4 PBMCs were seeded at a density of 1000 cells / well in white 96-well plates. Additionally, NUGC-4sub10cH11 subE10 Luci#2 cells (8000 cells / well) were seeded as a positive control in all assays. Cells were cultured for 4–6 h and PBMCs were purified before antibody addition.

[0468] Peripheral blood mononuclear cells (PBMCs) were prepared from the serotonin layer of fresh human erythrocyte sedimentation rate (ESR) obtained from healthy donors. Approximately 3 x 20–25 ml of blood was diluted with PBS (1:2) and carefully separated into layers on 4 x 15 ml Ficol-Paque Plus (GE Healthcare) in 50 ml Falcon tubes. Gradient centrifugation was performed (25 min, 700 g). After centrifugation, peripheral blood mononuclear cells (PBMCs) were collected from the intermediate layer, washed in PBS / 2 mM EDTA, centrifuged (5 min, 468 g), resuspended in PBS / 2 mM EDTA, and centrifuged (10 min, 208 g) to remove platelets. The pellet was resuspended in 50 ml PBS / 2 mM EDTA and the cells were counted. PBMCs were centrifuged (5 min, 468 g) and, for the addition of pancreatic cells, centrifuged at 1.6 x 10⁻⁶. 7 Resuspended in X-Vivo-15 medium at a concentration of 1.28 x 10^6 cells / ml for NUGC-4sub 10cH11subE10 Luci#2 cells. 7 The cells were resuspended in X-Vivo-15 medium at a concentration of 1 cell / ml.

[0469] The antibody (IMAB362 and isotype control antibody ch78H11 1H6) was serially diluted (4.5-fold) 10 times to produce a concentration range of 200 μg / ml to 0.26 ng / ml. 25 μl of each dilution was added to target cells in quadruplicate. Antibody-free PBS was added to the culture medium and lysis control wells. Subsequently, 25 μl of PBMC (E:T ratio = 40:1) was added to each well, and the plate was incubated at 37°C, 5% CO2 for 24 h ± 1 h.

[0470] On the second 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, 1xPBS, 3.84 mg / ml D-luciferin (BD Biosciences)) was added to each well, and the plate was incubated in the dark at RT for 80 min. The luminescence induced by the oxidation of luciferase by fluorescein in live cells was measured using a microtiter plate reader (Infinite200, Tecan, Switzerland). The percentage of cytotoxicity was calculated using the following formula:

[0471] Specific lethality (%) = 100 – [(RLU) 样品 –RLU triton ) / (RLU 培养基对照 –RLU triton )x100]

[0472] 10. CDC

[0473] The following is a CDC procedure.

[0474] Target cells (CHO-K1 p740 MACS / FACS(24H5)p3151 Luci#2A5) were seeded in 50 μl of assay medium (10,000 cells / well) in 96-well white assay plates and incubated at 37°C, 7.5% CO2, and 95% rH for 24 h + 20 min before sample addition. Each 96-well assay plate included a total of three different negative controls (heat-inactivated serum, serum containing and without IMAB362, and serum with an isotype control antibody (rituximab)) and a positive control from a healthy human serum bank containing 500 ng / ml IMAB362 (lot #31032011). At the end of the reaction, an additional positive control was formed by inducing complete lysis by adding 0.8% Triton X100 to the second medium control wells. One of the 96-well assay plates includes a functional positive control formed by seven serial 3.16-fold dilutions of IMAB362 (10,000–31.8 ng / ml). This control produces S-shaped dose-dependent lysis of target cells. All samples (200 μl each) are prepared simultaneously in the 96-well deep-dilution plate. Samples are taken three times from each well by reverse pipetting to generate triplicate in the assay plate. After adding 50 μl of each test and control sample to the assay plate, the plate is incubated at 37°C, 7.5% CO2, and 95% RH for 80 ± 5 min.

[0475] Add 10 μl of PBS to each well except for the Triton-lysis control well. Add 10 μl of 0.8% Triton / PBS solution to each Triton-lysis control well. Prepare the fluorescein substrate solution (6114 μl Aqua bidest, 2496 μl HEPES (1M), 1998 μl 1x DPBS, 4992 μl D-fluorescein stock solution (12 mg / ml)). Add 50 μl of fluorescein substrate solution to each well. Incubate the plate at 37 °C, 7.5% CO2, and 95% rH for 45 min. Measure the plate using a microtiter plate reader.

[0476] • Calculate complement-dependent cleavage using the following formula:

[0477] Specific lysis (%) = 100 – [(RLU sample – RLUtriton) / (RLUHSCM – RLUtriton) x 100)]

[0478] Improvements in testing pancreatic cancer cell lines:

[0479] • Transfect pancreatic cancer cells with luciferase RNA under optimal conditions. For each cell line tested, seed 1.5 x 10⁶ cells per well. 4 Each cell.

[0480] Since most pancreatic cancer cell lines are difficult to isolate and monopolize, trypsin was used on day 1.

[0481] • The pancreatic cancer cells in the assay plate were cultured at 37°C in 5% CO2.

[0482] • CDC assays of cells pretreated with chemotherapeutic agents were performed using the following concentrations of IMAB362 or as an allotype control antibody ch78H11 1H6: 640,000, 160,000, 40,000, 10,000, 2,500, 625, 156 and 39 ng / ml.

[0483] 11. Inhibition of proliferation

[0484] To analyze the dose-response curves of each chemotherapeutic agent, proliferation assays were performed.

[0485] Table 6: Analysis of the efficacy of gemcitabine or oxaliplatin in pancreatic cancer cell lines. Different concentrations of gemcitabine or oxaliplatin were applied to each pancreatic cancer cell line to inhibit proliferation.

[0486] cell lines Inoculated cells / well BxPC3-LVT 5000 BxPC3 5000 Panc05.04 5000 BxPC3-LVT 5000 CAPAN1-LVT 5000 DANG 2000 MiaPaCa-2-LVT 7000 Patu8988S 10000 Patu8988Sp3151#6 15000

[0487] Cells were seeded in 96-well plates, and gemcitabine or oxaliplatin was added at the following concentrations after 4–6 hours: 1000 ng / ml, 500 ng / ml, 250 ng / ml, 100 ng / ml, and 20 ng / ml. Proliferation assays were performed by incubation 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 was measured using Tecan Safire after 3 and 4 hours (cells plus supernatant). Inhibition of proliferation was calculated compared to the culture medium value set at 100%. EC50 of gemcitabine and oxaliplatin was calculated using the GraphPad Prism program. 50 value.

[0488] 12. Culture pancreatic cancer cell lines ADCC or CDC using chemotherapy drugs.

[0489] 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+07Patu8988S cells were seeded and cultured in medium with or without 10 ng / ml gemcitabine or in combination with 100 ng / ml oxaliplatin and 10 ng / ml gemcitabine.

[0490] On the day ADCC began, the protocol described above was followed and CLDN18 cell surface expression was measured in FACS analysis as described above.

[0491] 13. Cell cycle analysis

[0492] Cells were seeded in six-well plates and incubated with chemotherapeutic agents for 24, 48, or 3 days after 5–6 hours. Cells floating in the medium were combined with a trypsinized adherent cell layer. Cells were washed. Cell cycle analysis was initiated directly or cell surface staining was performed prior to initiating cell cycle analysis as described above. Cells were resuspended in 1 ml PBS with 3 ml 4% PFA. After fixing the cells at room temperature for 15 min, the cells were precipitated and washed. RNase-treated cells were resuspended in 200 μl RNase (10000 U / ml) with 0.05% Triton X-100 and incubated at 37°C for 30 min. 1 ml PBS was added, the sample was centrifuged, and resuspended in 200 μl PBS / PJ 50 μg / ml. After at least 30 min, the sample was ready for flow cytometry analysis. FlowJo software was used to analyze DNA content histograms to determine cell cycle phase distribution.

[0493] 14. Apoptosis assay

[0494] Following the specified treatment, apoptosis was measured by annexin V binding (Assay Kit I) or by DNA fragmentation assay (Apo-Direct), as recommended by the manufacturer (PharMingen, San Diego, CA). Briefly, cells floating in the supernatant were combined with trypsinized adhesion fractions and then washed. Aliquots of 5E+05 cells were incubated with annexin V-APC and PI at room temperature in the dark for 15 min. Cells were analyzed immediately by flow cytometry. Viable cells were excluded by annexin V-APC and PI. Early apoptotic cells were annexin V-APC-positive and PI-negative; however, cells no longer viable due to apoptosis or necrosis were stained 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).

[0495] Apoptosis assays based on DNA fragmentation were performed as follows. Treated cells (adherent and floating) were fixed overnight in 70% ice-cold EtOH. After washing, 10... 6Fixed cells were incubated with terminal deoxyribonucleoside transferase (TdT) and FITC-dUTP at 37°C for 90 min to label DNA breaks. Cells were washed and incubated in RNase A / propidium iodide at room temperature in the dark for 30 min to stain total DNA, followed by analysis by flow cytometry. Cell bimodalities and clumps caused by gating were eliminated from the analysis.

