RU2792932C2 - Combination therapy using antibodies against claudin 18.2 for cancer treatment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine; it can be used for the treatment of precancerous lesion of the pancreas, characterized by pancreas cells expressing claudin 18 splicing variant 2 (hereinafter – CLDN18.2), in a patient. For this purpose, the patient is administered with (i) an antibody having a capability of binding to CLDN18.2 and (ii) an agent stabilizing or increasing CLDN18.2 expression. The antibody binds to CLDN18.2 and provides destruction of cells expressing CLDN18.2, wherein the antibody contains an antibody heavy chain containing an amino acid sequence represented with SEQ ID NO: 32 and an antibody light chain containing an amino acid sequence represented with SEQ ID NO: 39. The agent stabilizing or increasing CLDN18.2 expression is a nucleoside analogue selected from a group consisting of gemcitabine and its composite ester or salt. An option of the specified method, including additional administration to the patient of bisphosphonate, is also disclosed.

EFFECT: group of inventions provides effective treatment of precancerous lesion of the pancreas.

11 cl, 45 dwg, 21 tbl, 10 ex

Description

State of the art

Pancreatic cancer is one of the most lethal types of cancer. Mortality reaches 100% due to the tendency to early metastasis and due to the fact that the disease is very resistant to radiation and chemotherapy. With 27,000 new cases diagnosed each year in North America and 68,000 in Europe, there is an urgent need to develop new treatment strategies to reduce mortality in patients with pancreatic cancer.

Splice variant 2 of the tight junction molecule claudin 18 (claudin 18.2, or CLDN18.2) is a member of the claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa transmembrane protein containing four transmembrane domains with two small extracellular loops. In normal tissues, CLDN18.2 expression was not detected by RT-PCR, with the exception of the stomach. Immunohistochemistry using antibodies specific for CLDN18.2 showed that the only tissue, the stomach, gives a positive reaction. CLDN18.2 is a highly selective gastric lineage antigen that is exclusively expressed in short-lived differentiated gastric epithelial cells. CLDN18.2 is conserved during malignant transformation and therefore often protrudes on the surface of human gastric cancer cells. In addition, this pan-tumor antigen is activated ectopically at a significant level in adenocarcinoma cells of the esophagus, pancreas, and lungs.

Ganymed Pharmaceuticals AG has developed a chimeric IgG1 type antibody IMAB362 directed against CLDN18.2. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to other members of the family, including the closely related splice variant 1 claudin 18 (CLDN18.1). IMAB362 exhibits strong specificity for tumor cells and combines four independent highly active mechanisms of action. Once bound to the target, IMAB362 mediates cell killing through ADCC, CDC and induction of apoptosis induced by cross-linking of the target on the surface of tumor cells and direct inhibition of proliferation. At the same time, IMAB362 effectively induces the lysis of CLDN18.2-positive cells, including cells of human gastric cancer lines in vitro and in vivo.

The toxicity and PK/TK profile of IMAB362 has been extensively studied in mice and cynomolgus monkeys, including dose ranging studies, 28-day repeated dose toxicity studies in macaques, and 3-month repeated dose toxicity studies in mice. Both mice (maximum weekly treatment duration 3 months, maximum dose level 400 mg/kg) and cynomolgus monkeys (up to 5 weekly doses up to 100 mg/kg) tolerated repeated doses of IMAB362 IV well. There were no signs of systemic or local toxicity. In particular, no toxicity studies reported gastric toxicity. IMAB362 does not induce immune activation and release of cytokines. There were no negative effects on the reproductive organs in males or females. IMAB362 does not bind to non-targeted tissues. Biodistribution studies in mice have shown that the lack of gastric toxicity is most likely due to tight junction compartmentalization on the luminal side of normal gastric epithelium, which greatly impedes access to IMAB362 epitopes.

IMAB362 is in early clinical trials. Conducted phase I clinical trials in humans. 3 patients in 5 dose groups (33 mg/m ² , 100 mg/m ² , 300 mg/m ² , 600 mg/m ² , 1000 mg/m ² ) received single intravenous doses of IMAB362 and were observed for 28 days. IMAB362 was very well tolerated with no significant patient safety concerns. In one patient, all measurable tumor markers decreased significantly over 4 weeks post-treatment. In the current phase IIa clinical trial, IMAB362 is administered multiple times.

Further, data are provided herein indicating that chemotherapeutic agents can stabilize or enhance the expression of CLDN18.2 on the surface of pancreatic cancer cells, leading to an increase in the availability of CLDN18.2 for antibodies against CLDN18.2 type IMAB362. A synergistic effect of anti-CLDN18.2 antibodies of the IMAB362 type has been observed with certain chemotherapy drugs, in particular with chemotherapy drugs used in the treatment of pancreatic cancer. With chemotherapeutic treatment, human cancer cells become more susceptible to antibody-induced target-specific killing. In mouse tumor models, tumor control with anti-CLDN18.2 antibodies plus chemotherapy is superior to that with anti-CLDN18.2 antibodies alone as a single agent.

The essence of the invention

The present invention generally provides a combination therapy 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, as well as their metastases, in particular metastases of gastric cancer such as Krukenberg tumors, metastases in the peritoneum and metastases in the lymph nodes. Particularly preferred cancers are pancreatic cancer and its metastases.

In one aspect, the present invention provides a method for treating or preventing pancreatic cancer in a patient, comprising administering to the patient (i) an antibody having the ability to bind to CLDN18.2, and (ii) an agent that stabilizes or enhances expression, i.e. level CLDN18.2. Expression of CLDN18.2 preferably occurs on the cell surface of cancer cells. An agent that stabilizes or enhances the expression of CLDN18.2 may be administered before, simultaneously with, or after administration of an antibody having the ability to bind to CLDN18.2, or in combination with it.

The agent that stabilizes or enhances the expression of CLDN18.2 may be a cytotoxic and/or cytostatic agent. In one embodiment, an agent that stabilizes or enhances CLDN18.2 expression includes an agent that induces cell cycle arrest or accumulation of cells in one or more cell cycle phases, preferably one or more cell cycle phases other than G1 phase, such as S phase, a G2 phase or a combination thereof, or a combination of an S phase or a G2 phase with a G1 phase. An agent that stabilizes or enhances CLDN18.2 expression may include an agent selected from the group consisting of nucleoside analogs, platinum compounds, camptothecin analogs, and taxanes, prodrugs, salts, and combinations thereof. Nucleoside analogs can be selected from the group consisting of gemcitabine, 5-fluorouracil, their prodrugs and salts. The platinum compounds can be selected from the group consisting of oxaliplatin, cisplatin, their prodrugs and salts. Camptothecin analogs can be selected from the group consisting of irinotecan, topotecan, their prodrugs and salts. Taxanes can be selected from the group consisting of paclitaxel, docetaxel, their prodrugs and salts. An agent that stabilizes or enhances the expression of CLDN18.2 may include an agent selected from the group consisting of gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, their prodrugs, salts and combinations. An agent that stabilizes or enhances the expression of CLDN18.2 may include a combination of oxaliplatin and 5-fluorouracil or their prodrugs, a combination of cisplatin and 5-fluorouracil or their prodrugs, a combination of at least one taxane and oxaliplatin, a combination of at least at least one taxane and cisplatin, a combination of at least one taxane and 5-fluorouracil or prodrugs thereof, or a combination of at least one camptothecin analog and 5-fluorouracil or prodrugs thereof. An agent that stabilizes or enhances the expression of CLDN18.2 may include a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of oxaliplatin, 5-fluorouracil or prodrugs thereof, and irinotecan. Accordingly, the method of the 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 prodrugs thereof and irinotecan. In one embodiment, the method of the invention comprises administering folinic acid, 5-fluorouracil or prodrugs thereof, irinotecan, and oxaliplatin. An agent that stabilizes or enhances the expression of CLDN18.2 may include an agent that induces immunogenic cell death. The immunogenic cell death inducing agent may include oxaliplatin.

In another aspect of the present invention, there is provided a method for treating or preventing cancer in a patient, comprising administering to the patient (i) an antibody having the ability to bind to CLDN18.2 and (ii) gemcitabine. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases. The cancer may be a Krukenberg tumor, peritoneal metastases, and/or lymph node metastases. In one embodiment, the cancer is an adenocarcinoma, in particular an advanced adenocarcinoma. In one embodiment, the cancer is pancreatic cancer.

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

The method of the invention may further include administering at least one other chemotherapeutic agent, which may be a cytotoxic agent.

An antibody capable of binding to CLDN18.2 can bind to native CLDN18.2 epitopes on the surface of living cells. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, an antibody capable of binding to CLDN18.2 mediates cell killing by one or more of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cell cytotoxicity (ADCC) mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment, the antibody having the ability to bind to CLDN18.2 is a monoclonal, chimeric, or humanized antibody or antibody fragment. In one embodiment, the antibody mediates cell killing upon binding to cellular CLDN18.2, in particular CLDN18.2, which is expressed by cells on their cell surface, the cells preferably being cancer cells of the type described herein. In one embodiment, the antibody having the ability to bind to CLDN18.2 is an antibody selected from the group consisting of (i) antibodies generated and/or derived from a clone deposited under accession number DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) antibodies that are chimerized or humanized forms of the antibodies of (i) ), (iii) antibodies having the antibody specificity of claim (i), and (iv) antibodies containing an antigen-binding site or an antigen-binding site, in particular the variable region of the antibodies of claim (i), and preferably having antibody specificity of claim .(i). In one embodiment, the antibody is conjugated to a therapeutic agent, such as a toxin, radioisotope, drug, or cytotoxic agent.

In one embodiment, the method of the invention comprises administering an antibody capable of binding to CLDN18.2 at a dose of up to 1000 mg/m ² . In one embodiment, the method of the invention comprises repeated administration of an antibody capable of binding to CLDN18.2 at a dose of 300 to 600 mg/m ² .

According to the invention, CLDN18.2 preferably has the amino acid sequence according to SEQ ID NO: 1.

In one embodiment, the cancer described here is CLDN18.2 positive. In one embodiment, the cancer cells of the cancer described herein are CLDN18.2 positive. In one embodiment, cancer cells of the cancer described here express CLDN18.2 on their cell surface.

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 methods of the invention are for the simultaneous treatment of primary cancer and metastatic cancer, such as primary pancreatic cancer and metastatic pancreatic cancer. In one embodiment, metastatic cancer includes lymph node, ovarian, liver, or lung metastases, or combinations thereof. In one embodiment, the pancreatic cancer includes cancer of the pancreatic ducts. 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, pancreatic cancer is partially or completely refractory to treatment with gemcitabine such as gemcitabine monotherapy. In one embodiment, prevention of pancreatic cancer includes preventing the recurrence of pancreatic cancer.

In one embodiment, the patient being treated according to the invention has had surgery for pancreatic cancer. In one embodiment, the patient has precancerous lesions of the pancreas, in particular, precancerous lesions of the pancreas, including the occurrence of malignant histological changes in the pancreatic ducts. In these embodiments, the methods of the invention are preferably directed to preventing the development of malignant pancreatic cancer.

In another aspect of the present invention, there is provided a medicament for the treatment or prevention of pancreatic cancer, comprising: (i) an antibody having the ability to bind to CLDN18.2, and (ii) an agent that stabilizes or enhances the expression of CLDN18.2. The medicaments of the present invention may further comprise a γδ T-cell stimulating agent. The antibody capable of binding to CLDN18.2 and the agent that stabilizes or enhances the expression of CLDN18.2, and optionally the agent that stimulates γδ T-cells, may be present in the drug formulation as a mixture or separately from each other. The drug may be presented as a kit comprising a first container containing an antibody capable of binding to CLDN18.2 and a second container containing an agent that stabilizes or enhances the expression of CLDN18.2, and optionally a container containing an agent stimulating T-cells γδ. The medicament may further include printed instructions for the use of the drug in the treatment or prevention of pancreatic cancer, in particular for the use of the drug in the method of the invention. Various embodiments of drugs, in particular, antibodies with the ability to bind to CLDN18.2, agents that stabilize or enhance the expression of CLDN18.2, and agents that stimulate γδ T cells, have already been described above for the methods of the invention.

In a separate aspect of the present invention, medicinal preparations are provided, including: (i) an antibody having the ability to bind to CLDN18.2, and (ii) gemcitabine. The medicaments of the present invention may further comprise a γδ T-cell stimulating agent. The CLDN18.2 binding antibody and gemcitabine, and optionally the γδ T-cell stimulating agent, may be present in the drug formulation as a mixture or separately from each other. The drug may be used to treat or prevent cancer, such as pancreatic cancer. The drug may be presented as a kit comprising a first container containing an antibody capable of binding to CLDN18.2 and a second container containing gemcitabine, and optionally a container containing a γδ T-cell stimulating agent. The medicament may further include printed instructions for the use of the drug in the treatment or prevention of a cancer type of pancreatic cancer, in particular for the use of the drug in the method of the invention. Various embodiments of drugs, in particular antibodies with the ability to bind to CLDN18.2, agents that stabilize or enhance the expression of CLDN18.2, and agents that stimulate γδ T cells, have already been described above for the methods of the invention.

The present invention also provides agents described herein, such as antibodies having the ability to bind to CLDN18.2 and/or agents that stabilize or enhance the expression of CLDN18.2, for use in the methods described herein. For example, the present invention also provides antibodies capable of binding to CLDN18.2 for administration in combination with a gemcitabine-type agent that stabilizes or enhances CLDN18.2 expression, and optionally a γδ T-cell stimulating agent.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of the figures

Fig. 1. Lentiviral vector used for transduction of pancreatic cancer cells. Human CLDN18.2 was cloned downstream of the EF1α promoter. The expression cassette was inserted between the long terminal repeats (5' and 3-LTR) that facilitate packaging and reverse transcription of the viral mRNA. RSV: Rous sarcoma virus provides Tat-independent production of viral mRNA. Amp: ampicillin resistance gene. PGKp: blasticidin promoter. WPRE: woodchuck post-transcriptional regulatory element; increases the expression of the transgene. LTR: long terminal repeat, promotes virus packaging. SV40A promotes transcription termination and mRNA polyadenylation. pUC: bacterial vector backbone. Bla: ampicillin promoter.

Fig. 2. Analysis of pancreatic cell metastasis in mouse lungs. Scheme of lung opening in mice after intravenous administration of pancreatic cancer cells.

Fig. 3. Expression of CLDN18.2 in normal and cancerous pancreatic tissues. Staining of formalin-fixed paraffin-embedded (FFPE) normal pancreatic tissue (A) and pancreatic adenocarcinoma tissue (B) with mouse monoclonal antibody 35-22A (0.2 μg/ml). Counterstaining with hematoxylin (2:00 min). Magnification ×200.

Fig. 4. Expression of CLDN18.2 in normal and precancerous pancreatic tissues. 43-14A staining of various normal precancerous structures (A) and PanIN1; PanIN2 (B); PanIN3 (C). Magnification ×200.

Fig. 5. Pilot study. Correlation between CLDN18.2 signal intensity and the number of positive tumor cells in the analysis of primary pancreatic tumors. Each dot represents a case of primary pancreatic cancer as analyzed by staining FFPE sections with mouse monoclonal antibody 35-22A (0.2 μg/ml). The dotted line marks the value of 10%.

Fig. 6. Pilot study. Expression of CLDN18.2 in tissues of primary and metastatic pancreatic tumors. Staining of FFPE tissue sections (3 μm) with mouse monoclonal antibody 35-22A in primary adenocarcinoma tumor (A) and lymph node metastases (B). Counterstained with hematoxylin (Mayers).

Fig. 7. Main study. Correlation between CLDN18.2 signal intensity and the number of positive tumor cells in the analysis of primary pancreatic tumors. Each dot represents a case of primary pancreatic ductal adenocarcinoma tumor (black circles) or primary neuroendocrine tumor (light circles) as analyzed by staining FFPE sections with mouse monoclonal antibody 43-14A (0.2 μg/ml).

Fig. 8. Correlation between CLDN18.2 signal intensity and the number of positive tumor cells in the analysis of pancreatic metastases. Each dot represents a case of pancreatic cancer metastases in the lymph nodes (black circles) or liver (light circles) as analyzed by staining FFPE sections with mouse monoclonal antibody 43-14A (0.2 μg/ml). The dotted line marks the value of 10%.

Fig. 9. Expression of CLDN18.2 in tissues of primary and metastatic pancreatic tumors. Staining of FFPE tissue sections (3 μm) with mouse monoclonal antibody 43-14A in primary adenocarcinoma tumors (A, C, E) and their lymph node metastases (B, D, F). Sections counterstained with hematoxylin (Mayers).

Fig. 10. Graphical analysis. Expression of CLDN18.2 in tissues of primary pancreatic tumors and corresponding lymph node metastases.

Fig. 11. Expression of CLDN18.2 in tissues of primary pancreatic tumors and corresponding metastases. Staining of FFPE tissue sections (3 μm) from primary adenocarcinoma (A), liver metastases (B), and lymph node metastases (C) with mouse monoclonal antibody 43-14A. Sections counterstained with hematoxylin (Mayers). Magnification ×200.

Fig. 12. CLDN18.2 mRNA level in pancreatic carcinoma cell lines. (A) Expression analysis by quantitative PCR in various pancreatic CA cell lines, lentivirus (LVT) transduced cell lines (gray bars), gastric cancer cell line KATO-III (positive control) and breast cancer cell line SKBR-3 (negative). control). CLDN18.2 transcripts were amplified with gene-specific primers. Endogenous cell lines giving a relative expression level greater than 1×10 ⁵ were scored as CLDN18.2 positive (hatched bars). NTC: control sample with H ₂ O. Error bars: mean + SD. (BD) Analysis of passage-dependent expression of CLDN18.2 in Patu8988S (B), Panc05.04 (C) cells and indicated LVT cell lines (D). The passage number is indicated under each column.

Fig. 13. Level of CLDN18.2 protein in cell lysates of pancreatic carcinoma cell lines. Proteins were separated on 12.5% SDS-PAGE. Western blotting was performed with the CLDN18 antibody recognizing the C-terminal region of CLDN18.1 and CLDN18.2 (Zymed-MID) and with a control antibody recognizing β-actin. The exposure time is 140 sec (Pierce SuperSignal West Dura) and 20 sec (Pierce SuperSignal West Pico), respectively. (A) Detection of CLDN18 in pancreatic cell line lysates, positive control (HEK293-p740) and negative control (SKBR-3) cell lysates. (B) Comparison of CLDN18.2 expression between lysates of non-transduced parental cells and lysates of lentivirus (LVT) transduced cell lines. Patu8988S and SKBR-3 cells were added as positive and negative controls, respectively.

Fig. 14. Detection and cellular localization of CLDN18 expression in pancreatic cancer cells. Staining of pancreatic cancer cells grown on coverslips. Antibody: 35-22A (×20 magnification, exposure time indicated under each image). DAPI was used to stain the nuclei (blue). A: AsPC1; B: BxPC3; C: CFPAC; D: DANG; E: HPAF-II; F: HUP-T3; G: HUP-T4; H: KCI-M3; I: Panc1; J: Panc05.04; K: Panc02.04; L: Panc04.03; M: Patu8902; N: Patu8988S; A: Su86.86; P: Suit-2, Q: SW-1990; R: YAPC; S: gastric cancer control cell line KATO-III.

Fig. 15. Detection and cellular localization of CLDN18 expression in CLDN18.2 transduced pancreatic cancer cells. Detection of CLDN18 in lentivirus (LVT) transduced pancreatic cancer cells with 35-22A antibody after fixation and permeabilization. Secondary antibodies labeled with Alexa488 or Alexa555 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-CKO; H: YAPC-LVT.