[0496] 15. In vivo studies

[0497] All in vivo experiments are conducted in accordance with national regulations and ethical guidelines for laboratory animal research.

[0498] 15.1 Treatment of Xenotransplantation

[0499] By subcutaneously injecting 200 μl of PBS containing tumor cells into female Hsd: Athymic-Foxn1 nu Xenograft tumors were inoculated into the flanks of nude mice. Tumor-bearing mice were treated with weekly intravenous (IV) or alternating IV / IP (half-weekly) injections of 0 μg, 200 μg, 400 μg, or 800 μg antibodies. Chemotherapy agents were administered weekly or half-weekly via IP. Tumor size and animal health were monitored every half-week. At the end of chemotherapy treatment, antibody administration continued until the tumor volume reached >1400 mm². 3 Or until the tumor becomes ulcerated. Cryopreserve the tumor sample or fix it in 4% formalin for subsequent analysis.

[0500] 15.2 Transfer determination

[0501] Following intravenous administration of cells to nude mice, the ability of different pancreatic cancer cell lines to form metastases was first analyzed. For these implantation analyses, 1x10⁻⁶ cells were used. 6 and / or 2x10 6 Injecting cells into a group of 5-10 mice and sacrificing individual mice at different time points to identify the time points of transfer, implantation, and growth.

[0502] Each treatment group used 10-12 Hsd: Athymoma-Foxn1 mice. nu Nude mice were used for transfer therapy. Using 2x10... 6 Administer 1 cell (Patu8988S or Suit2-LVT) intravenously. Once the first symptom of metastatic disease appears (weight loss, weakness, shortness of breath) or the first mouse dies, all mice are euthanized simultaneously.

[0503] Tissue preparation: For implantation studies, mice were sacrificed at different time points, or once they exhibited obvious physiological signs of metastatic disease (weight loss, weakness, shortness of breath). All organs were used for macroscopic analysis of metastases. Only Patu8988S and Suit-2 cells, lung, and lung / liver showed macroscopically visible metastases. These organs were cut into four equal pieces, and two pieces (lung: right upper lobe and left lower lobe) were preserved for genomic DNA isolation. The other two pieces were fixed in formalin and preserved for IHC analysis. Figure 2 ).

[0504] Genomic DNA preparation and Q-PCR strategy: Genomic DNA was extracted from lung or liver tissue. As controls, genomic DNA was also isolated from human pancreatic cancer cells Patu8988S and uninjected negative control mice.

[0505] Q-PCR strategy is based on the amplification of human DNA present in the transfer. The relative detection level of human DNA in mouse lung samples is directly related to the amount and / or size of the transfer. Because this method is biased by the fact that the transfer does not spread uniformly in the lung, and sometimes one lung lobe is more affected than another, it is advisable to mix two different regions of the lung in a single DNA preparation. Figure 2 ).

[0506] Q-PCR was performed using primer pairs #5861 5'-GGGATAATTTCAGCTGACTAAACAG-3' and #5862 5'-TTCCGTTTAGTTAGGTGCAGTTATC-3' to specifically amplify α-satellite DNA present in human chromosome 17 but absent in mouse DNA. To generate a standard curve and serve as a positive control, Patu8988 SDNA was mixed with mouse DNA and diluted 5-fold to produce 100%, 20%, 4%, 0.8%, 0.16%, 0.032%, and 0.0064% human DNA in mouse DNA. The curves were used to calculate (linear regression) the amount of human transfer DNA present in mouse lung tissue. The Q-PCR reaction was 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 positive primer (10 μM), 1.6 μl negative primer, and 1.8 μl H2O.

[0507] Example 2: Expression of CLDN18.2 in normal human pancreatic tissue and tumor-bearing pancreatic tissue

[0508] To analyze the expression level and pattern of CLDN18.2 in normal tissues and pancreatic tumor tissues, histological staining of FFPE sections was performed using two mouse monoclonal antibody reagents. Figure 3 ).

[0509] Exploratory preliminary experiments were conducted using the prototype antibody 35-22A on tissue microarrays (TMA). A major drawback of TMA is the variable tissue quality and small size of the spots, making them unrepresentative of the sample. This, along with an inadequately optimized staining protocol, can lead to an underestimation of positive cases.

[0510] The main experiments were performed using antibody 43-14A. These stainings were performed on tissue sections (larger than TMA sections) to pre-assess the presence of tumor cells.

[0511] Precancerous lesions originating from the pancreatic duct can be ranked according to the international pancreatic intraepithelial neoplasia (PanIN) system (PanIN-1A, -1B, -2, -3 subtypes).

[0512] PanIN-1 damage ( Figure 4 A) is flattened, composed of tall columnar cells with basally positioned nuclei and abundant nuclear mucin. The nuclei are small and round to oval in shape, oriented perpendicular to the basement membrane. There is histological overlap between non-tumor flattened proliferative lesions and non-atypogenic flattened tumor lesions.

[0513] The lesions of subtype PanIN-1B have papillary, micropapillary or basal pseudostratified structures, while in other respects they are the same as those of PanIN-1A (Hruban et al. Am J Surg Pathol. May 2001; 25(5):579-86.).

[0514] PanIN-2 damage ( Figure 4 B) The cells are flattened or papillary, with typical nuclear abnormalities, including loss of polarity, nuclear crowding, enlarged nuclei, pseudolaminarization, and excessive pigmentation. Mitosis is rare, but when present, it is non-luminal (non-apical) and typical (Hruban et al. Am J Surg Pathol. May 2001; 25(5):579-86.).

[0515] PanIN-3 damage ( Figure 4 C) They are usually papillary or micropapillary; however, they are rarely flattened. Small clusters of true cribriform epithelial cells budding into the lumen and necrosis of the lumen suggest a diagnosis of PanIN-3. The lesion is characterized by loss of nuclear polarity, malnourished goblet cells (goblet cells with nuclei facing the lumen and mucinous cytoplasm facing the basement membrane), occasionally abnormal mitosis, irregular nuclei, and protruding (giant) nucleoli (Hruban et al. Am JSurg Pathol. May 2001; 25(5):579-86.).

[0516] The expression of CLDN18.2 in precancerous tissues was analyzed using tissue samples from different sources with the 43-14A antibody.

[0517] CLDN18.2 was frequently detected in the PanIN structures of subtypes PanIN-1, -2, and -3, demonstrating the early expression of CLDN18.2 in precancerous lesions. Figure 4 It is conserved in later stages. In contrast, expression was not observed in normal pancreatic tissue samples (including pancreatic ductal structures).

[0518] In summary, CLDN18.2 is an early marker of the onset of malignant histological changes in the pancreatic duct.

[0519] Two studies were conducted to evaluate CLDN18.2 expression in primary pancreatic cancer. For the preliminary study, several TMAs from a total of 141 primary pancreatic cancer cases were stained with monoclonal CLDN18.2-specific antibody 35-22A. The overall quality of the analyzed TMAs was unsatisfactory. Many spots were partially lost during repair, and uneven counterstaining with hematoxylin suggested suboptimal tissue treatment for FFPE tissue.

[0520] Overall, >48.9% of stained cases were positive for CLDN18.2, including 49.2% (65 / 132) of ductal adenocarcinomas, 50% (1 / 2) of acinar cell carcinomas, and 3 / 7 of neuroendocrine carcinomas (Table 7). Tumor cell membranes were stained against any background free of other cell types. Figure 6 ).

[0521] Furthermore, we observed a correlation between the expression intensity of CLDN18.2 within the tumor and the fraction of stained tumor cells (Table 8). Figure 5 ).

[0522] Table 7: Preliminary Study: Number of CLDN18.2-positive cases dividing in pancreatic cancer subtypes. Tissues were stained and CLDN18.2-positive tumor cells were examined using monoclonal mouse 35-22A (0, 2 μg / ml) antibody.

[0523] Primary pancreatic cancer total Positive percentage ≥ 1% Staining strength ≥2+[%) total 141 69[48.9] 62[43.9] ductal adenocarcinoma (PDAC) 132 65[49.2] 58[44.3] acinar cell carcinoma 2 1[50.0] 1[50.0] Neuroendocrine carcinoma 7 3[42.8] 3[42.8]

[0524] Table 8: Preliminary study: Correlation between CLDN18.2 signal intensity and the number of positive tumor cells in the analyzed primary pancreatic tumors.

[0525] The percentage of positive primary tumor cases was correlated with staining intensity. Cases were divided into six groups based on the amount of well-visible positive tumor cells.

[0526] Table 9: Preliminary study: Grading of CLDN18.2 positive tumor cases.

[0527] Tumor cell grading describes the cell appearance and level of differentiation. Grade 1 describes well-differentiated cells; Grade 2 describes moderately differentiated cells; and Grade 3 describes poorly differentiated cells.

[0528] grade Total cases Positive percentage ≥ 1% Staining strength ≥2+[%) 1 15 13[86.7] 12[80.0] 2 71 39[54.9] 36[50.7] 3 35 9[25.7] 7[20.0]

[0529] The second study was conducted using quality-controlled tissue sections and an optimized staining protocol with highly sensitive antibody 43-14A.

[0530] Table 10: Number of CLDN18.2 positive cases in the subtypes of pancreatic cancer in the primary study. Tissues were stained and CLDN18.2 positive tumor cells were examined using mouse monoclonal 43-14A (0.2 μg / ml) antibody.

[0531] Primary CA Total cases Positive percentage ≥ 1% Staining strength ≥2+[%) total 61 40[65.6] 39[63.9] ductal adenocarcinoma (PDAC) 42 38[90.5] 37[88.1] acinar cell carcinoma 1 0 0 Neuroendocrine carcinoma 18 2[11.1] 2[11.1]

[0532] Table A

[0533] Primary biliary CA total Positive Positive% Biliary tract cancer 15 9 60

[0534] Table B

[0535] A total of 42 primary ductal pancreatic carcinoma samples were analyzed. Approximately 90% of these (38 out of 42 cases) were positive for CLDN18.2 (Table 10), with the majority (>60%) showing a strong signal intensity of +++. Figure 7 (Table 11). A correlation was also observed between CLDN18.2 expression levels and the number of positive tumor cells. The majority of the analyzed cases (62%) were grade 3 tumors (Table 12). Table 11: Main Study: Correlation between CLDN18.2 signal intensity and the number of positive tumor cells for the analyzed primary pancreatic tumors. The percentage of positive primary tumor cases correlated with staining intensity. Cases were divided into six groups based on the number of well-visible positive tumor cells.

[0536]

[0537] Table 12: Grading of primary study - CLDN18.2 positive tumor cases.

[0538] For most of the tumor cases analyzed, grading by the relevant pathologist was available. Tumor cell grading measures the cell appearance and level of cell differentiation. Grade 1 describes well-differentiated cells; Grade 2 describes moderately differentiated cells; and Grade 3 describes poorly differentiated cells.

[0539] grade Total cases Positive percentage ≥ 1% Staining strength ≥2+[%) 1 7 1[14.3] 1[14.3] 2 25 15[60.0] 15[60.0] 3 28 24[85.7] 23[82.1]

[0540] Most patients are diagnosed with pancreatic cancer at an advanced stage. The tumors have metastasized to lymph nodes and other organs, particularly the liver. In the primary study, 79 FFPE tissue samples with lymph node and liver metastases from pancreatic cancer were analyzed in immunohistochemical assays using a CLDN18.2-specific 43-14A antibody. 70.5% of lymph node metastases (31 / 44 cases) and 68.6% of distant liver metastases (24 / 3 cases) showed significant tumor cell staining for CLDN18.2 (Table 13). The staining pattern of positive tumor cells was membranous, with additional weaker cytoplasmic signal in some cases. Figure 9 Based on the results of primary tumor analysis, there was a correlation between CLDN18.2 expression levels in metastatic samples and the proportion of CLDN18.2-positive tumor cells. Figure 8 ).

[0541] No correlation was found between the grade of the tumors analyzed and the expression level of CLDN18.2 or the positive tumor cell fraction.

[0542] Table 13: Number of CLDN18.2 positive metastatic cases grouped by target organ.

[0543] The tissue was stained with monoclonal mouse 43-14A (0.2 μg / ml) antibody and CLDN18.2 positive tumor cells were examined.

[0544] Transfer total Positive percentage ≥ 1% Staining strength ≥2+[%) total 79 55[69.6] 52[65.8] Pancreas to lymph nodes 44 31[70.5] 28[63.6] Pancreas to Liver 35 24[68.6] 24[68.6]

[0545] To test whether CLDN18.2 expression in positive primary tumor cases was conserved in metastases within the same patient, antibody 43-14A was used to screen paired primary cancer / lymph node metastasis pairs. Table 14: CLDN18.2 expression in paired pancreatic primary tumor samples and lymph node metastasis tumor samples - Analysis of CLDN18.2 expression in paired primary adenocarcinoma samples and lymph node (LN) metastasis samples in tumor cells.

[0546]

[0547]

[0548] Of the 27 paired cases analyzed, 25 (92.5%) showed that both the primary tumor and lymph nodes were positive for CLDN18.2. In one single case, both tissues were negative, and in another case, the primary tumor was positive for CLDN18.2 while the metastasis was negative.

[0549] Of the 26 positive duplexes tested, 21 (80.7%) had primary and metastatic tumor cells with the same signal intensity. In 5 cases, the signal intensity decreased from +++ to ++.

[0550] Of the 25 paired tissues, 11 (44%) had a lower number of positive tumor cells in metastases compared to the primary tumor (Table 14).

[0551] In summary, CLDN18.2 expression appears to be conserved as primary tumor cells progress to the metastatic stage. The overall intensity and the percentage of positive tumor cells in lymph node metastases were only slightly lower compared to primary tumors. Figure 10 ).

[0552] For a small number of patient tissue samples derived from primary tumors, lymph node and liver metastases were obtained. These paired triplets were stained to test the conservation of CLDN18.2 expression in distant metastases. Six paired triplets were analyzed using antibody 43-14A.

[0553] Table 15: CLDN18.2 expression in paired pancreatic primary tumor and metastatic tumor samples - Analysis of CLDN18.2 expression in paired primary adenocarcinoma, liver metastasis, and lymph node (LN) metastasis samples in tumor cells.

[0554] Patient ID Primary tumor Lymph node metastasis liver metastasis H / 2011 / 17191-VA-VI-ISS +++(60％) +++(70％) +++(1％) H / 2010 / 14296XA +++(5-10％) - - H / 2010 / 4157VIIA ++(60％) ++(30％) +++(90％) H / 2009 / 23598VII B +++(40％) - - H / 2011 / 3590-III SS-VIIC-I ++(20％) - - H / 2008 / 10701 +++(90％) +++(100％) +++(90％)

[0555] Of the six triplets, three triplets showed that all three tissue samples were relevant to their target

[0556] Positive scores for CLDN18.2 are comparable. Figure 11 In three cases, some tumor cells were CLDN18.2 positive in primary lesions, but not in metastatic lesions (Table 15).

[0557] Example 3: Target expression of human pancreatic cancer cell lines in in vitro and in vivo models and pancreatic cancer models.

[0558] Source of cell lines

[0559] The primary objective of this preclinical evaluation study was to analyze the inhibitory effect of IMAB362 treatment in a suitable model system. To identify CLDN18.2-positive cell lines suitable for characterizing IMAB362 action in vitro and in vivo, a panel of 26 commercially available pancreatic cancer cell lines for CLDN18.2 expression was screened and characterized in detail. Cell banks for experimental use were prepared immediately upon arrival of each cell line. They were derived from primary pancreatic cancer (6 of the 10 primary pancreatic cancers were mucinous adenocarcinomas), primary cancer (4), pancreatic cancer metastasized to the liver (5) or spleen (1), or isolated from ascites (5) (see Table 16). Several of these cell lines (8) were transduced with lentivirus to express CLDN18.2.

[0560]

[0561]

[0562]

[0563] Expression of CLDN18.2 transcript in human pancreatic cancer cell lines

[0564] To identify pancreatic cell lines expressing CLDN18.2, transcript levels were determined using quantitative real-time PCR (RT-PCR) with a forward primer binding to exon 1 of CLDN18.2 and a reverse primer binding to exon 3 of CLDN18. The human gastric cancer cell line KATO-III, endogenously expressing CLDN18.2, and the CLDN18.2-negative breast cancer cell line SKBR-3 were included as positive and negative controls, respectively. RT-PCR revealed relative levels exceeding 1 x 10-1. 5 Significant endogenous CLDN18.2 expression was observed in pancreatic cancer cell lines DANG, Panc03.27, Panc05.04, Patu8988S, and YAPC. Interestingly, Patu8988S cells showed CLDN18.2 expression levels comparable to those of gastric CAKATO-III cells (~1x10⁻⁶). 8 ()( Figure 12 A). In summary, we detected robust expression of CLDN18.2 in 5 out of 22 pancreatic cancer cell lines.

[0565] In addition to endogenous cell lines, LVT cell lines ectopically expressing CLDN18.2 were analyzed at the transcript level. Figure 12 A). Of the eight LVT cell lines, six showed a relative CLDN18.2 expression level greater than 1 x 10⁻⁶. 8 Only in HAPC-LVT and Suit2-LVT cells was the expression level at 1x10. 5 Above.