Fig. 16. IMAB362 binding to the cell surface of CLDN18.2-positive pancreatic CA cell lines (pharmacodynamics). IF analysis of pancreatic cancer cell lines (A, B, D, E), lentivirus-transduced pancreatic cell lines (GL), and control KATO-III gastric cancer cells (C, F) expressing CLDN18.2. Cells were stained with IMAB362 under native conditions (D-E) and, for comparison, with 35-22A after cell fixation and permeabilization (A-C). DAPI was used to stain the nuclei. The exposure time is indicated on each panel. G: BxPC3-LVT; H: CAPAN1-LVT; I: DANG-LVT; J: MiaPaCa2-LVT; K: Patu8902-LVT; L: Suit2-LVT.

Fig. 17. Expression of CLDN18.2 in grafted tumors from various cell lines. Expression of CLDN18.2 in grafted tumors 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). Tissue staining was performed with the Zymed-MID antibody. Lens magnification ×10 (A, C, E, G, J, L) and ×20 (B, D, F, H, K, M).

Fig. 18. Checking the engraftment of cells of cancer lines Suit-2 and MiaPaCa2 of the pancreas. Cells were injected into the tail vein of nude mice. Animals were sacrificed 45 (A), 52 (B), 59 (C) days after administration of Suit-2 (A-C) or 59 (D), 66 (E), 73 (F) days after administration of MiaPaCa2 (D-F). Lungs were dissected and stained with anti-MHC class I antibodies (anti-human MHC I, clone EPR1394Y) to detect human cells in mouse tissues.

Fig. 19. Analysis of engraftment of metastases Patu8988S. Patu8988S cells were intravenously injected into Nu/Nu mice at 1×10 ⁶ or 2×10 ⁶ cells, and at different time points the lungs (A) and liver (B) were removed from the mice, as indicated under the x-axis. To calculate the % presence of human DNA in each tissue preparation, a standard curve was generated by mixing human and mouse DNA and making 7 x 5-fold dilutions to obtain from 100% (1) to 0.0064% (7) of human DNA.

Fig. 20. IHC analysis of Patu8988S metastases in mouse lung tissues. Mice were injected with Patu8988S cells in the tail vein and at various time intervals (A-D = 70 days, E-H = 86 days) were sacrificed, lung tissues were isolated and stained with anti-MHC-I antibody (EPR1394Y) (A, B, E, F ) at a 1:1000 dilution or against claudin 18 (Zymed-Mid) (C, D, G, H) at 0.2 µg/ml. Magnification: A, C, E, G = ×10 and B, D, F, H = ×20.

Fig. 21. IMAB362 mediated apoptosis of gemcitabine-treated pancreatic cancer cells. Apoptosis was induced by cross-linking CLDN18.2 on BxPC3~CLDN18 cells after 48 hours. BxPC3~CLDN18 cells were cultured in medium without or + 100 ng/ml gemcitabine. The proportion of apoptotic cells in the fraction of mononuclear cells shifted. A similar shift was observed when tumor cells were incubated with camptothecin.

Fig. 22. Severity of IMAB362-induced ADCC activity on pancreatic cancer cells. (A) ADCC was performed on CLDN18.2 positive pancreatic cancer lines using PBMCs from different donors. (B-F) ADCC was performed with LVT pancreatic cancer cell lines ectopically expressing CLDN18.2 and corresponding parental cells. (G): scatter plot.

Fig. 23. Severity of IMAB362-induced CDC activity on pancreatic cancer cells. (A) CDC was performed with pooled healthy human serum as complement source, IMAB362 and CLDN18.2-positive pancreatic control CDOK1-p740 cells in 4 independent experiments. (B) CDC was performed with CLDN18.2 positive (Patu8988S, DANG, Panc05.04) and CLDN18.2 negative (CAPAN1, Suit2, BxPC3, YAPC) pancreatic cell lines. (C) CDC was performed with ectopically expressing LVT cell lines. (D) Scatter plot showing concentrations of IMAB362 causing half-maximal lysis (EC ₅₀ ) of pancreatic cancer cell lines. (E) Maximum cell killing obtained with IMAB362 on pancreatic cancer cell lines.

Fig. 24. Effect of IMAB362 treatment on subcutaneous MiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumors were grafted by injecting 1x10 ⁷ MiaPaCa2-LVT cells subcutaneously into the flank of 15 female Hsd:Foxn1 ^nu nude mice per treatment group. On the third day after the introduction of cancer cells began treatment with 200 μg of IMAB362 or controls, respectively. The treatment was continued 2 times a week, alternating intravenously and intravenously until the animals were slaughtered. (A) Effect of IMAB362 treatment on tumor growth. The size of SC tumors was measured 2 times a week (mean + SEM). (B) Kaplan-Meier survival plots. Mice were sacrificed when the volume of the tumors reached 1400 mm ³ or the tumors became ulcerated.

Fig. 25. IMAB362 treatment of subcutaneous BxPC3-LVT xenografts. BxPC3-LVT xenograft tumors were grafted by injecting 1×10 ⁷ BxPC3-LVT cells subcutaneously into the flank of 15 female nude Hsd:Foxn1 ^nu nude mice per treatment group. On the third day after the introduction of cancer cells began treatment with 200 μg of IMAB362 or controls, respectively. The treatment was continued 2 times a week, alternating intravenously and intravenously until the animals were slaughtered. (A) Effect of IMAB362 treatment on tumor growth. The size of SC tumors was measured 2 times a week (mean + SEM). (B) Kaplan-Meier survival plots. Mice were sacrificed when the volume of the tumors reached 1400 mm ³ or the tumors became ulcerated.

Fig. 26. Effect of IMAB362 treatment on the growth of Suit2-LVT pancreatic metastases. 2×10 ⁶ Suit2-LVT cancer cells were intravenously injected into the tail vein of 12 female nude Hsd:Foxn1 ^nu nude mice per treatment group. On the third day after the introduction of cancer cells, treatment with 200 μg of IMAB362, 200 μg of isotype control, or an equal volume of PBS was started. Animals were killed on the 42nd day after transplantation. (A) Quantitative PCR analysis (mean of 2-4 reactions per sample) to determine the percentage of human DNA in mouse lung samples. (B) Percentage of human lung surface in mice as measured by planimetry. Immunohistochemical staining of human cells on tissue sections with anti-human MHC class I antibody. *p<0.05 (Kruskal-Wallis test). Error bars: mean ± SD.

Fig. 27. Analysis by quantitative PCR and IHC of Patu8988S lung metastases. Mice were injected with 2×10 ⁶ Patu8988S cells per mouse. After 65 days, the animals were slaughtered. Open circles: mice were sacrificed after 63 days. (A) Mice were treated twice a week with 200 µg of IMAB362 or control saline. The amount of human DNA (ng) as determined by quantitative PCR was calculated from Ct values. (B) Repeat the q-PCR experiment as described in A. In this case, the percentage of human DNA in mouse DNA was calculated from Ct values. (C) Mice were treated with IMAB362 and the same isotype control antibody (rituximab). The percentage of human DNA in the lungs of mice was calculated from the Ct values. The IMAB362 group had one outlier (light triangle). Significance is given both with and without outlier values. (D/E) Same experiment as in C. In this case, the area of metastases was determined using the Image J program. In the scatter plots, the significance of IMAB362 inhibition is presented both with the inclusion (D) and the exclusion (E) of the outlier value. p-values: unpaired t-test. Error bars: mean ± SD.

Fig. 28. Dose-response curves for gemcitabine. Cells of pancreatic cancer lines show very large differences in sensitivity to gemcitabine. Cell lines were exposed to various concentrations of gemcitabine for 4 days and inhibition of proliferation was determined by viability assay.

Fig. 29. Dose-response curves for oxaliplatin. Cells of pancreatic cancer lines show very large differences in sensitivity to oxaliplatin. Cell lines were exposed to various concentrations of oxaliplatin for 4 days and inhibition of proliferation was determined by viability assay.

Fig. 30. Effect of treatment with chemotherapeutic agents on the expression of CLDN18.2 (RNA). RNA from untreated and pre-treated with Gem (1 ng/mL) or GemOx (Gem 1 ng/mL + Ox 10 ng/mL) DANG cells (2 days) (A) or Patu8988S (B) when treated for 3 days with Gem ( 10 ng/mL or GemOx (Gem 10 ng/mL + Ox 100 ng/mL) RNA was converted into cDNA and the level of CLDN18.2 transcripts was determined by quantitative real-time PCR. HPRT farms.

Fig. 31. Effect of chemotherapy on the level of CLDN18.2 protein in pancreatic carcinoma cells. CLDN18.2 expression was analyzed for total protein from cell lysates of untreated (med) and pre-treated with Gem (1 ng/mL) or GemOx (GEM 1 ng/mL + Ox 10 ng/mL) DANG or Patu8988S cells when detected by polyclonal antisera Zymed C-term. Actin was used to check for uniform protein deposition.

Fig. 32. FACS analysis of CLDN18.2 expression on the cell surface. CLDN18 expression (dark histogram) when cultured with Patu8988S in medium (left) and treated with Gem (right) is plotted overlaying the isotype control. Patu8988S cells were treated with gemcitabine (10 ng/ml) for 3 days.

Fig. 33. Cell cycle analysis of DANG cells treated or not treated with gemcitabine (GEM; 2 ng/mL) or gemcitabine + oxaliplatin (GemOx; 1 ng/mL + 10 ng/mL) for two days. (A) Treatment with gemcitabine results in cell cycle arrest of cells in S phase. The area of each bar is subdivided to show the percentage of cells in G0/G1, S, and G2 phases. (B) Western blotting showed upregulation of CLDN18 after Gem treatment.

Fig. 34. Effect of gemcitabine on cell cycle (A) and CLDN18.2 expression (B, C) in Patu8988S cells. Patu8988S cells were treated or not treated with gemcitabine (10 ng/ml) for 2 days. (A) The area of each bar is subdivided to show the percentage of cells in the G0/G1, S, and G2 phases. The graph shows the density of CLDN18.2 (on the x-axis) versus the number of cells (on the y-axis). (B) Expression of CLDN18.2 without treatment (dashed line) compared to Gem treatment (solid line). (C) CLDN18.2 expression in Gem-treated Patu8988S cells in G0/G1 phase (dashed line) compared to cells in S phase (solid line).

Fig. 35. Effect of chemotherapy on gastric cancer cells. Cultivation of KATO-III cells for 96 h results in cell cycle arrest in the G0/G1 phase (a) and downregulation of CLDN18.2 (c). Cytostatic compounds that induce cell cycle arrest at various phases of the cell cycle stabilize CLDN18.2 expression (c).

Fig. 36. Effect of chemotherapy on gastric cancer cells. Cytostatic compounds that cause cell cycle arrest in various phases of the cell cycle: S/G2 phase (irinotecan) or G2 phase (docetaxel). The area of each bar is subdivided to show the percentage of cells in the G0/G1, S, and G2 phases.

Fig. 37. Dose response curves for IMAB362 mediated ADCC following chemotherapeutic treatment of DANG cells. (A) Dose response curves from one representative donor after pretreatment of pancreatic DANG cancer cells with Gem or GemOx for 40 h. (B) EC ₅₀ values (mean) for IMAB362 mediated ADCC. p-values: unpaired t-test.

Fig. 38. Effect of chemotherapy on gastric cancer cells. a: Cells treated with irinotecan, docetaxel, or cisplatin show a lower level of viable cells compared to cultured target cells. c/d: Expression of CLDN18.2 in cells treated with irinotecan, docetaxel or cisplatin increased compared to cells cultured in medium. c/d: Treatment of cells with irinotecan, docetaxel, or cisplatin enhances the ability of IMAB362 to induce ADCC.

Fig. 39. Effect of chemotherapeutic agents on IMAB362 mediated CDC in MiaPaCa2-LVT cells. Dose-response curves from 2 independent experiments. MiaPaCa2-LVT was cultured in either medium with Gem (10 ng/mL) or GemOx (10 ng/mL Gem + 100 ng/mL Ox) for 70 h.

Fig. 40. Effect of chemotherapy on IMAB362-induced CDC.

Fig. 41. Effect of IMAB362 treatment in combination with Gem or GemOx on BxPC3-LVT xenografts. BxPC3-LVT xenograft tumors were grafted by injection of 8.5×10 ⁶ BxPC3-LVT cells subcutaneously into the flank of 10 female Hsd:Foxn1 ^nu nude mice per treatment group. On the third day after the injection of cancer cells, chemotherapy was started (50 mg/kg gemcitabine ip or 50 mg/kg gemcitabine plus 5 mg/kg oxaliplatin ip, respectively) and continued once a week for 6 weeks. 24 hours after the administration of chemotherapeutic agents, 800 μg of IMAB362 or control was administered intravenously into the tail vein. Treatment with IMAB362 was continued once a week until the mice were sacrificed. (A) Growth curves of subcutaneous BxPC3-LVT xenografts. The size of SC tumors was measured twice a week (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when the volume of the tumors reached 1400 mm ³ or the tumors became ulcerated.

Fig. 42. Increased antitumor efficacy when gemcitabine is combined with IMAB362. BxPC3-LVT xenograft tumors were grafted by injection of 8.5×10 ⁶ BxPC3-LVT cells subcutaneously into the flank of 10 female Hsd:Foxn1 ^nu nude mice per treatment group. On the third day after the injection of cancer cells, chemotherapy was started (100 mg/kg gemcitabine ip or 100 mg/kg gemcitabine plus 5 mg/kg oxaliplatin ip, respectively) and continued once a week for 6 weeks. 24 hours after the administration of chemotherapeutic agents, 200 μg (1/2 dose) or 400 μg (full dose) of IMAB362 were injected intravenously into the tail vein. Treatment with IMAB362 was continued 2 times a week, alternating i.p. and i.v. administration until the mice were sacrificed. (A) Growth curves of subcutaneous BxPC3-LVT xenografts. The size of SC tumors was measured twice a week (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when the volume of the tumors reached 1400 mm ³ or the tumors became ulcerated.

Fig. 43. Effect of IMAB362 treatment in combination with gemcitabine on MiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumors were grafted by injection of 5×10 ⁶ MiaPaCa2-LVT cells subcutaneously into the flank of 10 female nude Hsd:Foxn1 ^nu nude mice per treatment group. Four days after the injection of cancer cells, chemotherapy (50 mg/kg gemcitabine ip) was started and continued once a week for 6 weeks. 24 hours after the administration of chemotherapeutic agents, 200 μg of IMAB362 or control was administered intravenously into the tail vein. Treatment with IMAB362 was continued 2 times a week, alternating i.p. and i.v. administration until the mice were sacrificed. (A) Growth of subcutaneous xenografts. Tumor size was measured twice a week (mean + SEM). (B) Kaplan-Meier survival curves. Mice were sacrificed when the volume of the tumors reached 1400 mm ³ or the tumors became ulcerated.

Fig. 44. Effect of IMAB362 treatment in combination with gemcitabine on grafted MiaPaCa2-LVT xenograft tumors. MiaPaCa2-LVT xenograft tumors were grafted by injection of 1×10 ⁷ MiaPaCa2-LVT cells subcutaneously into the flank of female nude Hsd:Foxn1 ^nu nude mice. Nine days after subcutaneous inoculation, tumor-bearing mice were divided into uniform treatment groups of 8 animals per group and treatment was started. Mice were injected with 150 mg/kg of gemcitabine ip 2 times a week for 4 weeks. 24 hours after the administration of gemcitabine, 200 μg of IMAB362 or control was administered intravenously into the tail vein. Treatment with IMAB362 was continued 2 times a week, alternating i.p. and i.v. administration until the mice were sacrificed. (A) The size of subcutaneous tumors was measured twice a week (mean + SEM; ** = p<0.01). (B) Kaplan-Meier survival curves. Mice were sacrificed when tumor volume reached 1400 mm ³ or tumors became ulcerated (Mantel-Cox log-rank test; **=p<0.01).

Fig. 45. Effect of IMAB362 in combination with gemcitabine on lung metastases in a Patu8988S xenotransplantation model. 2×10 ⁶ Patu8988S cancer cells were intravenously injected into the tail vein of 12 female nude Hsd:Foxn1 ^nu nude mice per treatment group. 2 weeks after intravenous administration of cancer cells, treatment with IMAB362 200 μg 2 times a week (alternating iv-ip) was started in combination with the introduction of 100 mg/kg gemcitabine ip 2 times a week for 4 weeks. The control group received 200 μg of the control antibody of the same isotype in combination with 100 mg/kg of gemcitabine twice a week. Animals were sacrificed on the 70th day after transplantation. (A) PCR quantitation (average of 3 reactions per sample) of human DNA in lung samples from IMAB362 and isotype antibody treated mice. (B) Percentage of human lung surface covered in mice was determined based on computer analysis. Immunohistochemical staining was performed with anti-human MHC-I antibody (clone EPR1394Y) on paraffin-embedded lung tissues (mean ± SEM; p = 0.0003, Mann-Whitney test). C and D: Examples of immunohistochemical staining with anti-human MHC-I antibody of Patu8988S lung metastases in mice treated with IMAB362 + gemcitabine (C) or isotype antibody + gemcitabine (D).

Disclosure of the essence of the invention

Although the present invention is described in detail below, it should be borne in mind that it is not limited to the specific methods, techniques and reagents described here, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and should not limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as are widely known to those of ordinary skill in the art.

Next, all elements of the present invention will be described. These elements are given along with specific embodiments, however, it should be borne in mind that they can be combined in any way and in any quantity to create additional embodiments. The examples and preferred embodiments given are not to be construed as limiting the present invention to the embodiments expressly described. Such description is to be understood as supporting and covering such embodiments in which the expressly described embodiments are combined with any number of recited and/or preferred elements. Moreover, any permutations and combinations of all described elements in this application should be considered as disclosed by the description in this application, unless the context requires otherwise.

Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).Preferably, the terms used here are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, HGW Leuenberger, B. Nagel, and H.

Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

In the practice of the present invention, unless otherwise indicated, standard methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, as described in the literature in this field, should be used (for example, see Molecular Cloning: A Laboratory Manual, ^2nd Edition). , J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

Throughout the description and the claims that follow, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” should be understood to mean the inclusion of a specified element, number or step, or group of elements, numbers or steps, but not the exclusion of any other element, number or step or group of elements, numbers or steps, although in some embodiments such elements, numbers or steps or groups of elements, numbers or steps may be excluded, i. the question is to include a specified element, number, or stage, or a group of elements, numbers, or stages. Singular forms when describing the invention (especially in the context of a claim) should be understood to encompass both the singular and the plural, unless otherwise indicated expressly or clearly contrary to the context. Specifying ranges of values here is just a shorthand way of individually specifying all the individual values that fall within that range. Unless otherwise stated, each individual value is included in the description as if it were listed separately. All methods described here can be performed in any suitable order, unless otherwise noted or clearly inconsistent with the context. The use of any examples or typical expressions (such as "such as") provided herein serves only to better disclose the invention and does not limit the scope of the invention as claimed in the claims. No expressions in the description should be understood as indicating any unclaimed element necessary for the practice of the invention.

In the text of this description, some documents are cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, and the like), whether above or below, is hereby incorporated by reference in its entirety. Nothing, however, shall be construed as suggesting that the invention cannot anticipate such descriptions in view of a prior invention.