[0566] We investigated the stability of CLDN18.2 expression during in vitro culture. Patu8988S, Panc05.04 cells, and lentivirally transduced cell lines Suit2-LVT, MiaPaCa2-LVT, and Patu8902-LVT were passaged up to 15 times, and CLDN18.2 transcripts were analyzed. Figure 12 (BD). We observed loss of CLDN18.2 expression in both endogenous and transduced cells with higher passage numbers. The loss of expression was highest in transduced cells. Therefore, where possible, we used early passages and validated CLDN18.2 expression in tumor xenografts in implantation experiments as described below.

[0567] CLDN18.2 protein expression in human pancreatic cancer cell lines

[0568] Detection of CLDN18.2 in total cell lysates

[0569] In addition to transcriptomic analysis, CLDN18.2 expression was analyzed at the protein level by Western blotting and infusion (IF). For Western blotting analysis, cell lysates from 26 pancreatic cancer cell lines were studied by Western blotting (WB) using a CLDN18-specific antibody against dentin 18 (C-terminus). Lysates of SKBR-3 cells were again used as a negative control, while lysates of HEK293 cells stably transfected with CLDN18.2 (HEK293-p740) were used as a positive control. Here, we detected high protein expression in Patu8988S, DANG, and Panc05.04 cells, thus confirming the RNA data. Blurred bands were detected in Panc03.27 and BxPC3 cell lysates. YAPC cells identified as positive at the RNA level showed smaller, blurry bands in Western blotting. All other cell lines were negative. Figure 13 ).

[0570] CLDN18 expression in pancreatic cancer cells

[0571] To obtain supporting protein expression data, pancreatic cancer cell lines were studied by immunofluorescence (IF) after cell fixation and permeabilization, using antibody 35-22A for detection. IF analysis confirmed previous RNA and protein data showing that most pancreatic cancer cell lines were negative for CLDN18.2 staining. Figure 14Nuclear spots were observed in some cell lines (such as AsPC1, DANG, HUP-T3, HUP-T4, and Panc01), which most likely represent staining artifacts. DANG, Panc03.27, and BxPC3 cells, identified at the RNA and / or protein levels as characterized by low CLDN18.2, were negative in the IF analysis, which has low detection sensitivity. In contrast, the cell membranes and cytoplasm of Panc05.04, Patu8988S, and KATO-III gastric cancer control cells were strongly positive for CLDN18.2 staining. Staining intensity varied for each cell type, and negative cells were also detected within the population. Figure 14 J and N). In the LVT cell line, we found that over 80% of all cells had strong membrane staining.

[0572] CLDN18.2 expression was confirmed in pancreatic cancer cells.

[0573] To confirm CLDN18.2 expression and assess the amount of this target on the cell surface, endogenous cell lines Panc05.04 and Patu8988S, as well as LVT cell lines, were stained with IMAB362 using a non-denaturing (native) staining protocol. Although staining of Patu8988S, Panc05.04, and KATO-III gastric cancer control cells with IMAB362 was not very strong, the percentage of positive cells was lower compared to cells stained with 35-22A. Figure 16 AF), but IF analysis confirmed that CLDN18.2 is expressed on the surface of pancreatic cancer cells. For eight LVT pancreatic cancer cell lines ectopically expressing CLDN18.2, significant membrane staining (e.g., on almost all cells) was observed. Figure 16 (Six LVT cell lines in GL were shown).

[0574] In summary, CLDN18.2 expression analysis led to the identification of endogenous pancreatic cancer cell lines Panc05.04 and Patu8988S, as well as 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.

[0575] The formation of a model for pancreatic cancer xenograft and metastasis.

[0576] Implantation studies for identifying suitable subcutaneous pancreatic cancer tumor models

[0577] A total of 37 engraftment studies with different pancreatic cancer cell lines were conducted to identify suitable subcutaneous xenograft models for testing the in vivo efficacy of IMAB362. Among all tested cell lines, BxPC3-LVT, CAPAN1-LVT, MiaPaCa-2-LVT, HPAC-LVT, DANG-LVT, and YAPC-LVT cell lines expressing ectopic CLDN18.2 were selected for subcutaneous xenograft models, showing high engraftment rates and homogeneous tumor growth. Additionally, subcutaneous xenograft models using the Patu8988S and DANG cell lines expressing endogenous CLDN18.2 were selected for testing the in vivo efficacy of IMAB362. Subcutaneous injection of Panc05.04 cells did not induce subcutaneous tumor formation.

[0578] Table 17: Overview of implantation conditions tested for establishing subcutaneous xenograft models of pancreatic cancer

[0579]

[0580]

[0581]

[0582]

[0583]

[0584] Implantation studies for the identification of suitable transfer models

[0585] To investigate the role of IMAB362 in metastasis formation, a metastatic cancer model was established in nude mice. The metastatic ability of pancreatic cancer cell lines after intravenous administration was analyzed. As described by Mohanty and Xu 2010, CAPAN1-LVT, MiaPaCa-2, Patu8988S, Patu8902, and Suit-2 cells were injected into the tail vein of nude mice. To determine the time point of metastatic implantation and growth rate, mice were sacrificed at different time points (Table 18).

[0586] Table 18: Analysis of metastasis and implantation of pancreatic cancer cell lines

[0587]

[0588]

[0589] Implantation analysis of Patu8902 cells and CAPAN1-LVT was not feasible because most mice died almost immediately. After 72 days, no macroscopically visible metastases were detected in the lungs and livers of the five surviving mice challenged with CAPAN-LVT cells. In contrast, injections of Suit-2 and MiaPaCa2 cells were well tolerated. Lung tissue from these mice was analyzed by IHC at different time points after injection. In mice challenged with MiaPaCa-2 cells, no metastases were detected in the lungs for up to 73 days, therefore this cell line was not selected as an IMAB362 treatment model. Suit-2 cancer cells metastasized to the lungs of mice. Multiple lesions were detected throughout the tissue. Therefore, the Suit-2-LVT cell line, transduced with lentivirus on CLDN18.2, was selected as the model system to analyze the role of IMAB362 treatment in metastasis formation.

[0590] In addition to Suit-2, the ability of Patu8988S cells endogenously expressing CLDN18.2 to form metastases was also analyzed. Two different cell numbers (1x10⁻²) were administered intravenously to each mouse. 6 2x10 6 Implantation was performed using [a specific method / mechanism]. Lungs and livers were isolated at different time points, as shown in Table 18. First, the different tissues obtained were analyzed using Q-PCR. Lungs and livers obtained over 70 days were analyzed by amplifying α-satellite DNA of human chromosome 17. The results for the lungs showed that the percentage of human DNA in the mouse lungs increased significantly over time, regardless of the number of injected cells. [The procedure was performed] by intravenous application of 1x10 [a specific method / mechanism]. 6 Or 2x10 6 After 70 days, 5.8% and 3.7% of human DNA could be detected in the cells, respectively. Figure 19 In the liver, almost no human DNA was amplified. After 70 days, the percentage increased slightly, but remained below 0.005%.

[0591] To validate CLDN18.2 expression in Patu8988S metastases, immunohistochemical staining was performed on lung tissue using anti-human MHC class I antibody and anti-mid protein 18 (Mid) antibody, both used to detect human cells in mouse tissues. MHC-I staining showed obvious metastatic lesions in mouse lung tissue sections, but none were detected in liver sections. Figure 20 Furthermore, staining of cell membranes in these lesions with anti-mid protein 18 (Mid) antibody revealed significant expression of the IMAB362 target protein in these cells. Therefore, in addition to the Suit2-LVT model, this endogenous metastasis model was chosen for IMAB362 treatment research.

[0592] Example 4: IMAB362-mediated cell killing

[0593] IMAB362 cross-linking effectively induces apoptosis.

[0594] Antibodies binding to cell surface targets can trigger aberrant signaling that directly leads to cell death. Such signaling events can depend on the target epitope, the binding valence, and whether the binding is associated with a cross-linking of the target. For several CD20-positive lymphoma cell lines, for example, rituximab-induced apoptosis has been observed only 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.

[0595] As measured by TUNEL assays, IMAB362 crosslinking induced direct apoptosis in human gastric cancer cells NUGC-4 and KATO-III within 18–42 hours. The magnitude of apoptosis was correlated with the antibody dose and the target expression level of the cancer cells. Gemcitabine treatment led to cell cycle arrest in tumor cells, followed by apoptotic cell death. Figure 21 The study showed apoptosis in gemcitabine-treated pancreatic tumor cells.

[0596] IMAB362-mediated ADCC activity against pancreatic cancer cells

[0597] IMAB362 is highly effective in recruiting and activating Fcγ-receptor-positive immune effector cells, such as natural killer cells. Binding of IMAB362 to target cells induces antibody-dependent cytotoxicity (ADCC) through granzymes and perforins secreted by effector cells after their Fcγ receptors bind to the antibody. The effects of this mechanism of action have previously been demonstrated by incubating luciferase- and CLDN18.2-positive gastric CA cells (such as NUGC-4 and KATO-III) with IMAB362 for 24 hours in the presence of human peripheral blood mononuclear cells (PBMCs) (effectant to target ratio = 40:1). Application of up to 200 μg / ml of IMAB362 resulted in a maximum lysis rate of 80–100%.