The term “CLDN18” refers to claudin 18 and includes any variants including splicing variant 1 of claudin 18 (claudin 18.1 (CLDN18.1)) and splicing variant 2 of claudin 18 (claudin 18.2 (CLDN18.2)).

The term "CLDN18.2" preferably refers to human CLDN18.2, in particular a protein containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 from a sequence listing or variant of that amino acid sequence.

The term “CLDN18.1” preferably refers to human CLDN18.1, in particular a protein containing, preferably consisting of, the amino acid sequence of SEQ ID NO: 2 from a sequence listing or variant of that amino acid sequence.

The term "variant" according to the invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those that occur in nature. An allelic variant means a change in the normal sequence of a gene, the meaning of which is often unclear. Whole gene sequencing often identifies multiple allelic variants of a given gene. Species homologue means that a nucleotide or amino acid sequence is from a different species than the given nucleotide or amino acid sequence. The term "variant" also encompasses any post-translationally modified variants and conformational variants.

In accordance with the invention, the term "CLDN18.2-positive cancer" means a cancer involving cancer cells expressing CLDN18.2, preferably on the surface of these cancer cells.

"Cell surface" is used in accordance with its usual meaning in this area, so that it includes the outer surface of the cell, which is available for binding to proteins and other molecules. For example, a transmembrane protein having one or more extracellular regions is considered to be expressed on the cell surface.

CLDN18.2 is expressed on the cell surface if it resides on the surface of these cells and is available for binding to CLDN18.2-specific antibodies when added to the cells.

In accordance with the invention, CLDN18.2 is practically not expressed in cells if its expression level is lower compared to expression in cells of the stomach or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of the expression level in gastric cells or gastric tissue, or even lower. Preferably, CLDN18.2 is not substantially expressed in cells if its expression level exceeds the expression level in another non-cancerous tissue other than the stomach by no more than 2 times, preferably 1.5 times, and preferably does not exceed the expression level in this non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in cells if its expression level is below the detection limit and/or if the expression level is too low to bind CLDN18.2-specific antibodies when added to cells.

According to the invention, CLDN18.2 is expressed in cells if its expression level exceeds that in other non-cancerous tissue other than the stomach, preferably more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10,000-fold. Preferably, CLDN18.2 is expressed in cells if its expression level is above the detection limit and/or if the expression level is high enough to bind CLDN18.2-specific antibodies when added to the cells. Preferably, when CLDN18.2 is expressed in cells, it is expressed or exposed on the surface of these cells.

In accordance with the invention, the term "disease" means any pathological condition, including cancer, in particular the forms of cancer described here. Any reference to cancer or certain forms of cancer also includes their metastases. In a preferred embodiment, the disease to be treated according to the present application involves cells expressing CLDN18.2.

"Diseases associated with cells expressing CLDN18.2" or similar expressions mean in accordance with the invention that CLDN18.2 is expressed in cells of diseased tissues or organs. In one embodiment, the expression of CLDN18.2 in the cells of the affected tissues or organs is increased compared to a healthy tissue or organ. Increase means an increase of at least 10%, in particular at least 20%, 50%, 100%, 200%, 500%, 1000%, 10000% or more. In one embodiment, expression occurs only in diseased tissue, while expression is downregulated in the corresponding healthy tissue. For example, CLDN18.2 is expressed in pancreatic cancer tissue, but it is not detected in non-cancerous pancreatic tissue. In accordance with the invention, diseases associated with cells expressing CLDN18.2 include cancers. In addition, according to the invention, cancers are preferably those in which CLDN18.2 is expressed in cancer cells.

In the present invention, "cancer disease" or "cancer" includes those diseases that are characterized by abnormal regulation of cell growth, proliferation, differentiation, adhesion and/or migration. By "cancer cells" we mean abnormal cells that grow in a process of rapid, uncontrolled cell proliferation and continue to grow after the stimulus that caused the tumor to grow is stopped. Preferably, the "cancer disease" is characterized by the presence of cells expressing CLDN18.2, wherein the cancer cells express CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, preferably the cancers described here.

In accordance with the invention, "carcinoma" means a malignant tumor derived from epithelial cells.

“Adenocarcinoma” means a cancer that originates in glandular tissue. Such tissues are also included in a larger category of tissues known as epithelial tissues. Epithelial tissue includes the skin, glands, and a variety of other tissues that line the cavities and organs of the body. Embryologically, the epithelium is derived from the ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, cells do not have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. A well-differentiated adenocarcinoma usually resembles the glandular tissue from which it originates, while a poorly differentiated one may not. By staining the cells from the biopsy, the pathologist must determine whether the tumor is an adenocarcinoma or belongs to some other type of cancer. Adenocarcinoma can occur in many body tissues due to the ubiquitous nature of glands in the body. Although not every gland can secrete the same substance, however, if the cells perform exocrine functions, then they are considered glandular, and their malignant forms are called adenocarcinomas. Malignant adenocarcinomas invade other tissues and often metastasize if given enough time to do so.

The pancreas is an organ of endodermal origin and is a key regulator of protein and carbohydrate breakdown and glucose homeostasis. The exocrine part of the pancreas (80% of the mass of the entire organ) consists of an extensive network of acinar cells and duct cells that produce and deliver digestive enzymes to the gastrointestinal tract. Acinar cells, which form functional units along the network of ducts, synthesize and secrete enzymes into the lumen of the ducts in response to signals from the stomach and duodenum. In the acinar links near the ducts are centroacinar cells. The endocrine portion of the pancreas, which regulates glucose metabolism and homeostasis by releasing hormones into the blood, is made up of four specialized types of endocrine cells arranged in clusters called the islets of Langerhans.

Pancreatic cancer is a malignant neoplasm derived from transformed cells that originate in the tissues that make up the pancreas. Pancreatic cancer is the fourth leading cause of cancer deaths in the United States and eighth worldwide. Initially, pancreatic cancer often causes no symptoms, and later symptoms are usually non-specific and variable. Therefore, pancreatic cancer is often not detected until advanced stages. Pancreatic cancer has a poor prognosis: at all stages combined, the 1-year and 5-year survival rates are 25% and 6%, respectively. With localized disease, the 5-year survival rate is approximately 20%, while the median survival for localized advanced disease and metastatic disease, which together account for more than 80% of patients, is about 10 and 6 months, respectively.

Pancreatic cancers include adenocarcinomas (tumors exhibiting glandular architecture) arising from the exocrine portion of the pancreas and neuroendocrine carcinomas arising from islet cells.

The most common form of pancreatic cancer, ductal adenocarcinoma, is usually characterized by moderately or poorly differentiated glandular structures on microscopic examination. Pancreatic ductal adenocarcinoma (PDAC) usually occurs in the head of the pancreas with infiltration into surrounding tissues, including the lymph nodes, spleen, and abdomen, and metastases to the liver and lungs. PDAC mainly exhibits a glandular profile with duct-like structures and varying degrees of atypia and cell differentiation. Less common subtypes of PDAC include colloidal, squamous glandular, or sarcomatoid histology. There are often regional differences in histology, grade, and degree of differentiation within an individual tumor. Even the smallest primary lesions usually show perineural and lymphovascular invasion, suggesting a tendency to spread early and far.

The second most common type of exocrine pancreatic cancer is mucosal. Mucinous adenocarcinoma produces a large amount of mucin, resulting in a cystic appearance on CT scans.

Neuroendocrine tumors of the pancreas arise from the hormone-producing cells (islet cells) of the pancreas. Acinar cell neoplasms arise from the acinar cells of the pancreas.

In accordance with the invention, the term "cancer" also includes cancer metastases from primary tumors such as primary pancreatic cancer. Thus, if, for example, pancreatic cancer is mentioned, then this also includes metastases of pancreatic cancer, for example, metastases in the lungs, liver and/or lymph nodes.

“Metastasis” refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastases is a very complex process and depends on the separation of malignant cells from the primary tumor, invasion into the extracellular matrix, penetration through the basement membranes of the endothelium to enter the body cavities and vessels, and then, after transfer with the blood, infiltration of target organs. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or their components may remain and create a metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "remote metastases", which means metastases remote from the primary tumor and the regional lymph node system. In one embodiment, the term "metastases" according to the invention refers to metastases in the lymph nodes. One particular type of metastasis that can be treated with the therapy of the invention is those originating from pancreatic cancer as the primary site. In preferred embodiments, such pancreatic cancer metastases are lymph node metastases, lung metastases, and/or liver metastases.

Krukenberg's tumor is a rare metastatic ovarian tumor accounting for 1% to 2% of all ovarian tumors. Krukenberg's tumor is a metastatic ovarian cricoid adenocarcinoma. In most cases of Krukenberg tumors (70%), the primary site is the stomach. The next most common primary sites are colon, appendix, and breast carcinomas (mainly invasive lobular carcinoma). Rare cases of Krukenberg tumors originating from carcinoma of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder/urinary duct have been reported.

Refractory is a cancer for which a particular treatment is ineffective, and it is either initially untreatable or ceases to be treatable over time.

By "treatment" is meant administering to a subject a compound or composition, or a combination of compounds or compositions, to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in the subject; stopping or slowing the disease in the subject; suppressing or slowing the development of a new disease in the subject; reducing the frequency or severity of symptoms and/or relapses in a subject who now or previously had the disease; and/or renewal, i.e. increasing the lifespan of the subject.

In particular, the term “treating a disease” includes treating, shortening, ameliorating, preventing, slowing or suppressing the development or aggravation of, or preventing or delaying the onset of a disease or its symptoms.

The term "patient" in accordance with the invention means the subject to be treated, in particular, sick subjects, including humans, primates or other animals, in particular mammals such as cows, horses, pigs, sheep, goats, dogs, cats or such rodents, like mice and rats. In a particularly preferred embodiment, the patient is a human.

The term "an agent that stabilizes or increases the expression of CLDN18.2" refers to such agents or combinations of agents, the administration of which to cells results in an increase in the level of RNA and/or protein of CLDN18.2 in these cells, preferably in an increase in the level of CLDN18.2 protein on the cell surface compared to the situation when the cells do not receive this agent or combination of agents. Preferably, the cells are cancer cells, in particular cancer cells expressing CLDN18.2, so they are the target of antibodies that bind CLDN18.2, such as cells of the types of cancer described here, in particular pancreatic cancer. The term "an agent that stabilizes or increases the expression of CLDN18.2", in particular, refers to such agents or combinations of agents, the administration of which to cells leads to an increase in the density of CLDN18.2 on the surface of these cells compared to the situation when the cells do not receive this agent or combinations of funds. “Stabilization of CLDN18.2 expression” specifically includes situations where the agent or combination of agents prevents or reduces the reduction in CLDN18.2 expression, e.g., CLDN18.2 expression will decrease without providing the agent or combination of agents, and providing the agent or the combination of agents prevents or reduces such a decrease in CLDN18.2 expression. “Upregulation of CLDN18.2 expression” specifically includes situations where an agent or combination of agents increases the expression of CLDN18.2, for example, CLDN18.2 expression will decrease, remain substantially constant, or increase without providing the agent or combination of agents, and providing the agent or combination of agents increases the expression of CLDN18.2 compared to the situation without providing the agent or combination of agents, resulting in higher expression compared to a situation where the expression of CLDN18.2 could decrease, remain almost constant or increase without providing the agent or combination of agents.

In accordance with the invention, the term "an agent that stabilizes or increases the expression of CLDN18.2" includes chemotherapeutic agents or combinations of chemotherapeutic agents such as cytostatics. Chemotherapeutic agents can affect cells in one of the following ways: (1) damage the DNA in cells so that they can no longer reproduce, (2) inhibit the synthesis of new DNA strands so that cells cannot replicate, (3) stop mitotic processes in cells so that the cells cannot divide into two cells.

According to the invention, the term "an agent that stabilizes or increases the expression of CLDN18.2" preferably refers to such agents or combinations of agents, such as cytostatic compounds or combinations of cytostatic compounds, the provision of which to cells, in particular cancer cells, causes the cells to stop or accumulate in one or more cell cycle phases, preferably in one or more cell cycle phases other than the G1 and G0 phases, preferably other than the G1 phase, preferably in one or more of the G2 or S phases of the cell cycle, such as phases G1/G2, S/G2, G2 or S of the cell cycle. The term "cells stop or accumulate in one or more phases of the cell cycle" means that the percentage of cells that are in one or more phases of the cell cycle increases. Each cell for its reproduction goes through a cycle consisting of four phases. The first phase is called G1, when the cell prepares to reproduce its chromosomes. The second stage is called S, and in this phase, DNA synthesis and DNA duplication occurs. The next phase is the G2 phase, when duplication of RNA and proteins occurs. The final stage is stage M, which consists in the actual cell division. In this final stage, the duplicated DNA and RNA separate and move to different poles of the cell, and the cell actually divides into two identical functional cells. Chemotherapy drugs that cause DNA damage usually result in cell accumulation
in phase G1 and/or G2. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, typically cause cells to accumulate in the S phase. Examples of such drugs are gemcitabine, 6-mercaptopurine, and 5-fluorouracil.

According to the invention, the term "an agent that stabilizes or increases the expression of CLDN18.2" includes nucleoside analogs such as gemcitabine, 5-fluorouracil or their prodrugs, platinum compounds such as cisplatin and oxaliplatin, taxanes such as paclitaxel and docetaxel, and camptothecin analogues such as irinotecan and topotecan, and combination preparations, such as combination preparations containing one or more of gemcitabine, oxaliplatin and 5-fluorouracil, such as combination preparations containing gemcitabine and oxaliplatin, gemcitabine and 5-fluorouracil , oxaliplatin and 5-fluorouracil, or other drug combinations described here. According to the invention, references to an agent that stabilizes or increases the expression of CLDN18.2, such as references to a nucleoside analog, a platinum compound or a camptothecin analog or a taxane, for example, references to gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan or paclitaxel, should also include their prodrug forms, such as esters, salts, or derivatives such as conjugates of these agents. Examples of these are conjugates of these agents with carrier substances, for example protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salts of these agents are pharmaceutically acceptable.

In one preferred embodiment, "an agent that stabilizes or increases the expression of CLDN18.2" is or includes an "immunogenic cell death inducing agent".

Under certain circumstances, cancer cells can enter a lethal stress pathway associated with the supply of a spatially and temporally specified pattern of signals that is decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140: 798-804). In this scenario, cancer cells are activated and produce signals that are sensed by innate immune effectors such as dendritic cells and trigger a cognate immune response that includes CD8+ T cells and IFN-γ signaling so that cancer cell death can elicit a productive anti-cancer immune response. These signals include pre-apoptotic effects on the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface, pre-apoptotic ATP secretion, and post-apoptotic release of the HMGB1 nuclear protein. In general, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and γ-irradiation are capable of inducing all of the signals that define ICD, whereas for cisplatin, for example, which is unable to induce CRT translocation from the ER to the surface of dying cells, i.e. process requiring ER stress, the assistance of thapsigargin, an ER stress inducer, is needed.

According to the invention, the term “immunogenic cell death inducing agent” refers to those agents or combinations of agents which, when given to cells, in particular cancer cells, are capable of inducing the entry of cells into a lethal stress pathway that ultimately elicits tumor-specific immune responses. . In particular, agents that induce immunogenic cell death, when provided to cells, cause the cells to supply a combination of signals specified in space and time, including, in particular, a preapoptotic effect on the endoplasmic reticulum (ER) chaperone - calreticulin (CRT) on the cell surface, preapoptotic secretion ATP and post-apoptotic release of the HMGB1 nuclear protein.

According to the invention, the term “immunogenic cell death inducing agent” includes anthracyclines and oxaliplatin.

The term “nucleoside analog” refers to structural nucleoside analogs, a category that includes both purine analogs and pyrimidine analogs.

The term "gemcitabine" means a compound which is a nucleoside analog of the following formula:

.

.

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

In accordance with the invention, gemcitabine is preferably administered intravenously. Preferably, gemcitabine is administered at doses of 0.5 to 2 g/m ² , preferably 0.8 to 1.5 g/m ² , more preferably 1 to 1.2 g/m ² body surface area. For example, gemcitabine can be administered at a dose of 1000 mg per square meter once a week for 7 out of 8 weeks, and then once a week for 3 out of 4 weeks.

The term “nucleoside analog” 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) means a compound that is a pyrimidine analog of the following formula:

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

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

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

According to the invention, the term “platinum compound” refers to compounds containing platinum in their platinum complex type structure and includes compounds such as cisplatin, carboplatin and oxaliplatin.

The term “cisplatin” or “cisplatin” refers to a cis-diaminochloroplatinum(II) (CDDP) compound of the following formula:

The term “carboplatin” refers to a cis-diamine-(1,1-cyclobutanedicarboxylate)platinum(II) compound of the following formula:

The term "oxaliplatin" refers to a compound which is a platinum compound complexed with a diaminocyclohexane-bearing ligand according to the following formula:

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

Taxanes are a class of diterpene compounds that were first obtained from natural sources such as plants from the genus Taxus, but some have been artificially synthesized. The main mechanism of action of drugs of the taxane class is a violation of the function of microtubules, which inhibits the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

According to the invention, the term "docetaxel" refers to a compound having the following formula:

.

.

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

According to the invention, the term "paclitaxel" refers to a compound having the following formula:

.

.

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

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

In accordance with the invention, the preferred analogues of camptothecin are inhibitors of the enzyme DNA topoisomerase I (topo I). Preferred analogues of camptothecin according to the invention are irinotecan and topotecan.

Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. From a chemical point of view, it is a semi-synthetic analogue of the natural alkaloid camptothecin, which has the following formula:

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

Topotecan is a topoisomerase inhibitor with the formula:

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

Anthracyclines are a class of drugs that are widely used in cancer chemotherapy and are also antibiotics. Structurally, all anthracyclines share the common 4-ring structure of 7,8,9,10-tetrahydrotetracen-5,12-quinone and generally require glycosylation at specific sites.

Anthracyclines preferably act by one or more of the following mechanisms of action: 1) inhibition of DNA and RNA synthesis by intercalation between base pairs in the DNA/RNA strand, thereby preventing the replication of rapidly growing cancer cells; 2) inhibition of the enzyme topoisomerase II, which prevents the unwinding of supercoiled DNA and thereby blocks transcription and DNA replication; 3) iron-mediated generation of oxygen free radicals that damage DNA and cell membranes.

According to the invention, the term "anthracycline" preferably refers to such agents, preferably anti-cancer agents, which induce apoptosis, preferably by inhibiting DNA unwinding by topoisomerase II.

Preferably, according to the invention, the term "anthracycline" generally refers to a class of compounds having the following ring structure

including their analogues and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs.

Examples of anthracyclines and anthracycline analogues include, without limitation, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin, N-trifluoroacetyl-doxorubicin-14-valerate, aclacinomycin, morpholinodoxorubicin (morpholino-DOX) , cyanomorpholinodoxorubicin (cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and aclacinomycin (acclarubicin). Mitoxantrone is a member of the class of anthracenedione compounds, which are anthracycline analogs that do not contain the saccharide moiety of anthracyclines but retain the planar structure of the polycyclic aromatic ring, which allows intercalation into DNA.

Particularly preferred anthracyclines according to the invention are compounds of the following formula:

where: R ₁ is selected from the group consisting of H and OH, R ₂ from the group consisting of H and OMe, R ₃ from the group consisting of H and OH, and R ₄ from the group consisting of H and OH .

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

A particularly preferred anthracycline in the present invention is epirubicin, an anthracycline preparation which has the following formula:

and is marketed under the brand name Ellence in the US and Pharmorubicin or Epirubicin Ebewe in other countries. In particular, the term "epirubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6 ,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracen-5,12-dione. Epirubicin is preferred over doxorubicin, the most popular anthracycline, in some chemotherapy regimens because it causes fewer side effects.