[0598] Here, we determined the ADCC activity of IMAB362 against pancreatic cancer cell lines. Increasing concentrations of IMAB362 were incubated with different cell lines at an E:T ratio of 40:1. PBMCs from different donors were added in each experiment. Results for all cell lines are summarized in Table 19. Among the five initially identified CLDN18.2-positive pancreatic cell lines, only Patu8988S, Panc05.04, and DANG(…) were effectively killed by the addition of IMAB362 and PBMCs. Figure 22A). Although CLDN18.2 surface expression was undetectable in FACS for Panc05.04 and DANG, expression levels were significantly sufficient to induce effector cell-dependent killing (EC). 50 Patu8988S: 0.01–1.4 μg / ml, DANG / Pan05.04: 0.1–38 μg / ml). From these data, it can be inferred that only those expressing relative RNA levels >5.5 x 10⁻⁶ are effective. 5 The cells were effectively lysed.

[0599] ADCC analysis was also performed using LVT pancreatic cancer cell lines and their corresponding parental cell lines. Figure 22 (BF). ADCC is strictly dependent on the specific binding of IMAB362 to the target, as only CLDN18.2-positive target cells are killed by IMAB362 and PBMCs. The half-maximal kill and maximum kill rate induced by IMAB362 in human pancreatic cancer cells vary between PBMC donors and also depend on the number of passages of cells that affect CLDN18.2 expression levels.

[0600] Figure 22 The GH figure shows the half-maximal kill rate (MCC) of target cells and the IMAB362 concentration at which the maximum kill rate was achieved. LVT pancreatic CA cell lines were killed after adding small amounts of antibody at high ratios; however, for DANG and Panc05, the highest antibody concentrations were required to achieve a maximum kill rate of ~50%. For Panc05.04, results obtained with subclone 15D3 (a CLDN18.2-positive clone selected by limiting dilution of Panc05.04 and FACS) are included in the figure showing ADCC lysis rates comparable to those with LVT cell lines. Unfortunately, CLDN18.2 expression in this clone was rapidly silenced in vitro after cell subculture, therefore this clone was not used for further experiments.

[0601] IMAB362-mediated CDC activity against pancreatic cancer cells

[0602] The sensitivity of pancreatic cancer cells killed by IMAB362 in ADCC assays to the complement-dependent cleavage activity of IMAB362 was analyzed. Additionally, LVT cell lines and parental lines were tested in CDC assays.

[0603] CDC activity is activated via a complex of antigen and IgM or IgG antibody (classical pathway) or via the microbial surface (alternative pathway). In the classical pathway, complement C5 is converted to C5b. Anaphylatoxins C3a, C4a, and C5b are released and form the membrane attack complex (MAC) through the successive binding of C5b, C7, C8, and C9. This pathway is inhibited not only by soluble proteins but also by membrane-bound proteins (such as CR1, DAF, MCP, CD59, CD55, and CD46) protecting the tissue itself.

[0604] In each assay, CHO-K1 cells stably transfected with CLDN18.2 (p740) and luciferase were used as positive controls. Figure 23 A). Cell lines DANG, BxPC3, YAPC, Patu8988S, Panc05.04, CAPAN1, and Suit2 were not lysed by IMAB362 and added to a healthy human serum bank. Figure 23 B). Although DANG, Patu8988S, and Panc05.04 cells were CLDN18.2 positive, as shown in all previous experiments, these cells did not lyse in a complement-dependent manner. This is most likely due to the fact that tumor cells overexpress one or more cell membrane-bound complement-inhibiting proteins (such as CD46, CD55, and CD59) (Geis et al., Curr Cancer Drug Targets, 2010 10:922-931). However, if the expression of these inhibitory proteins on tumor cells affects the clinical outcomes of the antibody, the therapy remains paradoxical (Dzietczenia et al. Med. Oncol. 2010, 27:743-6; Weng and Levy, Blood 2001 98:1352-7).

[0605] In addition to endogenous cell lines, all LVT cell lines were tested in the CDC assay. For example, in Figure 23 The results showed that the addition of IMAB362 and serum to MiaPaCa-2-LVT, Suit2-LVT, and CAPAN1-LVT resulted in dose-dependent cleavage, with EC 1 folds. 50 The values ​​range from 0.3 μg / ml to 2.6 μg / ml.

[0606]

[0607]

[0608]

[0609] Example 5: Efficacy of IMAB362 in a pancreatic cancer xenograft model

[0610] Ten out of 41 tested pancreatic cancer xenograft models were selected to investigate the in vivo efficacy of IMAB362. Using a pancreatic xenograft model with high CLDN18.2 expression, IMAB362 treatment showed high antitumor activity. This was investigated by subcutaneously treating mice with BxPC3-LVT or MiaPaCa-2-LVT xenografts on the left side. Treatment began 3 days after tumor inoculation with 200 μg IMAB362 every half week. Mice treated with IMAB362 showed significant inhibition of tumor growth compared to mice treated with saline. Furthermore, the inhibition of tumor growth in mice treated with IMAB362 led to a prolonged median survival. Figure 24 and Figure 25 The efficacy of IMAB362 is related to the duration of treatment. To examine its effect on established tumors, initiating IMAB362 treatment at an earlier time point resulted in increased tumor growth inhibition compared to initiating treatment at a later time point. Furthermore, the antitumor effect of IMAB362 depends on the level of CLDN18.2 target expression. Compared to tumor growth inhibition using xenografts expressing high CLDN18.2, IMAB362-mediated growth inhibition was reduced in tumors expressing low CLDN18.2 (such as DANG and Patu8988S xenografts).

[0611] Example 6: Treatment of a mouse model of pancreatic metastasis

[0612] Table 20: Overview of the treatment efficacy of IMAB362 against pancreatic cancer metastases

[0613]

[0614] Suit2-LVT transfer model:

[0615] Use 2x10 6 Mice were intravenously injected with Suit2-LVT cells 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 (the isotype control group) died. Therefore, all mice were sacrificed on day 42, and their lungs and livers were harvested for IHC and Q-PCR analysis.

[0616] Q-PCR analysis of human DNA in mouse lungs was performed in triplicate at least twice. Compared with PBS and isotype control treatments, treatment with IMAB362 revealed a significant reduction in Suit2-LVT metastasis in the lungs when the percentage of human DNA calculated using the obtained Ct values ​​was calculated (P < 0.05). Figure 26A). To validate these results, tissue sections of lung samples were prepared and stained with MHC-I antibody. The surface area of ​​positively stained cells in the lung sections was calculated using the ImageJ Program. For IMAB362 treatment, significant inhibition was observed compared to PBS treatment (P<0.05), thus validating the results obtained using Q-PCR. However, no significant difference was observed for the isotype control antibody. Figure 26 B). This difference is most likely due to the different tissue processing: IHC treatment of tissue sections provides insights into only a very small portion of the lung, compared to Q-PCR analysis, where genomic DNA is extracted from half of the tissue.

[0617] Besides tissue treatment, the results may indicate an unexpected inhibitory effect from the allotype control antibody targeting CLDN6. To investigate this choice, CLDN6 expression and IMAB027 binding in Suit2-LVT cells were analyzed in FACS. Adding 200 μg / ml IMAB362 to Suit2-LVT cells confirmed strong binding, while adding 200 μg / ml IMAB027 resulted in weaker binding, indicating that CLDN6 is indeed weakly expressed in these cells. These results suggest that at least two factors (tissue treatment and weak IMAB027 inhibition) contribute to the observed differences compared to the allotype control antibody.

[0618] Patu8988S Transfer Model

[0619] To analyze the effect of IMAB362 treatment on the formation and growth of Patu8988S metastases in vivo, 2x10⁻¹⁰ μg / mL of IMAB362 was used. 6 Patu8988S cells were injected into 10 mice in each group. The first experiment was conducted by comparing IMAB362-treated mice with PBS-treated mice. In each group, one mouse died immediately after cell injection. In the other 18 mice, metastasis formation was much more rapid compared to the implantation experiment. After 63 days, the first two mice in the PBS group were sacrificed due to poor health. After 65 days, all other mice were sacrificed. Optical analysis of the lungs revealed large metastases throughout the lung tissue. The amount of metastasis was analyzed in a Q-PCR experiment. Figure 27 The results showed that IMAB362 inhibited the growth of metastases in lung tissue.