According to the invention, the agent that stabilizes or increases the expression of CLDN18.2 may be a chemotherapeutic agent, in particular a chemotherapeutic agent recognized for the treatment of cancer, which may be included in drug combinations such as combinations of drugs recognized for use in the treatment of cancer. Such drug combinations may be drug combinations used in chemotherapy, such as drug combinations used in the FOLFIRINOX chemotherapy regimen.

The drug combination used in FOLFIRINOX chemotherapy includes leucovorin, fluorouracil, irinotecan (such as irinotecan hydrochloride), and oxaliplatin. Oxaliplatin can be administered at a dose of 85 mg/m ² body surface area; irinotecan at a dose of 180 mg/m ² ; leucovorin at a dose of 400 mg/ ^m2 ; and fluorouracil at a dose of 400 mg/m ² as a bolus followed by the introduction of 5-fluorouracil at a dose of 2400 mg/m ² as a continuous infusion preferably over 46 hours, preferably every 2 weeks.

The term "folinic acid" or "leucovorin" refers to a compound that is used in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Thus, if the administration of 5-fluorouracil or a prodrug thereof is mentioned here, then such administration in one embodiment may include administration in combination with folinic acid. Folinic acid has the following formula:

In particular, this 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}pentadionic acid.

γδ T cells (gamma delta T cells) are a small subset of T cells that have specific T cell receptors (TCRs) on their surface. In most T cells, the TCRs consist of two glycoprotein chains called the TCR α- and β-chains. In contrast, in T cells, γδ TCRs consist of one γ chain and one δ chain. This group of T cells is usually much less common than αβ T cells. Human γδ T cells play an important role in stress surveillance responses such as infectious diseases and autoimmunity. Transformation-induced changes in tumors are also hypothesized to elicit stress surveillance responses mediated by γδ T-cells and enhance antitumor immunity. Importantly, antigen-activated γδ T cells at the site of injury provide cytokines (eg, INFγ, TNFα) and/or chemokines that mediate recruitment (recruitment) of other effector cells and exhibit direct effector functions such as cytotoxicity (via receptors death and cytolytic granule pathway) and ADCC.

Most γδ T cells in peripheral blood express the T cell receptor Vγ9Vδ2 (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates and are thought to play an early and critical role in the perception of “danger” when invaded by pathogens, as they greatly increase in many acute infections and can outnumber all other lymphocytes in a matter of days, for example, tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis and malaria.

γδ T cells respond to small non-peptide phosphorylated antigens (phosphoantigens) such as pyrophosphates synthesized in bacteria and isopentenyl pyrophosphates (IPPs) produced in mammalian cells via the mevalonate pathway. While IPP production in normal cells is insufficient to activate γδ T cells, dysregulation of the mevalonate pathway in tumor cells results in accumulation of IPP and activation of γδ T cells. IPPs can also be therapeutically increased by aminobisphosphonates, which inhibit the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). Among others, such aminobisphosphonates are zoledronic acid (ZA, zoledronate, Zometa™, Novartis), which is already used clinically in patients to treat osteoporosis and metastatic bone disease. Following in vitro treatment with PBMCs, ZA is especially taken up by monocytes. IPPs accumulate in monocytes, which differentiate into antigen-presenting cells that stimulate the development of γδ T cells. It is preferable to add interleukin-2 (IL-2) as a growth and survival factor for activated γδ T cells. Finally, some alkylated amines have been described to activate Vγ9Vδ2 T cells in vitro, but only at millimolar concentrations.

According to the invention, the term “γδ T-cell stimulating agent” refers to compounds that stimulate the development of γδ T-cells, in particular Vγ9Vδ2 T-cells, in vitro and/or in vivo, in particular by causing the activation and expansion of T- γδ cells. Preferably, the term refers to compounds which in vitro and/or in vivo increase the production of isopentenyl pyrophosphates (IPP) produced in mammalian cells, preferably by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS).

One preferred group of compounds that stimulate γδ T cells are the bisphosphonates, especially the nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

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

[1-Hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), i.e.
zoledronic acid, eg zoledronate;

(dichlorophosphonomethyl)phosphonic acid, eg clodronate;

{1-hydroxy-3-[methyl(pentyl)amino]propan-1,1-diyl}bis(phosphonic acid), i.e. ibandronic acid, eg ibandronate;

(3-amino-1-hydroxypropan-1,1-diyl)bis(phosphonic acid), i.e. pamidronic acid, eg pamidronate;

(1-Hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid, i.e. risedronic acid, such as risedronate;

(1-Hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, i.e. minodronic acid;

[3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), i.e. olpadronic acid;

[4-Amino-1-hydroxy-1-(hydroxy-oxydophosphoryl)butyl]phosphonic acid, i.e. alendronic acid, eg alendronate;

[(cycloheptylamino)methylene]bis(phosphonic acid), i.e. incadronic acid;

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), i.e. etidronic acid, eg etidronate; And

{[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), i.e. tiludronic acid.

According to the invention, a particularly preferred bisphosphonate is zoledronic acid (INN) or zoledronate (sold by Novartis under the trademarks Zometa, Zomera, Aclasta and Reclast). Zometa is used to prevent skeletal fractures in patients with cancers such as multiple myeloma and prostate cancer, and to treat osteoporosis. It may also be used to treat hypercalcemia in cancer and may be useful in the treatment of pain from bone metastases.

In one particularly preferred embodiment, the γδ T-cell stimulating agent of the invention is administered in combination with IL-2. This combination has been shown to be particularly effective in mediating the expansion and activation of γ9δ2 T cells.

Interleukin-2 (IL-2) is an interleukin, a cytokine type signaling molecule in the immune system. It is a protein that attracts lymphocytes and is part of the body's natural response to microbial infections, and is also involved in distinguishing between foreign (non-self) and self antigens. IL-2 mediates its effects by binding to IL-2 receptors that are expressed by lymphocytes.

When used according to the invention, IL-2 may be any IL-2 that maintains or promotes γδ T-cell stimulation and may be from any species, preferably human. IL-2 may be isolated, recombinantly produced or synthetic IL-2, and may also be naturally occurring or modified IL-2.

The term “antigen” refers to substances such as proteins or peptides containing an epitope against which an immune response is directed and/or to be directed. In a preferred embodiment, the antigen is a tumor antigen of type CLDN18.2, i.e. a component of cancer cells which may be derived from the cytoplasm, cell surface or cell nucleus, in particular an antigen which is produced, preferably in large quantities, within the cell or as a surface antigen on cancer cells.

In the present invention, the term "tumor-associated (tumor) antigen" preferably refers to those proteins that under normal conditions are specifically expressed in a limited number of tissues and/or organs or at certain stages of ontogeny and are expressed or abnormally expressed in one or more tumor or cancer cells. tissues. In the present invention, tumor antigens are preferably associated with the cell surface of cancer cells and are preferably not or rarely expressed in normal tissues.

The term "epitope" refers to antigenic determinants in molecules, i. that part of the molecules that is recognized by the immune system, for example, is recognized by an antibody. For example, epitopes are discrete, three-dimensional sites on antigens that are recognized by the immune system. Typically, epitopes consist of reactive surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes of proteins such as CLDN18.2 preferably include a continuous or discontinuous stretch of this protein, preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids long, e.g. may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids long.

The term "antibody" refers to glycoproteins containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any molecule containing antigen-binding sites. The term “antibody” includes monoclonal antibodies and antibody fragments or derivatives, including, but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies such as scFv, and antigen-binding antibody fragments such as Fab fragments and Fab', and also includes all recombinant forms of antibodies, for example, antibodies expressed in prokaryotes, non-glycosylated antibodies, as well as any antigen-binding fragments and derivatives of antibodies, as described here. Each heavy chain is composed of a heavy chain variable region (abbreviated as V _H ) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as V _L ) and a light chain constant region. The V _H and V _L regions can be further subdivided into regions of hypervariability, referred to as complementarity-determining regions (CDRs), which are interspersed with more conserved regions, referred to as framework regions (FRs). Each V _H and V _L region consists of three CDRs and four FRs, N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

Antibodies described herein may be human antibodies. The term "human antibody" in the present invention is used to refer to antibodies having variable and constant regions derived from human germline immunolobulin sequences. Human antibodies described herein may contain amino acid residues not encoded by germline human immunolobulin sequences (eg, mutations introduced at random or by site-directed mutagenesis in vitro or by somatic mutation in vivo).

The term "humanized antibody" refers to molecules in which the antigen-binding sites are primarily derived from immunoglobulins of other than human species, while the rest of the immunoglobulin structure of the molecule is based on the structure and/or sequence of human immunoglobulin. Antigen binding sites may either contain complete variable domains embedded in constant domains, or only hypervariable regions (CDRs) embedded in the corresponding framework regions of the variable domains. Antigen binding sites may be wild-type or modified with one or more amino acid substitutions, for example modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other forms, one or more CDR regions have been altered from the original antibody.

The term "chimeric antibody" refers to those antibodies in which one part of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequences in antibodies derived from a certain species or belonging to a certain class, and the remaining chain segments are homologous to the corresponding sequences in others. Typically, the variable regions of light and heavy chains reproduce the variable regions of antibodies derived from one mammalian species, and the constant regions are homologous to the sequences of antibodies derived from another. One of the obvious advantages of such chimeric forms is that the variable region can be easily obtained from currently known sources using readily available B cells or hybridomas from organisms other than humans, in combination with constant regions obtained, for example, from human cell preparations. While a variable region has the advantage of ease of preparation and source-independent specificity, a human-derived constant region is less likely to elicit an immune response in a human subject when administered with antibodies than if the constant region is from another source. from a source than a person. However, this definition is not limited to this specific example.

The terms “antigen-binding portion” of an antibody (or simply “binding portion”) or “antigen-binding fragment” of an antibody (or simply “binding fragment”) or similar terms refer to one or more fragments that retain the ability of the antibody to specifically bind to an antigen. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) Fab fragments, monovalent fragments consisting of V _L , V _H , _CL and _CH domains; (ii) F(ab') ₂ fragments, bivalent fragments containing two divalent Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of V _H and C _H domains; (iv) Fv fragments consisting of V _L and V _H domains on the same antibody arm; (v) dAb fragments (Ward et al. (1989) Nature 341: 544-546) which consist of a V _H domain; (vi) complementarity determined regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be linked via a synthetic linker. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be connected, recombinantly, using a synthetic linker that allows them to form a single protein chain that combines the V _L and V _H regions with formation of monovalent molecules (known as single chain Fv (scFv), for example, see 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 encompassed by the term "antigen-binding fragment" of an antibody. The following example is immunoglobulin binding domain fusion proteins comprising: (i) a binding domain polypeptide fused to an immunoglobulin hinge polypeptide, (ii) an immunoglobulin heavy chain C _H 2 constant region fused to the hinge, and (iii) a C _H constant region 3 immunoglobulin heavy chain fused to a C _H 2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Immunoglobulin binding domain fusion proteins are disclosed in more detail in US 2003/0118592 and US 2003/0133939. These antibody fragments are prepared by standard methods known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies.

The term "bispecific molecules" is used to refer to such agents, for example, proteins, peptides or protein or peptide complexes, which have two different binding specificities. For example, the molecule can bind to or interact with (a) an antigen on a cell surface and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is used to refer to those agents, for example, proteins, peptides or protein or peptide complexes, which have more than two different binding specificities. For example, the molecule can bind to or interact with (a) an antigen on a cell surface, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, without limitation, bispecific, trispecific, tetraspecific and other multispecific molecules that target CLDN18.2 and other targets such as Fc receptors on effector cells. The term "bispecific antibodies" also includes diabodies. Diabodies are bivalent bispecific antibodies in which the V _H and V _L domains are expressed on the same polypeptide chain, but with a linker that is too short to form a link between the two domains on the same chain, forcing the domains to bind with complementary domains on the other strand, forming two antigen-binding sites (eg, see Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak RJ et al. (1994) Structure 2 : 1121-1123).

An antibody can be conjugated to a therapeutic molecule or agent, such as a cytotoxin, a drug (eg, an immunosuppressant), or a radioisotope. Cytotoxins or cytotoxic agents includes any agents that cause harm and in particular kill cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, vincristine, tenoposide, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogues or homologues thereof. Suitable therapeutic agents for preparing antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP, cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC) and anti-mitotic 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 the following 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.

Antibodies can also be conjugated to radioisotopes such as iodine-131, yttrium-90, or indium-111 to form cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention may be used to modify a given biological response, and drug molecules should not be limited to classical chemical therapeutic agents. For example, the drug molecule may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or γ-interferon; 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.

Methods for conjugating such therapeutic molecules to antibodies are well known, for example see Arnon et al., “Monoclonal antibodies for immunotargeting of drugs in cancer therapy”, in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies for drug delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody carriers of cytotoxic agents in cancer therapy: a review”, in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, results, and future prospective of the therapeutic use of radiolabeled antibodies in cancer therapy”, in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation and cytotoxic properties of antibody-toxin conjugates”, Immunol. Rev. 62: 119-58 (1982).

In the present invention, an antibody is “derived from” a specific germline sequence if it is obtained from a system by immunizing an animal or screening an immunoglobulin gene library, wherein the selected antibody is at least 90% amino acid, more preferably 95%, even more preferably at least at least 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, an antibody derived from a particular germline sequence should show no more than 10 amino acid differences, more preferably no more than 5, or even more preferably no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence, germline encoded by the immunoglobulin gene.

In the present invention, the term "heteroantibodies" refers to two or more antibodies, their derivatives or antigen-binding sites connected to each other, at least two of them having different specificities. These different specificities include binding specificity for Fc receptors on effector cells and binding specificity for an antigen or epitope on target cells, such as tumor cells.

The antibodies described herein may be monoclonal antibodies. The term "monoclonal antibody" in the present invention refers to preparations of antibody molecules of the same molecular composition. A monoclonal antibody exhibits only one binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma that includes non-human animal-derived B cells, such as mice, fused with immortalized cells.

The antibodies described herein may be recombinant antibodies. The term "recombinant antibody" in the present invention includes all antibodies that are obtained, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from such animals (for example, mice) that are transgenic or transchromosomal with respect to immunoglobulin genes or the hybridoma from which they are derived, (b) antibodies isolated from host cells transformed to express antibodies, e.g., from a transfectoma, (c) antibodies isolated from a recombinant or combinatorial antibody library, and (d) antibodies obtained, expressed created or isolated by any other method that involves splicing immunoglobulin gene sequences with other DNA sequences.

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

The antibodies described herein include polyclonal and monoclonal antibodies and encompass antibodies of the IgA type such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM and IgD. In various embodiments, the antibodies are IgG1 type antibodies, more preferably kappa or lambda isotype IgG1 antibodies (i.e., IgG1, κ, λ), IgG2a type antibodies (e.g., IgG2a, κ, λ), IgG2b type antibodies (e.g., IgG2b, κ, λ), IgG3 type antibodies (eg IgG3, κ, λ) or IgG4 type antibodies (eg IgG4, κ, λ).

The term "transfectome" in the present invention encompasses recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant or fungal cells, including yeast cells.

In the present invention, the term "heterologous antibody" is defined in relation to transgenic organisms that produce such antibodies. The term refers to antibodies in which the amino acid sequence or nucleic acid coding sequence matches that found in the non-transgenic organism and is usually from a species other than the transgenic organism.

In the present invention, "heterohybrid antibody" means an antibody whose light and heavy chains are derived from different organisms. For example, an antibody in which a human heavy chain is linked to a mouse light chain is a heterohybrid antibody.

The invention encompasses all antibodies and antibody derivatives described herein, which for the purposes of the present invention are covered by the term "antibody". The term "derivatives" of antibodies refers to any modified form of antibodies, such as conjugates of antibodies with other molecules or antibodies or antibody fragments.

The antibodies described herein are preferably isolated. The term "isolated antibody" in the present invention is used to refer to those antibodies that are substantially free of other antibodies with different antigenic specificity (for example, an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind to other antigens, than CLDN18.2). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may be cross-reactive with other related antigens, eg from another species (eg, species homologues of CLDN18.2). Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies refers to antibodies having different specificities and combined in well-defined compositions or mixtures.

The term "binding" in accordance with the invention preferably refers to specific binding.

In accordance with the present invention, an antibody is capable of binding to a given target if it has significant affinity for that given target and binds to it in standard assays. “Affinity” or “binding affinity” is often measured by the equilibrium dissociation constant (K _D ). Preferably, the term “substantial affinity” means binding to a given target with a dissociation constant (K _D ) of 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less.

An antibody is (substantially) unable to bind to a target unless it has significant affinity for that target and does not bind substantially, in particular, binding to that target is not detected in standard assays. Preferably, binding of the antibody to this target is not detected if it is present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg/ml or more. Preferably, an antibody does not have significant affinity for a target if it binds to that target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times higher than the K _D to bind to a target with which the antibody is able to bind. For example, if the K _D for binding an antibody to a target for which the antibody is able to bind is 10 ^-7 M, then the K _D for binding to a target for which the antibody does not have significant affinity will be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

An antibody is specific for a given target if it is able to bind to that given target, but is not able to bind to other targets, ie. has no significant affinity for other targets and does not significantly bind to other targets in standard assays. In accordance with the invention, an antibody is specific for CLDN18.2 if it is able to bind to CLDN18.2 but is (practically) unable to bind to other targets. Preferably, the antibody is specific for CLDN18.2 if the affinity for or binding to such other targets is not substantially greater than the affinity for or binding to unrelated CLDN18.2 proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or with transmembrane proteins other than claudin, such as MHC molecules or transferrin receptors, or with any other such polypeptides. Preferably, an antibody is specific for a given target if it binds to that target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times lower than the K _D for binding to a target for which it is not specific. For example, if the K _D for binding of an antibody to a target for which it is specific is 10 ^-7 M, then the K _D for binding to a target for which it is not specific will be at least 10 ^-6 M, 10 ^{- 5} M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

Binding of an antibody to a target can be determined experimentally by any suitable method, for example, see Berzofsky et al., "Antibody-Antigen Interactions" in Fundamental Immunology, Paul WE, ed., Raven Press, New York, NY (1984); Kuby & Janis, Immunology, W.H. Freeman and Company, New York, NY (1992); as well as the methods described here. Affinity is easily determined by standard methods such as the equilibrium dialysis method; using the BIAcore 2000 instrument using the general procedures outlined by the manufacturer; radioimmunoassay method using a radioactive target antigen; or by other method known to those skilled in the art. Affinity data can be analyzed, for example, according to the method of Scatchard et al., Ann NY Acad. Sci, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, eg salt concentration, pH. Therefore, measurements of affinity and other antigen binding parameters, eg K _D , IC ₅₀ , are preferably carried out with antibody and antigen standard solutions and standardized buffer.

In the present invention, the term “isotype” refers to the class of antibodies (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

In the present invention, the term "isotype switch" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

The term "naturally occurring" or "natural" in the present invention in relation to an object means that the object occurs in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

The term "permutation" as used herein refers to a configuration of an immunoglobulin heavy chain or light chain locus such that the V segment is located immediately adjacent to the DJ or J segment in a conformation encoding a substantially complete V _H or V _L domain, respectively. Rearrangement of the immunoglobulin (antibody) gene locus is detected by comparison with germline DNA; upon rearrangement, the locus will contain at least one recombined heptamer/nonamer type homology element.