[0620] A second experiment was conducted in 11 mice per group, comparing IMAB362 treatment with an allotype control (rituximab). In this experiment, metastasis formation was slow, as observed during implantation. However, for comparability, this second experiment was terminated after 65 days. Reanalysis of lung tissue in Q-PCR again showed that IMAB362 again reduced metastasis growth. One mouse in the IMAB362 group was identified as an outlier, and excluding this outlier resulted in almost significant (P = 0.0588) inhibition. These data were validated by IHC surface analysis as described in the Suit2-LVT metastasis assay. Here, the same outlier was identified and omitted in the t-test; the inhibition by IMAB362 was also at the borderline of significant (for the same mouse) (P = 0.0691).

[0621] Example 7: Main pharmacodynamics of IMAB362 in combination with chemotherapy

[0622] Sensitivity of pancreatic cancer cells to gemcitabine and oxaliplatin

[0623] The mechanism of action of IMAB362 in combination with the chemotherapy agents oxaliplatin or gemcitabine was investigated using constitutively CLDN18.2-expressing pancreatic cancer cell lines (DANG, Patu8988S) and cells stably transduced with CLDN18.2 (MiaPaCa-2-LVT, BxPC3-LVT).

[0624] Chemically, gemcitabine (Gemzar, sold by Eli Lilly & Co.) is a nucleoside analog. Like 5-fluorouracil (5-FU) and other pyrimidine analogs, gemcitabine triphosphate analogs replace one of the structural units of nucleic acids during DNA replication. This process arrests tumor growth because only one other nucleoside can be attached to the "wrong" nucleoside, leading to apoptosis.

[0625] Oxaliplatin functions by forming inter- and intra-strand crosslinks in DNA. Crosslinks in DNA prevent DNA replication and transcription, thereby leading to cell death (Graham, Joanne; Mushin, Mohamed; Kirkpatrick, Peter (January 2004). "Oxaliplatin". Nature Reviews Drug Discovery 3(1):11-2.).

[0626] The dose-response curves for gemcitabine and oxaliplatin showed the different sensitivities of the pancreatic tumor cell lines tested. Figure 28 and Figure 29 ).

[0627] Table 21: IC50 values ​​of gemcitabine and oxaliplatin for pancreatic cancer cell lines.

[0628]

[0629]

[0630] High concentrations of gemcitabine (IC50 > 100 ng / ml) or oxaliplatin (IC50 > 500 ng / ml) are necessary to inhibit the proliferation of Patu8988S cells. DANG and BxPC3-LVT cells are highly sensitive to gemcitabine but insensitive to oxaliplatin. MiaPaCa-2-LVT cells are most sensitive to oxaliplatin but less sensitive to gemcitabine treatment. Figure 28 , Figure 29 (and Table 21).

[0631] Effects of chemotherapy agents on CLDN18.2 expression in pancreatic cancer cell lines

[0632] 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 a combination of gemcitabine and oxaliplatin (GemOx) resulted in increased mRNA and protein levels of CLDN18.2, which was confirmed by RT-PCR in untreated and chemotherapy-pretreated cells. Figure 30 ) and protein blotting ( Figure 31 Analysis showed that, as demonstrated by flow cytometry, the amount of CLDN18.2 protein, which can be targeted by IMAB362, was increased on the surface of pancreatic cancer cell lines pretreated with Gem or GemOx. Figure 32 ).

[0633] Treatment of DANG and Patu8988S with gemcitabine resulted in an upregulation of CLDN18.2. GemOx and Patu8988S showed a stronger upregulation of CLDN18.2, while GemOx and Patu8988S showed a weaker upregulation.

[0634] Effects of chemotherapy compounds on cell cycle and CLDN18.2 expression

[0635] The cell cycle refers to the sequence of events between one mitosis and another in a cell. Following the quiescent phase (G0 / G1) is the DNA synthesis phase (S), then the cell enlargement phase (G2) and DNA replication (M), after which the cell divides into two daughter cells. Any disruption to cellular mechanisms can inhibit all phases of the cell cycle at any given time. For example, specific chemotherapeutic agents can block the progression of G2 or M, or G2 and M (G2 / M).

[0636] Gemcitabine treatment with DANG or Patu8988S resulted in cell cycle arrest in S-phase. Figure 33 , Figure 34 Analysis was performed on Patu8988S cells cultured with Gem. Gemcitabine treatment not only induced cell cycle arrest but also altered the expression of CLDN18.2. Figure 34 B). When comparing proliferating cells in the S phase with quiescent cells in the G0 / G1 phase, the change in CLDN18.2 density after gemcitabine treatment was even greater ( Figure 34 C). In Patu8988S cells, CLDN18.2 is expressed at all stages of the cell cycle. Its expression is even increased after treatment with gemcitabine, with the highest levels of CLDN18.2 / cell found in the S phase cell population.

[0637] Perturbations in tumor cell phenotype have a significant impact on the bioavailability of therapeutic antibodies. ADCC and CDC are dose-related, therefore, an increase in the target structure CLDN18.2 provides a synergistic benefit for standard chemotherapy regimens.

[0638] Kato III cells (human gastric tumor cell line) were cultured at 37°C and 5% CO2 in RPMI 1640 medium (Invitrogen) containing 20% ​​FCS (Perbio) and 2 mM Glutamax (Invitrogen), with or without cell-inhibiting compounds. 5-FU (Neofluor from NeoCorp AG) was tested at concentrations of 10 or 100 ng / ml, and oxaliplatin (Hospira) was tested at concentrations of 50 or 500 ng / ml. At 37°C and 5% CO2, 8 x 10⁸ cells were cultured... 5 Kato III cells were cultured in 6-well tissue culture plates for 96 hours without changing the medium, or for 72 hours followed by 24 hours in standard medium to allow release from cell cycle arrest. Cells were harvested, washed, and analyzed using EDTA / trypsin.

[0639] For extracellular assays of CLDN18.2 cells, cells were stained with monoclonal anti-CLDN18.2 antibody IMAB362 (Ganymed) or an allotype-matched control antibody (Ganymed). A second reagent from Diano, goat-anti-huIgG-APC, was used.

[0640] Cell cycle stages are determined by measuring cellular DNA content. This allows differentiation between cells in the G1-, S-, or G2- phases of the cell cycle. DNA replication occurs in the S- phase, while in the G2- phase, cells grow and prepare for mitosis. Cell cycle analysis was performed using the CycleTEST PLUS DNA Kit from BD Biosciences, following the manufacturer's protocol. Flow cytometry was used for acquisition and analysis using BD FACS CantoII (BD Biosciences) and FlowJo (Tree Star) software.

[0641] Figure 35 The bar charts in a and b show the percentage of cells in each of the G1-, S-, or G2- phases of the cell cycle. Kato III cells cultured in the medium showed predominantly arrested cell cycle in the G1- phase. Cells treated with 5-FU showed predominantly arrested cell cycle in the S- phase. Oxaliplatin-treated Kato III cells showed enrichment primarily in the G1- and G2- phases. Figure 35 As seen in c, cell cycle arrest in S-phase or G2-phase leads to the stabilization or upregulation of CLDN18.2. Once cells are released from any phase of the cell cycle ( Figure 35 b) The expression of CLDN18.2 on the cell surface of Kato III cells is upregulated ( Figure 35 d).

[0642] Kato III cells were pretreated with irinotecan or docetaxel for 4 days, and CLDN18.2 expression and cell cycle arrest were analyzed. Irinotecan treatment induced dose-dependent inhibition of cell growth and cell cycle arrest in the S / G2 phase. Figure 36 Treatment of cells with docetaxel resulted in a dose-dependent inhibition of cell growth and cell cycle arrest in the G2 phase. Figure 36 ).

[0643] Effects of chemotherapy on IMAB362-induced antibody-dependent cytotoxicity (ADCC)

[0644] A series of experiments were conducted using the constitutively CLDN18.2-expressing pancreatic cancer cell lines Patu8988S and DANG to investigate the effects of gemcitabine (Gem) or gemcitabine + oxaliplatin (GemOx) on IMAB362-mediated ADCC. The dose-response curves of IMAB362-mediated cell lysis in pretreated cells were compared with the culture medium.

[0645] Compared to untreated target cells, the dose-response curve of DANG (2 days) pretreated with Gem (1 ng / ml) or GemOx (Gem 1 ng / ml + Ox 10 ng / ml) shifted upward and to the left. Figure 37 A). Treatment of tumor cells with Gem or GemOx resulted in upregulation of CLDN18.2 and increased susceptibility to IMAB362-mediated ADCC. We observed decreased EC50 values ​​in DANG cells and higher maximal cell lysis in IMAB362-mediated ADCC after chemotherapy. Figure 37 B).