The term "unrearranged" or "germ configuration" in the present invention in relation to the V segment refers to such a configuration in which the V segment has not undergone recombination and therefore is not immediately adjacent to the D or J segment.

According to the invention, an antibody having the ability to bind to CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, preferably an epitope located in the extracellular domains of CLDN18.2, in particular in the first extracellular domain, preferably at amino acid positions 29 to 78 in CLDN18.2. In certain embodiments, an antibody capable of binding to CLDN18.2 is an antibody capable of binding to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NOS: 3, 4 and 5, (ii ) an epitope located on loop 1 of CLDN18.2, preferably SEQ ID NO: 8, (iii) an epitope located on loop 2 of CLDN18.2, preferably SEQ ID NO: 10, (iv) an epitope located on loop D3 of CLDN18.2 , preferably SEQ ID NO: 11, (v) an epitope spanning loop 1 of CLDN18.2 and loop D3 of CLDN18.2, or (vi) a non-glycosylated epitope located on loop D3 of CLDN18.2, preferably SEQ ID NO: 9.

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

Preferably, binding of an antibody capable of binding to CLDN18.2 to cells expressing CLDN18.2 induces or mediates the killing of cells expressing CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, particularly selected from the group consisting of gastric, esophageal, pancreatic, lung, ovarian, colon, liver, head and neck, and gallbladder tumor cells. Preferably, the antibody induces or mediates cell killing by inducing one or more of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cell cytotoxicity (ADCC) mediated lysis, apoptosis, and inhibition of proliferation of cells expressing CLDN18.2. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, which in certain embodiments are selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs. Cell proliferation inhibition can be measured in vitro by determining cell proliferation by the bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) method. BrdU is a synthetic nucleoside that is analogous to thymidine and can be incorporated into newly synthesized DNA during cell replication (during the S phase of the cell cycle) instead of thymidine during DNA replication. Detection of its inclusion, for example, using antibodies specific for BrdU, indicates that active DNA replication occurred in the cells.

In preferred embodiments, the antibodies described herein are characterized by one or more of the following properties:

a) specificity for CLDN18.2;

b) the binding affinity for CLDN18.2 is about 100 nm or less, preferably 5-10 nm or less, more preferably 1-3 nm or less;

c) the ability to induce or mediate CDC on CLDN18.2 positive cells;

d) the ability to induce or mediate ADCC on CLDN18.2 positive cells;

e) the ability to inhibit the growth of CLDN18.2 positive cells;

f) the ability to induce apoptosis of CLDN18.2 positive cells.

In a particularly preferred embodiment, an antibody having the ability to
to bind to CLDN18.2 is produced by a hybridoma deposited with the DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) with the following designation and accession number:

a). 182-D1106-055, DSM access number ACC2737, deposited October 19, 2005,

b). 182-D1106-056, DSM access number ACC2738, deposited October 19, 2005,

c). 182-D1106-057, DSM access number ACC2739, deposited October 19, 2005,

d). 182-D1106-058, DSM access number ACC2740, deposited October 19, 2005,

e). 182-D1106-059, DSM access number ACC2741, deposited October 19, 2005,

f). 182-D1106-062, DSM access number ACC2742, deposited October 19, 2005,

g). 182-D1106-067, DSM access number ACC2743, deposited October 19, 2005,

h). 182-D758-035, DSM access number ACC2745, deposited November 17, 2005,

i). 182-D758-036, DSM access number ACC2746, deposited November 17, 2005,

j). 182-D758-040, DSM access number ACC2747, deposited November 17, 2005,

k). 182-D1106-061, DSM access number ACC2748, deposited November 17, 2005,

l). 182-D1106-279, DSM access number ACC2808, deposited October 26, 2006,

m). 182-D1106-294, DSM access number ACC2809, deposited October 26, 2006,

n). 182-D1106-362, DSM access number ACC2810, deposited October 26, 2006.

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

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

Clone mAb Isotype Variable region Chimeric antibody heavy chain 43A11 182-D1106-062 IgG2a SEQ ID NO:29 SEQ ID NO:14 163E12 182-D1106-294 IgG3 SEQ ID NO:30 SEQ ID NO:15 125E1 182-D1106-279 IgG2a SEQ ID NO:31 SEQ ID NO:16 166E2 182-D1106-308 IgG3 SEQ ID NO:33 SEQ ID NO:18 175D10 182-D1106-362 IgG1 SEQ ID NO:32 SEQ ID NO:17 45C1 182-D758-187 IgG2a SEQ ID NO:34 SEQ ID NO:19 light chain 43A11 182-D1106-062 IgK SEQ ID NO:36 SEQ ID NO:21 163E12 182-D1106-294 IgK SEQ ID NO:35 SEQ ID NO:20 125E1 182-D1106-279 IgK SEQ ID NO:37 SEQ ID NO:22 166E2 182-D1106-308 IgK SEQ ID NO:40 SEQ ID NO:25 175D10 182-D1106-362 IgK SEQ ID NO:39 SEQ ID NO:24 45C1 182-D758-187 IgK SEQ ID NO:38 SEQ ID NO:23 45C1 182-D758-187 IgK SEQ ID NO:41 SEQ ID NO:26 45C1 182-D758-187 IgK SEQ ID NO:42 SEQ ID NO:27 45C1 182-D758-187 IgK SEQ ID NO:43 SEQ ID NO:28

In preferred embodiments, the antibodies, in particular the chimerized forms of the antibodies of the invention, comprise antibodies comprising a heavy chain constant region ( _CH ) comprising an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO: 13 , or a fragment of it. In other preferred embodiments, antibodies, in particular chimerized forms of antibodies of the invention, include antibodies comprising a light chain constant region (C _L ) containing an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence represented by SEQ ID NO: 12, or a fragment thereof. In a particularly preferred embodiment, the antibodies, in particular the chimerized forms of the antibodies of the invention, comprise antibodies that contain a _CH containing an amino acid sequence derived from human _CH , such as the amino acid sequence shown in SEQ ID NO: 13, or a fragment thereof, and also contain C _L containing an amino acid sequence derived from human C _L , such as the amino acid sequence represented by SEQ ID NO: 12, or a fragment thereof.

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

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

In some preferred embodiments, chimerized forms of antibodies include antibodies containing a combination of heavy chains and light chains selected from the following options from (i) to (ix):

(i) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof,

(ii) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 15, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 20, or a fragment thereof,

(iii) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 16, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 22, or a fragment thereof,

(iv) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 18, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 25, or a fragment thereof,

(v) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 17, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 24, or a fragment thereof,

(vi) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 19, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 23, or a fragment thereof,

(vii) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 19, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 26, or a fragment thereof,

(viii) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 19, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 27, or a fragment thereof, and

(ix) the heavy chain contains the amino acid sequence represented by SEQ ID NO: 19, or a fragment thereof, and the light chain contains the amino acid sequence represented by SEQ ID NO: 28, or a fragment thereof.

A “fragment” or “amino acid sequence fragment” above refers to a portion of an antibody sequence, i.e. a sequence that is an antibody sequence truncated at the N- and/or C-terminus, which, when it replaces that sequence in an antibody, retains that antibody's binding to CLDN18.2 and preferably retains the functions of that antibody described herein, e.g., CDC mediated lysis; or ADCC mediated lysis. Preferably, an amino acid sequence fragment contains at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of amino acid residues from the amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28, preferably means that of this sequence deleted 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus.

In a preferred embodiment, an antibody capable of binding to CLDN18.2 comprises a heavy chain variable region (V _H ) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and their fragments.

In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 contains a light chain variable region (V _L ) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43 and their fragments.

In some preferred embodiments, an antibody capable of binding to CLDN18.2 comprises a combination of a heavy chain variable region (V _H ) and a light chain variable region (V _L ) selected from the following options (i) to (ix):

(i) V _H contains the amino acid sequence represented by SEQ ID NO: 29, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 36, or a fragment thereof,

(ii) V _H contains the amino acid sequence represented by SEQ ID NO: 30, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 35, or a fragment thereof,

(iii) V _H contains the amino acid sequence represented by SEQ ID NO: 31, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 37, or a fragment thereof,

(iv) V _H contains the amino acid sequence represented by SEQ ID NO: 33, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 40, or a fragment thereof,

(v) V _H contains the amino acid sequence represented by SEQ ID NO: 32, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 39, or a fragment thereof,

(vi) V _H contains the amino acid sequence represented by SEQ ID NO: 34, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 38, or a fragment thereof,

(vii) V _H contains the amino acid sequence represented by SEQ ID NO: 34, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 41, or a fragment thereof,

(viii) V _H contains the amino acid sequence represented by SEQ ID NO: 34, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 42, or a fragment thereof, and

(ix) V _H contains the amino acid sequence represented by SEQ ID NO: 34, or a fragment thereof, and V _L contains the amino acid sequence represented by SEQ ID NO: 43, or a fragment thereof.

In a preferred embodiment, an antibody capable of binding to CLDN18.2 contains a V _H region comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following options from (i) to (vi):

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

(ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15,

(iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16,

(iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17,

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

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

In a preferred embodiment, an antibody capable of binding to CLDN18.2 contains a V _L region comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following options from (i) to (ix):

(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,

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

(iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23,

(v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

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

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

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

In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 contains a combination of V _H and V _L that includes a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):

(i) V _H : CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14; V _L : CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO: 21;

(ii) V _H : CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15; V _L : CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20;

(iii) V _H : CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16; V _L : CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22;

(iv) V _H : CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18; V _L : CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25;

(v) V _H : CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17; V _L : CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24;

(vi) V _H : CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19; V _L : CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23;

(vii) V _H : CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19; V _L : CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26;

(viii) V _H : CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19; V _L : CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27; And

(ix) V _H : CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19; V _L : CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

In other preferred embodiments, the antibody having the ability to bind to CLDN18.2 preferably contains one or more complementarity determining regions (CDRs), preferably at least a CDR3 region from the heavy chain variable region (V _H ) and/or light chain variable region (V _L ) an anti-CLDN18.2 monoclonal antibody, preferably an anti-CLDN18.2 monoclonal antibody described herein, and preferably it contains one or more complementarity determining regions (CDRs), preferably at least a CDR3 region of the heavy chain variable regions described herein (V _H ) and/or light chain variable regions (V _L ). In one embodiment, these 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 having the ability to bind to CLDN18.2 preferably contains the complementarity determining regions CDR1, CDR2 and CDR3 from the heavy chain variable region (V _H ) and/or light chain variable region (V _L ) of an anti-CLDN18 monoclonal antibody. 2, preferably the anti-CLDN18.2 monoclonal antibody described herein, and preferably it contains the complementarity determining regions CDR1, CDR2 and CDR3 from the heavy chain variable regions (V _H ) and/or light chain variable regions (V _L ) described here.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of CDR sets as described herein contains these CDRs along with their interleaved framework regions. Preferably, this portion should also include at least 50% of one of the first and fourth framework regions, or both, with this 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The construction of antibodies by recombinant DNA techniques may result in the inclusion of residues at the N- or C-terminus of the variable regions encoded by linkers introduced to facilitate cloning or other processing steps, including the introduction of linkers to connect the variable regions of the invention to other protein sequences, including including heavy chains of immunoglobulins, other variable domains (for example, when receiving diabodies) or protein labels.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of CDR sets, as described herein, contains these CDRs in a human antibody framework.

Reference herein to antibodies containing, in relation to their heavy chain, a particular chain, or a particular region or sequence, preferably refers to such a situation where all the heavy chains of a given antibody contain that particular chain, region or sequence. This also applies to the light chain of antibodies, respectively.

The term "nucleic acid" in the present invention is used to refer to DNA and RNA. The nucleic acid may be single or double stranded, but is preferably double stranded DNA.

In accordance with the 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 partial expression of nucleic acids. In addition, expression can be transient or stable.

Information given herein with respect to certain amino acid sequences, for example, those presented in a sequence listing, should be understood so that they also apply to those variants of these specific sequences in which the sequences are functionally equivalent to this particular sequence, for example, their amino acid sequences exhibit identical properties or close to those of certain amino acid sequences. One of the important properties is to maintain the binding of the antibody to its target or to maintain the effector functions of the antibody. Preferably, a sequence that is a variant of a particular sequence when it replaces that particular sequence in an antibody retains that antibody's binding to CLDN18.2 and preferably that antibody's function as described herein, e.g., CDC-mediated lysis or ADCC-mediated lysis.

Specialists should be aware that, in particular, the sequences of CDR regions, hypervariable and variable regions can be modified without losing the ability to bind to CLDN18.2. For example, the CDR regions must be either identical or strongly homologous to the antibody regions shown here. By "high homology" is meant that there may be 1 to 5, preferably 1 to 4, such as 1 to 3, or 1 or 2 substitutions in a CDR. In addition, hypervariable and variable regions can be modified so that they show significant homology to the specific antibody regions shown here.

In the present invention, amino acid sequence “variants” include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid deletion variants that include deletions at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncated variants.

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

Amino acid addition variants include N- and/or C-terminal attachment of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

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

Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. Preference is given to modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides, and/or substitutions of amino acids for others with similar properties. Preferably, the amino acid substitutions in the protein variants are conservative substitutions, ie. these are substitutions for analogous charged or uncharged amino acids. Conservative amino acid substitutions include substitutions within the same family of amino acids that are similar in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified collectively as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence which is a variant of the given amino acid sequence should be at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably given for an amino acid region of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 100% of the entire length of the control amino acid sequence. For example, if the control amino acid sequence is 200 amino acids, then the degree of similarity or identity is preferably given for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180 or about 200 amino acids, preferably continuous amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Sequence alignment to determine similarity, preferably sequence identity, is performed using known tools, preferably by the best sequence alignment method, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" means the percentage of amino acids that are either identical or represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences means the percentage of amino acids that are identical between those sequences.

The term "degree of identity" refers to the percentage of identical amino acid residues between two compared sequences, obtained after the best alignment, and this percentage is purely statistical, and the differences between the two sequences are distributed randomly and along the entire length. Sequence comparisons between two amino acid sequences are typically made by comparing the sequences after they have been optimally aligned, and such comparison is done segment by segment or by “comparison windows” to identify and compare local sequence similarities. Optimal alignment of sequences for comparison can be done manually or using the local homology algorithm of Smith and Waterman, 1981, Ads. app. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, Pearson and Lipman, 1988, Proc. Natl. Acad. sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

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

The term "transgenic animal" refers to those animals whose genome contains one or more transgenes, preferably heavy and/or light chain transgenes or transchromosomes (whether or not integrated into the animal's natural genomic DNA), which are preferably capable of expressing those transgenes. For example, a transgenic mouse may contain a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome such that the mouse will produce human anti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and/or cells expressing CLDN18.2. The human heavy chain transgene may be inserted into the chromosomal DNA of the mouse, as in the case of transgenic mice, e.g., HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene may be present outside the chromosomes, as in the case of transchromosomal mice (e.g., KM mice) as described in WO 02/43478. Such transgenic and transchromosomal mice can produce multiple isotypes of anti-CLDN18.2 human monoclonal antibodies (eg, IgG, IgA, and/or IgE) by going through V-D-J recombination and isotype switching.

"Reduction", "reduction" or "inhibition" in the present invention means an overall reduction or the ability to cause an overall reduction in the level, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more and most preferably by 75% or more, for example, the level of expression or the level of cell proliferation.

Terms like "increase" or "increase" preferably mean an increase or increase of at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least at least 50%, even more preferably at least 80%, and most preferably at least 100%, 200%, 500%, 1000%, 10,000% or more.

Mechanisms of action of mAbs

Although the following are considerations regarding the mechanism underlying the therapeutic efficacy of the antibodies of the invention, this should in no way be construed as limiting the invention.

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

Antibody dependent cellular cytotoxicity (ADCC)

ADCC means cell killing capability in effector cells as described herein, in particular in lymphocytes, which preferably requires that the target cells be labeled with an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cells and Fc domains of antibodies engage Fc receptors (FcRs) on the surface of immune effector cells. Several families of Fc receptors have been identified, with certain cell populations expressing their characteristic Fc receptors. ADCC can be considered as a mechanism to directly induce varying degrees of immediate tumor destruction, leading to antigen presentation and induction of tumor-targeted T cell responses. Preferably, the induction of ADCC in vivo should elicit tumor-targeted T-cell responses and body antibody responses.

Complement dependent cytotoxicity (CDC)

CDC is another method of killing cells that is triggered by antibodies. The most effective complement activation isotype is IgM. Also IgG1 and IgG3 are both very efficient in triggering CDC through the classical complement pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the demasking of several proximate C1q binding sites on the C _H 2 domains of the involved IgG type antibody molecules (C1q is one of the three subcomponents of C1 complement). Preferably, these unmasked C1q binding sites convert a previously low affinity C1q-IgG interaction into a high affinity interaction that triggers a cascade of events involving
a number of other complement proteins and leads to the proteolytic release of chemotactic/activating effector cells of C3a and C5a molecules. Preferably, the complement cascade ends with the formation of a membrane attack complex that creates pores in the cell membrane to allow free passage of water and solutes into and out of the cell.

Obtaining and testing antibodies

The antibodies described herein can be prepared in a variety of ways, including standard monoclonal antibody methods, such as the standard somatic cell hybridization method Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization methods are preferred, other methods for producing monoclonal antibodies can in principle be used, such as viral or oncogenic transformation of B-lymphocytes, or phage display methods using antibody gene libraries.

The preferred animal system for producing monoclonal antibody-secreting hybridomas is the murine system. Obtaining hybridomas in mice is a very well established procedure. Known methods of immunization and methods for isolating immunized splenocytes for fusion. Fusion partners (eg mouse myeloma cells) and fusion procedures are also known.

Other preferred animal systems for producing monoclonal antibody-secreting hybridomas are those in rats and rabbits (for example, those described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995), also see Rossi et al., Am J Clin Pathol 124: 295 (2005)).

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

Another strategy for producing monoclonal antibodies is to directly isolate antibody-coding genes from lymphocytes that produce antibodies of a certain specificity, for example, see Babcock et al., 1996: A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes antibodies producing of defined specificities. For details on the construction of recombinant antibodies, see also Welschof and Kraus, Recombinant antibodies for cancer therapy: ISBN-0-89603-918-8; and Benny K.C. Lo, Antibody Engineering: ISBN 1-58829-092-1.

To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the antigen sequence, ie. the sequence against which the antibodies are to be directed, enriched in a preparation of recombinantly expressed antigen or fragments thereof, and/or cells expressing the antigen, as described. On the other hand, mice can be immunized with DNA encoding the antigen or fragments thereof. In the event that immunization with a purified or enriched antigen preparation does not produce antibodies, mice can also be immunized with cells expressing the antigen, such as a cell line, to enhance immune responses.

The immune response can be monitored during immunization with plasma and serum samples obtained from the tail vein or postorbital blood samples. Mice with sufficient immunoglobulin titer can be used for fusion. To increase the percentage of hybridomas secreting a particular antibody, mice can be revaccinated intraperitoneally or intravenously with cells expressing the antigen 3 days before slaughter and spleen removal.

To obtain hybridomas producing monoclonal antibodies, splenocytes and lymph node cells can be isolated from immunized mice and fused with an appropriate immortalized cell line such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Using cells expressing the antigen, antibodies with specificity for the antigen can be identified by immunofluorescence and FACS. Antibody-secreting hybridomas can be subcultured, screened again and, if still positive for monoclonal antibodies, subcloned by serial dilution. The stable subclones can then be cultured in vitro to generate antibodies in tissue culture medium for study.

Antibodies can also be generated in transfectoma cells, for example, by a combination of recombinant DNA techniques and gene transfection techniques, which are well known in the art (Morrison S. (1985) Science 229: 1202).

For example, in one embodiment, genes of interest, such as antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as that used in the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841, or other expression system well known in the art. The purified plasmid with cloned antibody genes can be introduced into host eukaryotic cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells, or other eukaryotic cells such as plant, fungal or yeast cells. The methods already described, such as electroporation, lipofection, lipofectamine, or others, can be used to introduce these genes. Once these antibody genes are introduced into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomes, which can then be expanded to the desired level of expression and production of antibodies. From the supernatants of these cultures and/or cells, recombinant antibodies can be isolated and purified.

On the other hand, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms such as E. coli. Moreover, antibodies can also be obtained in transgenic animals, for example, in milk from sheep and rabbits or eggs from chickens, or in transgenic plants; for example, see 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) J. Biol. Chem. 380: 825-839.

Chimerization

Mouse monoclonal antibodies can be used as therapeutic antibodies in humans if labeled with toxins or radioactive isotopes. Unlabeled mouse antibodies are highly immunogenic in humans, and repeated use leads to a decrease in therapeutic effect. In general, immunogenicity is mediated by heavy chain constant regions. The immunogenicity of mouse antibodies to humans can be reduced or eliminated if the corresponding antibodies are chimerized or humanized. Chimeric antibodies are those antibodies whose various parts are derived from different animal species, such as the variable region is derived from a mouse antibody and the constant region is derived from a human immunoglobulin. Antibody chimerization is accomplished by attaching the heavy and light chain variable regions of a mouse antibody to a human heavy and light chain constant region (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918 -8). In a preferred embodiment, chimeric antibodies are generated by fusion of a human kappa light chain constant region with a mouse light chain variable region. In another preferred embodiment, chimeric antibodies can be generated by fusing a human lambda light chain constant region with a mouse light chain variable region. Heavy chain constant regions of the IgG1, IgG3 and IgG4 types are preferred for making chimeric antibodies. Other preferred heavy chain constant regions for making chimeric antibodies are IgG2, IgA, IgD and IgM.

humanization

Antibodies interact with target antigens predominantly through those amino acid residues found in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDR are more diverse between individual antibodies than those outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of certain native antibodies by constructing expression vectors that include CDR sequences from that native antibody grafted onto framework sequences from another antibody with different properties (e.g., see 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. Sc. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they do not include the fully assembled variable region genes that result from V-(D)-J fusion during B cell maturation. The germline sequences of the genes also differ from the high affinity antibody sequences from the secondary repertoire at discrete points uniformly throughout the variable region.

The ability of antibodies to bind to an antigen can be determined by standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometry).

For antibody purification, selected hybridomas can be grown in 2 liter spin flasks for monoclonal antibody purification. On the other hand, antibodies can be produced in dialysis bioreactors. Supernatants are filtered and, if necessary, concentrated prior to affinity chromatography on Protein G Sepharose or Protein A Sepharose. The eluted IgG is tested for purity by gel electrophoresis and high performance liquid chromatography. The buffer solution is changed to PBS and the concentration is determined by OD ₂₈₀ using an extinction coefficient of 1.43. Monoclonal antibodies are divided into portions and stored at -80°C.

To determine whether selected monoclonal antibodies bind to unique epitopes, site-directed or multiple site-directed mutagenesis can be used.

To determine the isotype of antibodies, an isotype ELISA can be performed using various commercial kits (eg, Zymed, Roche Diagnostics). The microplate wells are coated with anti-mouse Ig. After blocking, the plate is reacted with monoclonal antibodies or purified isotype controls at room temperature for two hours. The wells are then reacted with peroxidase-conjugated probes specific for mouse IgG1, IgG2a, IgG2b or IgG3, mouse IgA or IgM. After washing, the plate is developed with ABTS substrate (1 mg/ml) and analyzed by OD _405-650 . Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, cat. no. 1493027) can be used as described by the manufacturer.

Flow cytometry can be used to show the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to living cells expressing the antigen. Cell lines expressing the antigen naturally or after transfection and negative controls not expressing the antigen (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and incubated at 4°C. within 30 min. After washing, Apc- or Alexa647-labeled anti-IgG antibodies are bound to the antigen-bound monoclonal antibody under the same conditions as when binding the primary antibody. Samples are analyzed by flow cytometry on a FACS instrument with the channel tuned to single living cells according to the properties of forward and side light scattering. In order to distinguish antigen-specific monoclonal antibodies from non-specific binding in a single measurement, the co-transfection method can be used. Cells transiently transfected with plasmids encoding an antigen and a fluorescent marker are stained as described above. Transfected cells are detected on a different fluorescence channel than antibody-stained cells. Because most transfected cells express both transgenes, antigen-specific monoclonal antibodies preferentially bind to cells expressing the fluorescent marker, while non-specific antibodies bind in comparable proportions to non-transfected cells. An alternative method using fluorescence microscopy can be used along with or instead of flow cytometry. Cells are stained exactly as described above and analyzed by fluorescence microscopy.

Immunofluorescence microscopy can be used to show the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to living cells expressing the antigen. For example, cell lines expressing antigen spontaneously or after transfection and negative controls not expressing antigen are cultured on well slides under standard growth conditions in DMEM/F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L- glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The cells are then fixed in methanol or paraformaldehyde or left untreated. The cells are then reacted with monoclonal antibodies against the antigen for 30 minutes at 25°C. After washing, the cells were reacted with an Alexa555 labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells are then examined by fluorescence microscopy.

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

Antibodies can also be tested for reactivity with antigen by immunohistochemistry methods well known in the art, for example, using paraformaldehyde or acetone-fixed cryosections or paraffin-embedded and paraformaldehyde-fixed tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from mice. carrying grafted tumors from cell lines expressing the antigen spontaneously or after transfection. For immunostaining, they are incubated with antigen-reactive antibodies and then with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit (DAKO) according to the manufacturer's instructions.

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

Antibody dependent cellular cytotoxicity (ADCC)

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells or other effector cells from healthy donors are purified by Ficoll Hypaque density gradient centrifugation followed by lysis of contaminating erythrocytes. Washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum or 5% heat-inactivated human serum and mixed with ⁵¹ Cr-labeled target cells expressing CLDN18.2 at different ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence enhancing ligand (BATDA). The highly fluorescent ligand-enhancing europium chelate that is released from dead cells is measured on a fluorimeter. Another alternative method uses transfection of target cells with luciferase. When Lucifer Yellow is added, it can only be oxidized by viable cells. Purified anti-CLDN18.2 IgG is then added at various concentrations. Foreign human IgG was used as a negative control. Analyzes are carried out within 4-20 hours at 37°C, depending on the type of effector cells used. Samples are examined for cytolysis by measuring ⁵¹ Cr release or the presence of EuTDA chelate in the culture supernatant. On the other hand, the luminescence resulting from the oxidation of Lucifer Yellow can serve as a measure of viable cells.

Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine if multiple monoclonal antibodies will enhance cytolysis.

Complement dependent cytotoxicity (CDC)

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

An alternative method determines CDC induction on adherent cells. In one embodiment of this method, cells are seeded 24 hours prior to analysis at 3×10 ⁴ /well in flat-bottom tissue culture microplates. The next day, the growth medium is removed and the cells are incubated in triplicate with antibodies. Control cells are incubated in growth medium or growth medium containing 0.2% saponin to determine background lysis and maximum lysis, respectively. After a 20 min incubation at room temperature, the supernatant is removed and 20% (v/v) human plasma or serum in DMEM (warmed to 37°C) is added to the cells and incubated for another 20 min at 37°C. All cells from each sample are added to a solution of propidium iodide (10 μg/ml). The supernatants are then replaced with PBS containing 2.5 μg/ml ethidium bromide and fluorescence measured at 600 nm with excitation at 520 nm on a Tecan Safire instrument. Specific lysis in percent is calculated as follows: specific lysis (%) = (fluorescence of the sample - background fluorescence) / (fluorescence at maximum lysis - background fluorescence) ×100.

Apoptosis Induction and Cell Proliferation Inhibition by Monoclonal Antibodies

To test for the ability to trigger apoptosis, anti-CLDN18.2 monoclonal antibodies can, for example, be incubated with CLDN18.2-positive cancer cells, such as CLDN18.2-transfected SNU-16, DANG, or KATO-III cancer cells, at 37° C. for approximately 20 hours. Cells are harvested, washed in Annexin-V binding buffer (BD Biosciences) and incubated with FITC or APC-conjugated Annexin V (BD Biosciences) for 15 min in the dark. All cells from each sample are added to a solution of PI (10 μg/ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). On the other hand, general inhibition of cell proliferation by monoclonal antibodies can be detected using commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, cat. no. AD0200) is a nonisotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells on microplates. BrdU incorporation is detected using europium-labeled monoclonal antibodies. To detect antibodies, the cells are fixed and the DNA is denatured with a Fix solution. Unbound antibodies are washed out and DELFIA Inducer is added, which displaces the europium ions from the labeled antibody into solution, where they form highly fluorescent chelates with the DELFIA Inducer components. Fluorescence is measured - by time-resolved fluorometry - which is proportional to DNA synthesis in cells for each well.

Preclinical studies

Monoclonal antibodies that bind to CLDN18.2 can also be tested in an in vivo model (e.g., immunodeficient mice bearing grafted tumors from cell lines expressing CLDN18.2, such as DANG, SNU-16, or KATO-III, or after transfection, eg HEK293) to determine their effectiveness in controlling the growth of CLDN18.2 expressing tumor cells.

In vivo studies following xenotransplantation of tumor cells expressing CLDN18.2 into mice or other immunocompromised animals can be performed using the antibodies described herein. Antibodies can be administered to tumor-deprived mice followed by tumor cells to measure the effects of antibodies in preventing tumor formation or associated symptoms. Antibodies can be administered to tumor-bearing mice to determine the therapeutic efficacy of the respective antibodies in reducing tumor growth, metastasis, or associated symptoms. The use of antibodies can be combined with the use of other agents such as cytotoxic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies to determine the synergistic efficacy and potential toxicity of the combinations. To analyze antibody-mediated toxic side effects, antibodies or control reagents can be inoculated into animals and closely examined for symptoms possibly associated with anti-CLDN18.2 antibody therapy. Possible side effects of in vivo use of antibodies to CLDN18.2 include, in particular, toxicity in expressing CLDN18.2 tissues, including the stomach. Antibodies recognizing CLDN18.2 in humans and other species such as mice are particularly useful in predicting possible side effects mediated by the use of anti-CLDN18.2 monoclonal antibodies in humans.

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

The compounds and agents described herein may be administered as any suitable pharmaceutical composition.

Pharmaceutical compositions are generally presented in unit dosage form and may be prepared by methods known per se. Pharmaceutical compositions, for example, may be in the form of a solution or suspension.

The pharmaceutical compositions may contain salts, buffers, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" means the absence of toxicity in a material that does not interfere with the action of the active ingredient of the pharmaceutical composition.

Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this type include, without limitation, salts derived from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acid, etc. Pharmaceutically acceptable salts may also be obtained in the form of alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

Suitable buffer substances for use in pharmaceutical compositions include acetic acid as a salt, citric acid as a salt, boric acid as a salt, and phosphoric acid as a salt.

Suitable preservatives for use in pharmaceutical compositions are benzalkonium chloride, paraben, chlorobutanol and thimerosal.

Formulations for injection may contain pharmaceutically acceptable excipients such as Ringer's lactate.

The term “carrier” refers to those organic or inorganic ingredients, of natural or synthetic origin, with which the active ingredient is combined in order to facilitate, enhance or enable administration. In accordance with the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to patients.

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

The term "filler" in the present invention is used to refer to all substances that may be present in a pharmaceutical composition, but are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings or colorants.

The agents and compositions described herein may be administered by any standard route, such as by parenteral administration, including injections or infusions. Parenteral administration is preferred, for example intravenously, intraarterially, subcutaneously, intradermally or intramuscularly.

Compositions suitable for parenteral administration generally contain a sterile aqueous or non-aqueous formulation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible vehicles and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils, usually sterile, are used as a medium for solutions or suspensions.

The agents and compositions described herein are administered in effective amounts. The term "effective amount" means that amount which causes the desired reaction or the desired effect by itself or together with subsequent doses. In the case of treating a specific disease or condition, the desired response preferably means inhibition of the development of the disease. This includes slowing down the progress of the disease, in particular stopping or reversing the progression of the disease. When treating a disease or condition, the desired response may be to delay the onset or prevent the onset of the disease or condition.

The effective amount of an agent or composition described herein will depend upon the disease being treated, the severity of the disease, individual patient parameters such as age, physiology, size and weight, duration of treatment, type of concomitant therapy (if any), particular route of administration, and similar factors. Accordingly, the administered doses of the agents described herein may depend on these various parameters. In the event that the patient's response is insufficient at the initial dose, higher doses may be used (or effectively higher doses achieved by a different, more localized route of administration).

The agents and compositions described herein can be administered to patients, for example, in vivo, for the treatment or prevention of various diseases such as those described herein. Preferred patients are ill individuals with conditions that can be corrected or alleviated by the administration of the agents and compositions described herein. This includes diseases involving cells characterized by an altered expression profile of CLDN18.2.

For example, in one embodiment, the antibodies described herein can be used
for the treatment of cancer patients, for example, cancers of the type described here, characterized by the presence of cancer cells expressing CLDN18.2.

Pharmaceutical compositions and methods of treatment described in accordance with the invention can also be used in immunization or vaccination to prevent the diseases described here.

Further, the present invention is disclosed in the following examples, which should not be taken as limiting the scope of the invention.

Examples

Example 1 Materials and Methods

1. Antibodies

Table 1. Antibodies used

Antibody Supplier (Cat. No.) Target Binding site clonality View Application IMAB362 Ganymed CLDN18.2 N-terminus monoclone. chimera ADCC, CDC, IF vs Claudine 18 (Mid) Zymed
#38-8100 CLDN18.1 CLDN18.2 C-terminus
a.k. 220-246 polyclone. rabbit IF, IHC vs claudin 18 (C-terminus) Zymed
#38-800 CLDN18.1 CLDN18.2 C-terminus
a.k. 240-262 polyclone. rabbit WB 43-14A Ganymed CLDN18.2 C-terminus
a.k. 248-262 monoclone. mouse IHC 35-22A Ganymed CLDN18.2 C-terminus
a.k. 248-262 monoclone. mouse IF, IHC against human EPR1394Y Antibody online human MHC-I - monoclone. rabbit IHC against β-actin Sigma actin - monoclone. mouse WB

2. Immunohistochemistry (IHC)

Tissue sections (4 µm thick) were stored at 2-8°C until use.

Before the deparaffinization process, the sections were incubated at 58-60°C in an oven for 1 hour to melt the paraffin and quantitatively remove water, thereby improving tissue adhesion to the slides (“baking”).

Dewaxing

After melting and drying, the glasses were subjected to xylene dewaxing in two stages (5 min each) and rehydration using a decreasing series of alcohol solutions (at an ambient temperature of 20–27°C):

- 5 (±1) min in xylene bath;

- again in a fresh xylene bath;

- remove excess liquid;

- 5 (±1) min in absolute ethanol;

- once again in a fresh bath;

- remove excess liquid;

- 5 (±1) min in 96% ethanol;

- once again in a fresh bath;

- remove excess liquid;

- 5 (±1) min in 80% ethanol;

- remove excess liquid;

- 5 (±1) min in 70% ethanol;

- remove excess liquid;

- 5 min in distilled or deionized water.

Epitope reproduction and quenching

After the paraffin was removed, the target epitopes were extracted using the thermally induced epitope extraction technique. For this, slides were placed in a staining vessel filled with 200 ml of reproducing buffer (10 mM citrate buffer, 0.05% Tween-20, pH 6) and incubated in a pressure cooker (PASCAL, Dako) at 120°C for 10 min. . After that, the vessel was removed from the pressure cooker and left to cool in the solution to reproduce the epitopes for 10 (±1) min at room temperature. Slides were washed with wash buffer (1×PBS).

After cooling, the sections were placed in a staining vessel filled with 200 ml quench solution (0.3% peroxidase in 1×PBS) and incubated 15 min at room temperature and then washed 2×5 min with fresh wash buffer.

Antibody blocking and incubation

Excess wash buffer was removed, slides were coated with 200 µl of blocking buffer (10% goat serum in 1×PBS) and incubated at room temperature for 30 min. The blocking buffer was removed and replaced with 200 μl of diluted antibody solution (blocking buffer dilution). Slides were incubated with primary antibody overnight at 2-8°C:

Table 2. Dilution of primary antibodies for histology - initial and final concentration of antibodies used in histological analysis

Dilution of primary antibodies Antibody original conc. subtype epitope epitope sequence final conc. mumAb 43-14A 1 mg/ml IgG2b CLDN18 C-conc. TEDEVQSYPSKHDYV 0.5 µg/ml mumAb 35-22A 1 mg/ml IgG2b CLDN18 C-conc. TEDEVQSYPSKHDYV 0.2 µg/ml

The next day, the primary antibody solution was removed and the sections were washed 3×5 min with wash buffer. Thereafter, excess wash buffer was removed and 200 μl of ready-to-use secondary antibody solution (Power Vision goat-versus-mouse with HRP; Immunologic, NL) was added. Slides were incubated for 30 min at room temperature. Excess liquid was removed and the slides were washed 3×5 min with fresh wash buffer.

Substrate reaction and counterstaining

After removing excess wash buffer, the sections were covered with approx. 50-150 µl of freshly prepared chromogen substrate solution (VectorRed; Vector Labs) over 2 min. Excess substrate was removed, and slides were incubated in vessels with deionized water for 1–5 min.

After that, counterstaining was performed by immersing the sections in vessels containing 200 ml of Mayer hematoxylin for 2 min. After that, the sections were placed in tap water for 5-10 min to blue the nuclei.

Dehydration and Conclusion of Drugs

After counterstaining, the sections were dehydrated with an ascending series of alcohol solutions:

- immersion in 70% ethanol (for 5-10 seconds);

- immersion in 80% ethanol (for 5-10 seconds);

- immersion in 96% ethanol (for 5-10 seconds);

- immersion in 96% ethanol (for 5-10 seconds);

- immersion in absolute ethanol (for 5-10 sec);

- 5 min in xylene;

- 5 min in xylene.

A non-aqueous embedding medium (X-TRA-Kit, Medite) was used to embed the preparations. Slides were poured directly after the last xylene-filled vial and dried in air at room temperature.

Table 3. Tissue microarrays for histological analysis

micromatrix Provider Number of samples Qty
samples Size
samples Thickness PA961; pancreatic carcinoma and normal tissues Biocat 96 96 1.5 mm 5 µm PA802; multiple tissue carcinoma of the pancreas,
1 sample per sample Biocat 78 80 1.5mm 5 µm BIC14011; neoplasia of the intraepithelial tissue of the pancreas Biocat 24 48 1.5 mm 5 µm

3. Cell culture

All pancreatic cancer cell lines and additional control cell lines used for the experiments presented here were cultured in media according to the original specifications and according to standard tissue culture techniques. The conditions are shown in Table 4. For all newly generated cell lines, the cells were checked for mycoplasma contamination and a master cell bank was prepared.