[0646] Peripheral blood mononuclear cells (PBMCs) from healthy human donors, including NK cells, monocytes, mononuclear cells, or other effector cells, were purified by Ficoll Hypaque density centrifugation. The washed effector cells were seeded in X-Vivo medium. In this setup, Kato III cells, endogenously expressing CLDN18.2 and originating from the stomach, were used as target cells. The target cells stably expressed luciferase (fluorescein), which was oxidized only by live cells. Purified anti-CLDN18.2 antibody IMAB362 was added at varying concentrations and used as an allotype control antibody, i.e., an unrelated chim hu IgG1 antibody. Cell lysis of the samples was determined by measuring the luminescence induced by the oxidation of fluorescein, representing the numerical value of the number of live cells remaining after IMAB362-induced cytotoxicity. Kato III cells pretreated for 3 days with irinotecan (1000 ng / ml), docetaxel (5 ng / ml), or cisplatin (2000 ng / ml) were compared with target cells cultured in untreated medium to quantify IMAB362-induced ADCC.

[0647] Kato III cells pretreated with irinotecan, docetaxel, or cisplatin for 3 days showed lower levels of viable cells compared to target cells cultured in culture medium. Figure 38 a); Compared with cells cultured in medium, cells pretreated with irinotecan, docetaxel, or cisplatin showed increased expression of dentin 18.2. Figure 38 b).

[0648] Furthermore, pretreatment of Kato III cells with irinotecan, docetaxel, or cisplatin increases the efficacy of IMAB362-induced ADCC. Figure 38 c,d).

[0649] Effects of chemotherapy on IMAB362-induced CDC

[0650] The CDC efficacy of IMAB362 has been characterized by incubation with target cells in the presence of human serum as a complement source.

[0651] MiaPaCa-2-LVT cultured in medium showed an EC50 value of 7665 ng / ml for specific cleavage of IMAB362. Treatment with Gem resulted in a decrease in EC50 to 4677 ng / ml compared to the maximum increase in cleavage. Figure 39 ).

[0652] The effect of chemotherapy 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. Figure 40 The figure shows a representative dose-response curve for IMAB362-induced CDC in KATO III cells pretreated with chemotherapy. Pretreatment of tumor cells for 48 hours increased the efficacy of IMAB362-induced CDC compared to untreated cells, resulting in higher maximum cell lysis in pretreated tumor cells.

[0653] Example 8: Efficacy of IMAB362 in combination with chemotherapy in a mouse tumor model

[0654] The antitumor activity of IMAB362 in combination with Gem or GemOx was examined in a subcutaneous pancreatic cancer xenograft model, and its efficacy was tested as IMAB362 as a single agent previously.

[0655] Compared with control mice treated with saline, BxPC3-LVT or MiaPaCa-2-LVT tumor-bearing nude mice treated with IMAB362 showed significant tumor growth arrest. Chemotherapy with up to 100 mg / kg gemcitabine in the absence of additional IMAB362 treatment did not show significant therapeutic effect on BxPC3-LVT or MiaPaCa-2-LVT xenografts. In contrast, combination therapy with 50–100 mg / kg gemcitabine plus IMAB362 resulted in significantly increased tumor growth inhibition and prolonged survival in tumor-bearing mice compared with chemotherapy alone. Figure 41 , Figure 42 , Figure 43 These observations suggest a synergistic therapeutic effect through the combination of gemcitabine and IMAB362 immunotherapy.

[0656] When high doses of gemcitabine (2 x 150 mg / kg / week) were used, established MiaPaCa-2-LVT xenograft tumors showed strong inhibition of tumor growth independent of IMAB362 treatment. Figure 44 A). However, compared with mice treated with gemcitabine as a single agent, mice treated with the combination therapy of IMAB362 and gemcitabine showed a highly significant prolongation of survival ( Figure 44 B).

[0657] Example 9: ZA / IL-2 treatment led to a large number of Vγ9Vδ2 T-cells expanding.

[0658] PBMCs were cultured for 14 days in RPMI medium supplemented with 300 U / ml IL-2 and with or without 1 μM zoledronic acid (ZA). On day 0 and day 14, the percentage of Vγ9+Vδ2+ T cells in the CD3+ lymphocyte population and the percentage of CD16+ cells in the CD3+Vγ9+Vδ2+ T cell population were determined by multicolor FACS.

[0659] The addition of IL-2 to PBMC cultures is essential for lymphocyte survival and growth. They expand efficiently in cultures supplied with 300 U / ml IL-2. FACS analysis using Vγ9 and Vδ2-specific antibodies revealed that ZA / IL-2 addition specifically induces the accumulation of Vγ9Vδ2 T cells. After 14 days, the CD3+ lymphocyte population could comprise up to 80% Vγ9Vδ2 T cells. A subset of Vγ9Vδ2 T cells express CD16, and these cells are enriched 10–700-fold within the CD3+ lymphocyte population, depending on the donor. CD16+Vγ9+Vδ2+ T cells are enriched 10–600-fold more in cultures grown without ZA. We conclude that in vitro ZA / IL-2 treatment of PBMCs results in a significant proportion of ADCC-mediated upregulation of the FcγIII receptor CD16 in γδT cells.

[0660] Similar to NK cells, ZA / IL-2-amplified Vγ9Vδ2T cells are CD16 positive, and cell-bound antibodies trigger ADCC via the FcγRIII receptor. A series of experiments were conducted to evaluate whether Vγ9Vδ2T cells could co-induce effective ADCC with IMAB362.

[0661] PBMCs derived from two different donors (#1 and #2) were cultured in medium containing 300 U / ml IL-2 and with or without 1 μM ZA. After 14 days, cells were harvested and gradually increased concentrations (0.26 ng / ml–200 μg / ml) of IMAB362 were added to NUGC-4 cells expressing CLDN18.2. Specific killing was determined by a luciferase assay. ADCC was performed using 27 donors grown in medium with or without ZA at 300 U / ml IL-2, with NUGC-4 cells used as target cells. For each donor, EC50 was calculated from dose-response curves. 50 The value was calculated and scored in a scatter plot to determine the maximum specific kill rate at a dose of 200 μg / ml IMAB362.

[0662] Strong IMAB362-dependent ADCC activity against CLDN18.2-positive NUGC-4 cells was observed in PBMCs cultured for 14 days with ZA / IL-2. ADCC in ZA / IL-2-treated PBMC cultures was dependent on the presence of Vγ9Vδ2T cells. ADCC activity was reduced for most donors when cells were cultured without ZA. Residual ADCC activity in these cultures was NK-cell dependent. ADCC assays, conducted on more than 20 donors, revealed that ZA / IL-2 treatment of PBMCs improved ECG. 50 And maximum specific kill rate.

[0663] Example 10: Efficacy of IMAB362 in combination with gemcitabine in a mouse transfer model

[0664] To analyze the effect of IMAB362 in combination with gemcitabine on lung metastases in Patu8988S, 2x10 6 Patu8988S cells were intravenously injected into the tail vein to treat 12 Hsd: athymic naked-Foxn1 mice per group. nu Mice. Fourteen days after tumor cell injection, mice were treated for four weeks with 200 μg IMAB362 or PBS as a control every half week (iv / ip) plus a weekly dose of 100 mg / kg gemcitabine ip. Treatment with IMAB362 or PBS continued until 70 days after tumor cell injection, at which point the mice were sacrificed. Xenograft tumors in the lungs were analyzed by qPCR of human DNA in prepared lung tissue and by optical analysis of immunohistochemical staining with anti-human MHC-I antibody (clone EPR1394Y). Results showed that mice treated with IMAB362 plus gemcitabine had significantly reduced amounts of human DNA in their lungs. Figure 45 A), and the surface area of ​​lung slices stained for human MHC-I complex was significantly smaller than that of lungs in mice treated with an irrelevant antibody plus gemcitabine. Figure 45 B). Both methods revealed a reduction in tumor load of Patu8988s xenografts in the lungs of mice treated with IMAB362 plus gemcitabine, demonstrating that the combination with IMAB362 was significantly superior to gemcitabine monotherapy.

[0665]

[0666] Instructions regarding microbial preservation or other biological materials

[0667] (PCT Rules 13-2)

[0668]

[0669]

[0670] PCT / RO / 134 form (July 1998, reprinted January 2004)

[0671] New international patent applications

[0672] Canimed Pharmaceuticals Co., Ltd., etc.

[0673] "Combination therapy involving anti-densin 18.2 antibodies for the treatment of cancer"

[0674] Our file number: 342-73PCT

[0675] Biomaterials Supplement

[0676] Other preservation certificates:

[0677] 1) The names and addresses of the depositary institutions for the deposits (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748) are as follows:

[0678] DSMZ - German Culture Collection

[0679] Mascheroder Weg 1b

[0680] 38124Braunschweig

[0681] DE

[0682] 2) The names and addresses of the depositary institutions for the deposits (DSM ACC2808, DSM ACC2809, DSM ACC2810) are as follows:

[0683] DSMZ - German Culture Collection

[0684] Inhoffenstr.7 B

[0685] 38124 Braunschweig

[0686] DE

[0687]

[0688] All other notes regarding the preservation mentioned above:

[0689] - Mouse (mice) myeloma P3X63Ag8U.1 fused with mouse (mice) spleen cells.

[0690] hybridomas that secrete antibodies against human micin-18A2.

[0691] 3) Depositor:

[0692] All the deposits mentioned above are submitted by the following:

[0693] Canimed Pharmaceuticals Inc.

[0694] Freiligrathstraβe 12

[0695] 55131 Mainz

[0696] DE sequence list <110> Canimed Pharmaceuticals Inc. TRON – Johannes Gutenberg University Mainz Translational Oncology Medical School Public Welfare Co., Ltd. <120> Combination therapy involving anti-densin 18.2 antibodies for the treatment of cancer <130> 342-73 PCT <150> PCT / EP2013 / 000505 <151> 2013-02-20 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 2 <211> 261 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn 1 5 10 <210> 4 <211> 11 <212> PRT <213> Homo sapiens <400> 4 Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln 1 5 10 <210> 5 <211> 14 <212> PRT <213> Homo sapiens <400> 5 Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe 1 5 10 <210> 6 <211> 13 <212> PRT <213> Homo sapiens <400> 6 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly 1 5 10 <210> 7 <211> 13 <212> PRT <213> Homo sapiens <400> 7 Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile 1 5 10 <210> 8 <211> 55 <212> PRT <213> Homo sapiens <400> 8 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala 1 5 10 15 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala 35 40 45 Met Leu Gln Ala Val Arg Ala 50 55 <210> 9 <211> 24 <212> PRT <213> Homo sapiens <400> 9 Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser Ala Lys 1 5 10 15 Ala Asn Met Thr Leu Thr Ser Gly 20 <210> 10 <211> 40 <212> PRT <213> Homo sapiens <400> 10 Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr 1 5 10 15 Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe 20 25 30 Gly Ala Ala Leu Phe Val Gly Trp 35 40 <210> 11 <211> 153 <212> PRT <213> Homo sapiens <400> 11 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala 1 5 10 15 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala 35 40 45 [[ID=зо]]Met Leu Gln Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly 50 55 60 Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile 65 70 75 80 Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly 85 90 95 Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val 100 105 110 Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met 115 120 125 Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 130 135 140 Phe Gly Ala Ala Leu Phe Val Gly Trp 145 150 <210> 12 <211> 107 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 13 <211> 326 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 13 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 1 5 10 15 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 20 25 30 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 35 40 45 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 50 55 60 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 65 70 75 80 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 85 90 95 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 100 105 110 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 14 <211> 466 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 14 Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe 35 40 45 Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 15 <211> 467 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 15 Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 16 <211> 465 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 16 Met Glu Trp Ile Trp Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 145 150 155 160 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 Lys 465 <210> 17 <211> 467 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 17 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 18 <211> 466 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 18 Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val 1 5 10 15 His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 20 25 30 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45 Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu 65 70 75 80 Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr 85 90 95 Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 100 105 110 Phe Cys Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 19 <211> 469 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 19 Met Asp Trp Ile Trp Ile Met Leu His Leu Leu Ala Ala Ala Thr Gly 1 5 10 15 Ile Gln Ser Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser 20 25 30 Pro Gly Ser Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val 35 40 45 Phe Pro Phe Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly 50 55 60 Phe Glu Trp Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr 65 70 75 80 Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser 85 90 95 Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala 100 105 110 Ile Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Lys 465 <210> 20 <211> 240 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 20 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> twenty one <211> 235 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> twenty one Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser 50 55 60 Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> twenty two <211> 234 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> twenty two Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser 20 25 30 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn 35 40 45 Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60 Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp 100 105 110 Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> twenty three <211> 240 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> twenty three Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> twenty four <211> 240 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> twenty four Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 25 <211> 239 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 25 Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 26 <211> 240 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 26 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr 100 105 110 His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 27 <211> 240 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 27 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 28 <211> 234 <212> PRT <213> artificial <220> <223> Description of artificial sequences: chimeric monoclonal antibodies <400> 28 Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr 1 5 10 15 Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser 20 25 30 Met Ser Val Gly Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn 35 40 45 Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro 50 55 60 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr 100 105 110 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 29 <211> 117 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 29 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 30 <211> 118 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 30 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 31 <211> 116 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 31 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 32 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 33 <211> 118 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 34 <211> 120 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: translation of PCR product <400> 34 Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe 20 25 30 Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp 35 40 45 Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys 50 55 60 Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala 65 70 75 80 Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 35 <211> 113 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 35 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 36 <211> 106 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 36 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 37 <211> 107 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 37 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 38 <211> 113 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 38 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 39 <211> 113 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 39 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 40 <211> 112 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 40 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 41 <211> 113 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 41 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln 85 90 95 Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 42 <211> 113 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 42 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 43 <211> 107 <212> PRT <213> artificial <220> <223> Description of artificial sequences: Translation of PCR products <400> 43 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 44 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 44 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr 1 5 10 15 <210> 45 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 45 Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 46 Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu 1 5 10 15 <210> 47 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 47 Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys 1 5 10 15 <210> 48 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 48 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser 1 5 10 15 <210> 49 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 49 Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr 1 5 10 15 <210> 50 <211> 15 <212> PRT <213> artificial <220> <223> Epitope <400> 50 Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg Gly 1 5 10 15 <210> 51 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 51 agagagctct ggcttcaccg agtg 24 <210> 52 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 52 ccagaagtta gtcaccagca tgttgg 26 <210> 53 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 53 gggataattt cagctgacta aacag 25 <210> 54 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 54 ttccgtttag ttaggtgcag ttatc 25

Claims

1. Use of a pharmaceutical combination in the preparation of a medicament for the treatment or prevention of CLDN18.2-positive pancreatic cancer in a patient, wherein the pharmaceutical combination comprises: (a) An antibody capable of binding to CLDN18.2 and mediating the killing of cells expressing CLDN18.2, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region having CDR1 at sites 45-52 of SEQ ID NO:17, CDR2 at sites 70-77 of SEQ ID NO:17, and CDR3 at sites 116-126 of SEQ ID NO:17, and the light chain variable region having CDR1 at sites 47-58 of SEQ ID NO:24, CDR2 at sites 76-78 of SEQ ID NO:24, and CDR3 at sites 115-123 of SEQ ID NO:24, and (b) Reagents that stabilize or increase CLDN18.2 expression, including gemcitabine or its salts. The antibody described therein is a chimeric mouse / human IgG1 monoclonal antibody comprising: κ, a mouse variable light chain; Human κ light chain constant region allotype Km(3); mouse heavy chain variable region; human IgG1 constant region, allotype G1m(3); The antibody capable of binding to CLDN18.2 and mediating the killing of cells expressing CLDN18.2 and the reagent stabilizing or increasing CLDN18.2 expression are formulated separately from each other, such that the reagent stabilizing or increasing CLDN18.2 expression can be applied before, simultaneously with, or after the application of the antibody capable of binding to CLDN18.2 and mediating the killing of cells expressing CLDN18.

2.

2. The use as described in claim 1, wherein the reagent for stabilizing or increasing CLDN18.2 expression further comprises taxane.

3. The use as described in claim 2, wherein the taxane comprises paclitaxel or albumin-bound paclitaxel.

4. The use as claimed in claim 1, further comprising an agent for stimulating γδT cells, wherein the agent for stimulating γδT cells is a bisphosphonate.

5. The use as claimed in claim 4, wherein the antibody capable of binding to CLDN18.2 and mediating the killing of cells expressing CLDN18.2, the reagent stabilizing or increasing CLDN18.2 expression, and the reagent stimulating γδT cells are formulated separately from each other.

6. The use as described in claim 4, wherein the reagent for stimulating γδT cells is a nitrogen-containing bisphosphonate.

7. The use as described in claim 4, wherein the bisphosphonate is an aminobisphosphonate.

8. The use as claimed in claim 4, wherein the reagent for stimulating γδT cells is selected from zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, opapadronic acid, alendronic acid, incadronic acid, and their salts.

9. The use as described in any one of claims 1-8, wherein the pancreatic cancer includes cancer of the pancreatic duct.

10. The use as described in any one of claims 1-8, wherein the pancreatic cancer is a malignant tumor originating from pancreatic epithelial cells.

11. The use as described in any one of claims 1-8, wherein the pancreatic cancer is a cancer originating from pancreatic gland tissue.

12. The use as described in any one of claims 1-8, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma, pancreatic mucinous adenocarcinoma, pancreatic neuroendocrine carcinoma, or pancreatic acinar cell carcinoma, or a combination thereof.

13. The use as described in any one of claims 1-8, wherein the pancreatic cancer is partially or completely untreatable with gemcitabine treatment.

14. The use as described in claim 13, wherein the gemcitabine treatment is gemcitabine monotherapy.

15. The use as described in any one of claims 1-8, wherein prevention of pancreatic cancer includes prevention of recurrence of pancreatic cancer.

16. The use as described in any one of claims 1-8, wherein the patient has undergone surgery for pancreatic cancer.

17. The use as claimed in any one of claims 1-8, wherein the patient has precancerous pancreatic lesions including malignant histological changes beginning in the pancreatic duct.

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