Table 4 Culture Conditions for Pancreatic Cancer Cell Lines and Control Cells

Cell line ¹ flasks/cups Wednesday Incubation Seeding density 2 days ² 3 days ² AsPC-1 T150 RPMI + 10% FCS 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ BxPC3 (ATCC) T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 4.5 g/L glucose (total) + 10% FCS Gold 5% _CO2 , 37°C 8×10 ⁶ 7×10 ⁶ BxPC3-LVT (ECACC) T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 1× sodium bicarbonate + 4.5 g/l glucose + 10% FCS + 1% pen/strept. + 0.5 µg/ml (fresh) blasticidin 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ BxPC3-LVT-Luciferase T150 RPMI + 10mM HEPES + 1mM sodium pyruvate + 1x sodium bicarbonate + 4.5g/l glucose + 10% FCS + 1% pen/strept. + 0.5 µg/ml blasticidin (fresh) + 40 µg/ml hygromycin (fresh) 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ CAPAN1 T150 RPMI + 20% FCS 5% _CO2 , 37°C 8×10 ⁶ 8×10 ⁶ CAPAN1-LVT T150 RPMI + 20% FCS + 1% pen/strept. + 2.5 µg/ml blasticidin (fresh) 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ CAPAN1-LVT T150 RPMI + 20% FCS + 1% pen/strept. + 2.5 µg/ml blasticidin (fresh) + 40 µg/ml hygromycin 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ CHO-K1p740 MACS/FACS (24H5) Luci#2A5 15 cm DMEM:F12 + 1% pen/strept. + 10% FCS + 1.5 mg/ml geneticin (G-418) 7.5% _CO2 , 37°C 1.6×10 ⁶ 7×10 ⁵ CFPAC-1 T150 Iscove's MDM + 10% FCS 5% _CO2 , 37°C 5×10 ⁶ 3×10 ⁶ DANG 1C5F2 15 cm RPMI + 1% pen/strept. +10% FCS 5% _CO2 , 37°C 4×10 ⁶ 2×10 ⁶ DANG 1C5F2 LVT 15 cm RPMI + 1% pen/strept. + 10% FCS + 1 µg/ml blasticidin (fresh) 5% _CO2 , 37°C 4×10 ⁶ 2×10 ⁶ HEK293p740 #A5 15 cm DMEM:F12 + 1% pen/strept. + 10% FCS + 1.5mg/ml Geneticin (G-418) 7.5% _CO2 , 37°C 8×10 ⁶ 5×10 ⁶ HPAC T150 DMEM:F12 + 15 mM HEPES + 0.002 mg/mL human insulin + 10 ng/mL EGF + 5% FCS 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ HPAC-LVT T150 DMEM:F12 + 15 mM HEPES + 0.002 mg/ml human insulin + 10 ng/ml EGF + 5% FCS + 1% pen/strept. + 3.5 µg/ml blasticidin (fresh) 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ HPAF-II T150 MEM + 10% FCS 5% _CO2 , 37°C 5×10 ⁶ 5×10 ⁶ HUP-T3 T150 MEM + 1× MEM NEAA + 1 mM sodium pyruvate + 10% FCS 5% _CO2 , 37°C 6×10 ⁶ 3×10 ⁶ HUP-T4 T150 MEM + 1× MEM NEAA + 1 mM sodium pyruvate + 20% FCS 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ KATO-III FGF-BP #12 adM ³ T150 RPMI + 1% pen/strept. + 4 mM glutamax (total) + 20% FCS 7.5% _CO2 , 37°C 8×10 ⁶ 5×10 ⁶ KP-2 T150 RPMI + 10% FCS 5% _CO2 , 37°C 8×10 ⁶ 610 ⁶ MiaPaCa-2 T150 MEM + 10% FCS 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ MiaPaCa-2-LVT T150 MEM + 10% FCS + 1% pen/strept. + 1.5 µg/ml blasticidin 5% _CO2 , 37°C 1×10 ⁷ 8×10 ⁶ NUGC-4 sub10cH11 subE10 Luci#2 T150 RPMI + 1% pen/strept. +10% FCS 5% _CO2 , 37°C 8×10 ⁶ 5×10 ⁶ Panc-1 T150 DMEM + 10% FCS 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ Panc03.27 T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 4.5 g/l glucose + 0.01 mg/ml insulin + 15% FCS 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ Panc05.04 T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 4.5 g/l glucose + 0.01 mg/ml insulin + 15% FCS 5% _CO2 , 37°C 6×10 ⁶ 4×10 ⁶ Sub-clones of Panc05.04 T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 4.5 g/l glucose + 0.01 mg/ml insulin + 15% FCS 5% _CO2 , 37°C 7×10 ⁶ 5×10 ⁶ Patu8902 T150 DMEM+ 10% FCS 5% _CO2 , 37°C 5×10 ⁶ 4×10 ⁶ Patu8902-LVT T150 DMEM+ 10% FCS + 1% pen/strept. + 9 µg/ml blasticidin (fresh) 5% _CO2 , 37°C 5×10 ⁶ 4×10 ⁶ Patu8988T T150 DMEM + 5% horse serum + 5% FCS 5% _CO2 , 37°C 3×10 ⁶ 1×10 ⁶ Patu8988S T150 DMEM + 5% horse serum + 5% FCS 5-7.5% CO ₂ , 37°C 1.5×10 ⁷ 1×10 ⁷ Su86.86 T150 RPMI + 10 mM HEPES + 1 mM sodium pyruvate + 4.5 g/l glucose + 10% FCS 5% _CO2 , 37°C 5×10 ⁶ 3×10 ⁶ suit-2 T150 RPMI + 10% FCS (use a new flask for each dropout) 5% _CO2 , 37°C 1.5×10 ⁷ 1.2×10 ⁷ Suit-2-LVT T150 RPMI + 10% FCS + 1% pen/strept. + 5 µg/ml blasticidin (fresh) (use a new flask for each drop) 5% _CO2 , 37°C 1.5×10 ⁷ 1.2×10 ⁷ SW1990 T150 Leibovitz's L-15 + 10% FCS 37°C 4×10 ⁶ 35×10 ⁶ YAPC T150 RPMI + 10% FCS Gold 5% _CO2 , 37°C 1.5×10 ⁷ 1×10 ⁷ YAPC-LVT T150 RPMI + 10% FCS Gold + 1% pen/strept. + 0.5 µg/ml blasticidin (fresh) 5% _CO2 , 37°C 1.5×10 ⁷ 1×10 ⁷

¹ LVT - cell lines stably transduced with lentivirus to express CLDN18.2;

² seed density in a given cell culture flask or dish for 2 or 3 days;

³ adM: cells recultivated from subcutaneous tumors.

4. Luciferase transfection of pancreatic cell lines

For ADCC analysis, pancreatic cancer cells were transfected with luciferase RNA. Luciferase RNA (vector Pst1-luc2mut-2hBgUTR-A121-EciI (pST1-109)) was prepared with an ARCA cap and dissolved in H ₂ O. This RNA was stored in 22 μl portions at -80°C. For all pancreatic cell lines, the optimal electroporation conditions were determined, giving the highest rates of transfection and cell viability. In each assay, cells were detached with PBS/5 mM EDTA, 2.5×10 ⁶ cells were dissolved in 250 μl X-Vivo and mixed in ice-cold cuvettes with 10 μg RNA. Cells were immediately electroporated (GenePulser Xcell, BioRad) and resuspended in warmed assay medium to a concentration of 5 x 10 ⁵ cells/ml. The tested electroporation conditions for all cell lines were:

EP1: 250V, 475uF

EP2: 200V, 300uF

EP3: 150V, 300uF

EP4: 200V, 400uF

EP5: 250V, 950uF

control: 0 V, 0 uF

Cell viability was determined immediately after electroporation using CASY or by staining the cells with trypan blue and determining the percentage of dead cells in a Neubauer chamber. Cells were plated in quadruplicates in white 96-well plates (2.5 x 10 ⁴ cells per well) and incubated for 24 hours. After that, luciferase activity was measured in a liminometer (Tecan Infinite200) after addition of the luciferin mixture for 90 minutes. Transfection was successful and ADCC measurable if RLU values were > 1000.

5. Quantitative real-time PCR reaction (quantitative PCR)

To isolate RNA from cells of pancreatic cancer lines, cells were seeded in 10 cm dishes and cultured for 2–3 days, up to 80% confluence. RNA was isolated according to the instructions supplied with the ^RNeasy® Mini Kit (Qiagen). cDNA preparation was performed according to the manufacturer's instructions included in the SuperScript ^® III First Strand Kit (Invitrogen). RNA and cDNA samples were stored at -80°C.

Quantitative analysis of CLDN18.2 transcripts was performed by amplification of oligo(dT)-primed cDNA in a 40-cycle PCR reaction using PCR primers #5054s (5'-AGAGAGCTCTGGCTTCACCGAGTG-3') and #5060as (5'-CCAGAAG‌TTAGTCACCAGCATGTTGG-3') that distinguish the CLDN18.1 and CLDN18.2 isoforms. The reaction mixture was prepared with SYBR Green dye (QuantiTect SYBR Green PCR Kit, Qiagen), which intercalates into double-stranded DNA. Reactions and measurements were performed on an ABI-PRISM7900 Sequence Detection System using software (Applied Biosystems).

Relative expression levels of CLDN18 transcripts were calculated from ΔΔCT values relative to the housekeeping gene HPRT.

6. Western blot analysis

To isolate proteins from cells of pancreatic cancer lines, cells were seeded in 10 cm dishes and cultured for 2–3 days, up to 80% confluence. Cells were lysed by adding 800 μl of 4x SDS sample buffer (34% glycine, 250 mM Tris pH 6.8, 5% β-mercaptoethanol, 8.2% SDS). To disrupt the genomic DNA, protein samples were sonicated under the following conditions: power output: level1, duty cycle: 70% for 20-25 sec. The protein concentration was measured on a spectrophotometer (absorbance at 280 nm) and the samples were stored at -80°C until use.

To detect CLDN18.2 expression on Western blots, a 12.5% polyacrylamide separation gel was prepared (for 2 small gels: 4.1 ml acrylamide/bisacrylamide 29:1, 100 µl 10% SDS, 2.5 ml Tris pH 8 ,8, 3.2 ml H ₂ O, 100 µl APS, 10 µl TEMED) between two fixed glass plates. After polymerization, a concentrating gel (1.5 ml acrylamide/bisacrylamide 29:1, 100 µl 10% SDS, 2.5 ml Tris pH 6.8, 5.8 ml H ₂ O, 100 µl APS, 10 µl TEMED) and a comb was inserted between the glass plates. After polymerization, 75 µg of each protein sample prepared by adding (1:20) 4× SDS sample buffer (250 mM Tris-HCl, 34% glycerol, 8.2% SDS, pH 6.8) and 7 .5 µl size marker mix (1.5 µl Magic Mark XP Western Standard along with 6 µl SeaBlue Plus2 Prestained Standard). Gels were run in 1× SDS running buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS) for 30 min at 80 V and 60 min at 180 V. Semi-dry blotting of the gel onto a nitrocellulose membrane was performed for 90 min at 160 mA in 1x transfer buffer (25 mM tris, 0.192 mM glycine, 20% methanol). Blots were first blocked in 5% milk powder in PBS, and then primary antibodies (0.25 μg/ml against claudin 18 (C-terminus) or 0.1 μg/ml against β-actin) in 1% powdered milk were added. at PBS. Blots were incubated overnight at 4°C, washed 3 times for 10 min in 1×PBS/0.05% Tween 20, and then incubated for 1 hour with labeled secondary antibodies at room temperature in 1% powdered milk in PBS (goat against rabbit IgG (FC) at a dilution of 1:1000). The blots were again washed 3 times for 10 min in 1× PBS/0.05% Tween 20, and then detected by adding 1-3 ml of detection solution (Pico and Dura Detection System (Pierce) for 1 min and scanning the blots in the detection chamber LAS-3000 (increment: 10 sec, interval time: 10 sec, sensitivity: high) according to GA_056_Chemolumineszenzentwickler LAS3000.

7. Flow Cytometry (FACS)

Cells from an exponentially growing culture at 70-85% confluence were harvested using PBS with 5 mM EDTA or trypsin with EDTA. Cells were counted, centrifuged for 5 min (468×g), and the pellet was resuspended in FACS buffer (2% FCS, 0.1% sodium azide in PBS), bringing the concentration to 2×10 ⁶ /ml. 100 µl of cells were seeded into round-bottom 96-well plates and centrifuged again (5 min, 468×g). Serial dilutions of IMAB362 (or isotype control - rituximab) from 0.1 to 200 μg/ml (11 dilution steps + control without antibodies) were made in 50 μl of FACS buffer and added to cells for 30 min at 4°C. Then 200 μl of FACS buffer was added to each well and the plates were centrifuged (5 min, 468×g). The supernatants were removed and washing was repeated. Secondary goat anti-human antibodies (FC-specific, F(ab') ₂ conjugated with APC (Dianova)) were diluted 1:100 in FACS buffer and added 30 μl to each well. The plates were incubated for 30 min at 4°C. After incubation, the plates were again washed twice with 200 µl of FACS buffer, and the pellet was finally resuspended in 100 µl of FACS buffer for measurement on the FACS Array Bioanalyzer (BD) according to GA_018_BD FACS Array Bioanalyzer.

8. Lentiviral transduction

Construction of a lentiviral vector. Lentiviruses are RNA viruses that stably integrate into the human genomic DNA of both dividing and non-dividing cells. The vector pLenti6.4 (Invitrogen) was taken as a basis. It contains the blasticidin gene for the selection of positively transduced cells. CLDN18.2 fused to the EF1α promoter was cloned into the recombinant region of the vector to give pL64B42E(EF1α-hCLaudin18.2)-Blasticidin (FIG. 1).

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

Determination of selection conditions for blasticidin. For all cell lines before transduction, it was determined what concentration of blasticidin is necessary for the selection of cells after lentiviral transduction. Pancreatic cancer cells were seeded in 6-well plates at high density giving a confluency of 80-90% after 24 hours. Blasticidin (initial: 10 mg/ml, Invitrogen) was added to the wells at increasing concentrations from 0.5 to 12 μg/ml (5 dilution steps + control without blasticidin). The medium was changed every 3-4 days, and the cells were analyzed under a microscope before removal of the medium. The number of dead cells and the state of living cells were noted. Cells were cultured for 14 days. To select cells transduced with lentivirus, the lowest concentration of blasticidin was used, which caused 100% cell apoptosis after 14 days. The required concentrations of blasticidin for each of the resulting LVT cell lines are shown in Table 4.

Shell selection. For lentiviral transduction, the control GFP-lentiviral vector pL64B42E-(EF1a-GFP)-blast was packaged into particles with different envelopes (VSV-G, GALV, RD114, Mokola-G and Rabies-G). Depending on the proteins present in the envelope and the composition of the cell membrane, attachment to the target cancer cells was more or less efficient. For all pancreatic cancer cell lines, the VSV-G coat produced the highest transduction efficiency (68.5-91.2%) (Table 5). Therefore, the CLDN18.2 expression vector pL64B42E (EF1α-hCLaudin18.2)-Blasticidin was packaged in VSV-G envelopes. Producer cells were infected and the virus was isolated from the medium at a high titer (3.86×10 ⁷ particles/ml). Viral supernatants were stored at -80°C.

Table 5. Generation of CLDN18.2 overexpressing pancreatic cancer cell lines by lentiviral transduction

cell line Envelope (control GFP virus vector) Transduction efficiency pL64B42E (EF1α-hCLaudin18.2) ¹ VSV-G GALV RD114a Mokola-G Rabies-G BxPC3-LVT 91.1 29.5 21.4 61.4 57.1 92.8% CAPAN1-LVT 83.7 12.3 31.7 24.3 23.1 89.6% DANG-LVT 91.2 41.3 13.8 33.7 41.4 87.5% HPAC-LVT 77.7 38.4 49.6 72.8 61.5 97.3% MiaPaCa-2-LVT But But But But But 96.3% Patu8902-LVT But But But But But 93.0% Suit2-LVT But But But But But 92.2% YAPC-LVT 68.5 41.3 13.8 33.7 41.4 82.2%

¹ Vector packaging efficiency in VSV-G coated particles as measured 2 days after transduction.

Lentiviral transduction of pancreatic cancer cells. To infect cells of the targeted pancreatic cancer lines, a 24-well plate was coated with 200 μl of 1× ^{retronectin®} (20 μg/ml, Takara Inc.), sealed with parafilm, and incubated for 3-16 hours at 4°C. The plates were washed with 200 ml PBS and blocked with PBS/2% BSA for 30 min at room temperature. The plates were washed again and 300 µl of viral supernatant were applied with centrifugation for 25 min at 2500 rpm at 15°C. The supernatant was removed and the application was repeated 3 times. Finally, the plates were washed once with PBS and targeted cells were plated in each well with a low number of passages. For all pancreatic cancer cell lines, 5×10 ⁵ -1×10 ⁷ cells were seeded in each of 24 wells. The plates were incubated for 2 days at 37°C. Thereafter, the cells were separated and the transduction efficiency was determined by FACS using FITC-labeled IMAB362 antibody. The cells were expanded and a master cell bank was prepared for each cell line.

9. ADCC analysis

The targeted pancreatic cancer cells were seeded into flasks two days in advance to obtain 80-90% confluent cultures on the day ADCC was started. Pancreatic cancer cells were transfected with luciferase RNA and seeded
in white 96-well plates at a density of 1×10 ⁴ cells per well in 50 μl of detection medium (the culture medium shown in Table 4 with 20 mM HEPES). In addition, NUGC-4 sub10cH11 subE10 Luci#2 cells (8000 cells/well) were seeded as a positive control in all assays. Cells were cultured 4-6 h before the addition of antibodies and purified PBMCs.

PBMCs were obtained from fresh human buffy coat obtained from healthy donors. Three 20-25 ml portions of blood were diluted 1:2 with PBS and carefully layered onto 4 15 ml portions of Ficol-Paque Plus (GE Healthcare) in 50 ml Falcon tubes. The gradients were centrifuged (25 min, 700×g). After centrifugation, peripheral blood mononuclear cells (PBMCs) were harvested from the interphase, washed in PBS with 2 mM EDTA, centrifuged (5 min, 468×g), resuspended in PBS with 2 mM EDTA, and centrifuged (10 min, 208×g), to remove platelets. The pellets were resuspended in 50 ml PBS with 2 mM EDTA and cell counts were performed. PBMCs were centrifuged (5 min, 468×g) and resuspended in X-Vivo-15 culture medium at a concentration of 1.6×10 ⁷ cells/ml to add to pancreatic cells or 1.28×10 ⁷ cells/ml to add to NUGC-4 sub 10cH11 sub E10 Luci#2 cells.

Antibodies (IMAB362 and ch78H11-1H6 antibody from isotype control) were subjected to serial (4.5-fold) dilution 10 times, obtaining concentrations from 200 μg/ml to 0.26 ng/ml. From each dilution, 25 μl was added in four repetitions to the target cells. PBS without antibodies was added to medium and lysis control wells. After that, 25 μl of PBMC were added to each well (E:T ratio = 40:1) and the plates were incubated for 24 h ± 1 h at 37°C, 5% CO ₂ .

The next day, 10 µl of 8% Triton X100/PBS solution was added to lysis control wells, and 10 µl of PBS was added to all other wells. Finally, 50 μl of freshly prepared luciferin solution (160 mM HEPES, 1×PBS, 3.84 mg/ml D-luciferin (BD Biosciences)) was added to each well and the plates were incubated for 80 min at room temperature in the dark. Luminescence was measured as a result of the oxidation of Lucifer Yellow with luciferase of viable cells on a microplate reader (Infinite200, Tecan, Switzerland). The degree of cellular cytotoxicity was calculated using the following formula:

specific kill (%) = 100 - [(RLU _sample - RLU _Triton ) / (RLU _{control on medium} - RLU _Triton ) ×100].

10. CDC

CDC analysis was performed as follows.

Target cells (CHO-K1 p740 MACS/FACS (24H5) p3151 Luci#2A5) were seeded in 50 µl of determination medium in white 96-well assay plates (10,000 cells per well) and cultured for 24 h + 20 min at 37 °C, 7.5% CO ₂ at 95% relative humidity before adding samples. Each 96-well plate contained a total of 3 different negative controls (heat-inactivated sera, sera with and without IMAB362, and sera with an antibody from an isotype control (Rituximab)) and a positive control from a pooled healthy human sera (lot #31032011) with 500 ng/ml IMAB362. An additional positive control was made at the end of the reaction by adding 0.8% Triton X100 to the second medium control well, which caused complete lysis. One of the 96-well plates contained a functional positive control prepared by 7 serial 3.16-fold dilutions of IMAB362 (10,000 to 31.8 ng/mL). This control gave a sigmoid cell lysis-dose relationship. All samples were prepared (200 µl) at the same time in a 96-well dilution plate. Samples were taken from each well 3 times with a pipette to obtain three repetitions in the assay plates. After adding 50 µl of test and control samples to the assay plates, they were incubated for 80+5 min at 37°C, 7.5% CO ₂ at 95% relative humidity.

10 μl of PBS was added to each well, except for the lysis control wells with Triton. 10 µl of a 0.8% Triton/PBS solution was added to each well of the Triton lysis control. Luciferin substrate solution was prepared (6114 µl bidist. water, 2496 µl HEPES (1M), 1998 µl 1xDPBS, 4992 µl D-luciferin stock solution (12 mg/ml)). In each well was added 50 μl of a solution of the substrate - luciferin. The plates were incubated at 37° C., 7.5% CO ₂ at 95% relative humidity for 45 minutes. Plates were measured on a microplate reader.

Complement-dependent lysis was calculated using the following formula:

specific lysis (%) = 100 - [(RLU _sample - RLU _Triton ) / (RLU _HSCM - RLU _Triton ) ×100]

Modifications for testing pancreatic cancer cell lines:

- pancreatic cancer cells were transfected with luciferase RNA under optimized conditions; for each cell line tested, 1.5×10 ⁴ cells per well were seeded;

- since most pancreatic cancer cell lines are difficult to separate and form single cells, trypsin was used on the 1st day;

- pancreatic cancer cells were cultured in assay plates at 37°C, 5% CO ₂ ;

- CDC analysis with pretreatment of cells with chemotherapeutic agents was performed at the following concentrations of IMAB362 or as an isotype control - antibodies ch78H11 1H6: 640,000, 160,000, 40,000, 10,000, 2,500, 625, 156, and 39 ng/ml.

11. Inhibition of proliferation

To analyze the dose-response curves for each chemotherapeutic agent, a proliferation assay was performed. Proliferation inhibition was analyzed after administration of gemcitabine or oxaliplatin at various concentrations for each pancreatic cancer cell line.

Table 6. Pancreatic cancer cell lines in the analysis of the effectiveness of gemcitabine or oxaliplatin

cell line Number of cells per well at seeding 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

Cells were seeded in 96-well plates and after 4-6 hours gemcitabine or oxaliplatin were added at the following concentrations: 1000, 500, 250, 100 and 20 ng/ml. For analysis of proliferation, cells were incubated for 4 days at 37°C and 5% CO ₂ . 50 μl of complete XTT reagent (50 parts of XTT + 1 part of crosslinking reagent) was added and incubated at 37°C. After 3 hours and 4 hours, absorbance was measured (cells plus supernatant) on Tecan Safire. The inhibition of proliferation was calculated as compared to the medium control taken as 100%. EC ₅₀ values for gemcitabine and oxaliplatin were calculated using the GraphPad Prism software.

12. Cultivation of pancreatic cancer cell lines with chemotherapy drugs for ADCC or CDC

Line DANG: seeded 4-6×10 ⁶ cells and cultured for 2 days in medium or in medium + 1 ng/ml gemcitabine or 1 ng/ml gemcitabine + 10 ng/ml oxaliplatin. Line Patu8988S: seeded 1-1.4×10 ⁷ cells and cultured in the absence or presence of 10 ng/ml gemcitabine or 10 ng/ml gemcitabine in combination with 100 ng/ml oxaliplatin.

On the day of initiation of ADCC, the above procedure was followed and cell surface expression of CLDN18 was determined by FACS as described above.

13. Cell cycle analysis

Cells were seeded in 6-well plates and after 5-6 hours chemotherapeutic agents were added for 24 hours, 48 hours or 3 days. Free-floating cells in the medium were combined with the cells of the adhesive layer, which was subjected to trypsinization. Cells were washed. Cell cycle analysis was either started immediately or cell surface staining was performed first as described above. Cells were resuspended in 1 ml PBS and inoculated into 3 ml 4% PFA. After fixing the cells for 15 min at room temperature, the cells were precipitated and washed. For RNase treatment, cells were resuspended in 200 μl of RNase (10,000 U/ml) plus 0.05% Triton X-100 and incubated for 30 min at 37°C. 1 ml PBS was added, samples were centrifuged and resuspended in 200 µl PBS with 50 µg/ml PJ. Within 30 minutes, the samples were ready for analysis by flow cytometry. The distribution of cell cycle phases was determined using the FlowJo software as histograms of DNA content.

14. Apoptosis assay

Following these procedures, apoptosis was measured by annexin V binding (detection kit I) or by DNA fragmentation (Apo-Direct) according to the manufacturer's recommendations (Pharmingen, San Diego, CA). Briefly, free-floating cells in the supernatant were combined with the cells of the adhesion layer, which was subjected to trypsinization and then washed. Portions of 5×10 ⁵ cells were incubated with annexin V-APC and PI for 15 min at room temperature in the dark. Cells were immediately analyzed by flow cytometry. Viable cells contain neither annexin V-APC nor PI. In the early stage of apoptosis, cells are positive for annexin V-APC and negative for PI, while cells that have lost viability due to apoptotic or necrotic cell death stain positive for both annexin V and PI. The percentage of stained cells in each quadrant was quantified using FlowJo software (BD Biosciences, Franklin Lakes, NJ).

Analysis of apoptosis by DNA fragmentation was performed as follows. Treated cells (attached and floating) were fixed in 70% ice-cold ethanol overnight. After washing, 10 ⁶ fixed cells were incubated with terminal deoxynucleotidyltransferase (TdT) enzyme and FITC-dUTP for 90 min at 37°C to mark DNA breaks. Cells were washed, incubated with RNase A and propidium iodide in the dark for 30 min at room temperature for total DNA staining, and then analyzed by flow cytometry. Double and adherent cells were excluded from the analysis when adjusting the channels.

15. In vivo studies

All in vivo experiments were performed in accordance with national regulations and ethical principles for experimental animal studies.

15.1. Processing of xenografts

Tumor xenografts were grafted by subcutaneous injection of cancer cells in 200 μl PBS into the flank of nude Hsd:Foxn1 ^nu female athymic mice. Tumor-bearing mice received 0 μg, 200 μg, 400 μg, or 800 μg of antibodies by intravenous injection once a week or alternating intravenous and intravenous administration 2 times a week. Chemotherapeutic agents were administered intravenously once a week or 2 times a week. The size of the tumors and the condition of the animals were monitored 2 times a week. At the end of chemotherapy, the administration of antibodies continued until the size of the tumors reached >1400 mm ³ or the tumors became ulcerated. Tumor samples were stored frozen or fixed in 4% formalin for later analysis.

15.2. Metastasis analysis

Various pancreatic cancer cell lines were first analyzed for their ability to form metastases after intravenous administration of these cells to nude mice. To analyze their engraftment, groups of 5-10 mice were injected with 1×10 ⁶ and/or 2×10 ⁶ cells and one mouse was killed at different time points to find the time points for engraftment and growth of metastases.

Metastasis treatment was performed on 10-12 nude Hsd:Foxn1 ^nu athymic mice in 1 treatment group. They were injected with 2×10 ⁶ cells (Patu8988S or Suit2-LVT) intravenously. All mice were sacrificed at the same time, as soon as the first symptoms of metastasis appeared (weight loss, weakness, shortness of breath) or the first mouse died.

Tissue dissection. In engraftment studies, mice were sacrificed at various time points or as soon as they showed clear physiological signs of metastasis (weight loss, weakness, dyspnea). All organs were subjected to macroscopic analysis for metastases. Only Patu8988S and Suit-2 cells showed macroscopically visible metastases in the lungs and lungs/liver, respectively. These organs were dissected into 4 equal pieces, of which two (lungs: upper right and lower left lobes) were stored for genomic DNA isolation. The other two pieces were fixed in formalin and stored for IHC analysis (FIG. 2).

Genomic DNA acquisition and quantitative PCR strategy. Genomic DNA was extracted from lung or liver tissue. As a control, genomic DNA was also isolated from human pancreatic cancer cells Patu8988S, as well as from uninjected negative control mice.

The quantitative PCR strategy is based on the amplification of human DNA present in metastases. The relative level of human DNA found in mouse lung samples directly correlates with the number and/or size of metastases. Since this method is subject to bias because lung metastases are unevenly distributed and sometimes one lobe is affected more than the other, two different lung regions were mixed for one DNA preparation (Fig. 2).

Quantitative PCR reactions were performed with a pair of primers #5861: 5'-GGGATAATTTCAGCTGACTAAACAG-3' and #5862: 5'-TTCCGTTTAGTTAGGTGCAGTTATC-3', which specifically amplify the alpha satellite DNA present on the 17th human chromosome, but not in mouse DNA. . To generate a standard curve and as a positive control, Patu8988S DNA was mixed with mouse DNA and 5-fold dilutions were made to give 100%, 20%, 4%, 0.8%, 0.16%, 0.032%, and 0.0064% DNA. human in mouse DNA. This curve was used to calculate (linear regression) the number of human DNA metastases in mouse lung tissue. Quantitative PCR reactions were performed in a final volume of 50 µl containing 20 µl (200 ng) mouse lung DNA, 25 µl SYBR Green (Qiagen), 1.6 µl sense primer (10 µM) and 1.6 µl antisense primer and 1.8 µl H ₂ O.

Example 2 Expression of CLDN18.2 in Normal and Tumor Tissues of the Human Pancreas

To analyze the level and profile of CLDN18.2 expression in normal and tumor pancreatic tissues, histological staining of FFPE sections was performed using two mouse monoclonal antibodies (Fig. 3).

Exploratory pilot experiments were performed using the original 35-22A antibody on tissue microarrays (TMAs). The main disadvantage of TMAs is the variable quality of the applied tissues and the small size of the spots, i.e. the unrepresentativeness of the samples. Along with a not fully optimized staining technique, this could lead to an underestimation of positive cases.

The main experiments were performed with antibody 43-14A. In this case, staining was performed on tissue sections, which (compared to TMAs) were larger and were subjected to a preliminary assessment for the presence of tumor cells.

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

PanIN-1 lesions (Fig. 4A) are flat, tall columnar cells with basal nuclei and abundant supranuclear mucin. The nuclei are small and round or oval in shape, oriented perpendicular to the basement membrane. There is a histological overlap between flat, non-neoplastic hyperplastic lesions and flat tumor lesions without atypia.

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

PanIN-2 lesions (Fig. 4B) are flat or papillary and have characteristic nuclear abnormalities including some loss of polarity, induration, enlargement, pseudostratification, and nuclear hyperchromatism. Mitoses are rare, but when present, they are not luminal (not apical) or atypical (Hruban et al., Am J Surg Pathol. 2001, 25(5):579-86).

PanIN-3 lesions (Fig. 4C) are usually papillary or micropapillary, but occasionally they may be flat. True striation, budding of small clusters of epithelial cells into the lumen, and luminal necrosis suggest a diagnosis of PanIN-3. Lesions are characterized by loss of nuclear polarity, dystrophic goblet cells (goblet cells with nuclei oriented toward the lumen and mucoid cytoplasm oriented toward the basement membrane), mitoses that can sometimes be abnormal, nuclear heterogeneity, and protruding (macro)-nucleoli (Hruban et al ., Am J Surg Pathol 2001, 25(5):579-86).

The expression of CLDN18.2 in precancerous tissues was analyzed using antibody 43-14A on tissue samples from various sources. CLDN18.2 was frequently found in the PanIN structures of the PanIN-1, -2 and -3 subtypes, indicating early expression of CLDN18.2 in precancerous lesions (Fig. 4) that persists into later stages. In contrast, expression was not observed in samples of normal pancreatic tissues, including pancreatic ducts. Thus, CLDN18.2 is an early marker of the onset of malignant histological changes in the pancreatic ducts.

Two studies were conducted to evaluate CLDN18.2 expression in primary pancreatic cancer. In a pilot study, several tissue microarrays (TMAs) including a total of 141 primary pancreatic cancers were stained with CLDNA18.2-specific monoclonal antibody 35-22A. In general, the quality of the analyzed TMAs was unsatisfactory. Many stains were partially lost during epitope rendering, and heterogeneous hematoxylin counterstaining is indicative of suboptimal FFPE tissue processing.

Overall, >48.9% of staining cases were positive for CLDN18.2, including 49.2% (65/132) of ductal adenocarcinomas, 50% (1/2) of acinar cell carcinomas, and 3 of 7 neuroendocrine carcinomas (table 7). Tumor cell membranes stained without any background on other cell types (FIG. 6). Moreover, a correlation was observed between the intensity of CLDN18.2 expression and the content of stained tumor cells in the tumor (Table 8, Fig. 5).

Table 7. Pilot study: breakdown of CLDN18.2-positive cases by pancreatic cancer subtype. Tissues were stained with murine monoclonal antibody 35-22A (0.2 μg/ml) and examined for the presence of CLDN18.2-positive tumor cells.

Primary pancreatic cancer Total The proportion of positive is ≥ 1% [%] Staining intensity is ≥2+ [%] Total 141 69 [48.9] 62 [43.9] Adenocarcinoma of the ducts 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]

Table 8. Pilot Study: Correlation between CLDN18.2 signal intensity and positive tumor cell count in primary pancreatic tumors assay. The percentage positive among primary tumors correlates with staining intensity. Cases are grouped into six fractions based on the number of positive tumor cells for clarity.

% posit. cells Signal intensity + ++ +++ Total Total 7 24 38 69 1-9% 3 [42.8] 6 [25.0] 1 [2,6] 10 [14.5] 10-39% 2 [28.6] 6 [25.0] 5 [13.2] 13 [18.8] 40-49% 0 0 1 [2,6] 1 [1,4] 50-59% 0 3 [12.5] 2[5,3] 5 [7,3] 60-69% 0 2[8,3] 5 [13.2] 7 [10.2] 70-100% 2 [28.6] 7 [29.2] 24 [63.1] 33 [47.8]

Table 9. Pilot study: grading of CLDN18.2-positive tumors. Gradation of tumor cells is carried out according to the appearance and degree of cell differentiation. In this case, the 1st degree means well differentiated cells, the 2nd degree - moderately differentiated and the 3rd degree - poorly differentiated cells.

Degree Total The proportion of positive is ≥1% [%] Staining intensity is ≥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]

The second study was performed using an optimized staining technique with highly sensitive antibody 43-14A using quality-checked tissue sections.

Table 10. Main study: breakdown of CLDN18.2-positive cases by pancreatic cancer subtype. The tissues were stained with the mouse monoclonal antibody 43-14A (0.2 μg/ml) and examined for the presence of CLDN18.2-positive tumor cells.

Primary pancreatic cancer Total The proportion of positive is ≥1% [%] Staining intensity is ≥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]

^{table A}

primary cholangiocarcinoma Total positive % positive cholangiocarcinoma 15 9 60

^{table B}

A total of 42 samples of primary pancreatic duct cancer were analyzed. About 90% (38 of 42 cases) of these were positive for CLDN18.2 (Table 10), with the majority (>60%) showing a high +++ signal intensity (FIG. 7, Table 11). At the same time, a correlation was also observed between the level of expression of CLDN18.2 and the content of positive tumor cells. The majority of analyzed cases (62%) were grade 3 tumors (Table 12).

Table 11. Main Study: Correlation between CLDN18.2 Signal Intensity and Number of Positive Tumor Cells in the Analysis of Primary Pancreatic Tumors. The percentage positive among primary tumors correlates with staining intensity. Cases are grouped into six fractions based on the number of positive tumor cells for clarity.

% positive cells Signal intensity + ++ +++ Total Total 1 13 26 40 1-9% 1 2 [15.4] 3 [11.5] 6 [15.0] 10-39% 0 3 [23.0] 4 [15.4] 7 [17.5] 40-49% 0 2 [15.4] 2 [7,7] 4[10.0] 50-59% 0 2 [15.4] 4 [15.4] 6 [15.0] 60-69% 0 2 [15.4] 1 [3,8] 3 [7.5] 70-100% 0 2 [15.4] 12 [46.2] 14 [35.0]

Table 12. Main study: gradation of CLDN18.2-positive tumors. For most of the tumors analyzed, grading was available by the appropriate pathologist. Gradation of tumor cells is carried out according to the appearance and degree of cell differentiation. In this case, the 1st degree means well differentiated cells, the 2nd degree - moderately differentiated and the 3rd degree - poorly differentiated cells.

Degree Total The proportion of positive is ≥1% [%] Staining intensity is ≥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]

Most patients with pancreatic cancer are diagnosed at an advanced stage. Tumors in patients have already metastasized to the lymph nodes and other organs, in particular, to the liver. In the main study, 79 FFPE lymph node and liver tissue samples with metastatic pancreatic cancer were analyzed by immunohistochemistry using CLDN18.2-specific antibody 43-14A.

70.5% of lymph node metastases (31/44 cases) and 68.6% of distant liver metastases (24/35 cases) showed clear tumor cell staining for CLDN18.2 (Table 13). The staining profile of the positive tumor cells was membranous, in some cases with additional weak cytoplasmic signals (Fig. 9). In accordance with the results of the analysis of primary tumors, a correlation was found between the level of expression of CLDN18.2 and the content of CLDN18.2-positive tumor cells in the samples of metastases (Fig. 8).

No correlation was found between the gradation of the analyzed tumors and the expression level of CLDN18.2 or the content of positive tumor cells.

Table 13 Number of CLDN18.2-positive metastases by organ affected. The tissues were stained with the mouse monoclonal antibody 43-14A (0.2 μg/ml) and examined for the presence of CLDN18.2-positive tumor cells.

Metastases Total The proportion of positive is ≥1% [%] Staining intensity is ≥2+ [%] Total 79 55 [69.6] 52 [65.8] From the pancreas to the lymph nodes 44 31 [70.5] 28 [63.6] From pancreas to liver 35 24 [68.6] 24 [68.6]

To test whether CLDN18.2 expression is maintained in metastases from positive primary tumors in the same patient, paired samples of primary cancer and lymph node metastases were screened with the 43-14A antibody.

Table 14 Expression of CLDN18.2 in paired samples of primary pancreatic tumors and their metastases in the lymph nodes. Paired samples of primary adenocarcinoma and lymph node metastases were analyzed for CLDN18.2 expression in tumor cells.

Patient primary tumor Metastases to lymph nodes H/2011/775 3S & 8D +++ (15%) +++ (25%) H/2011/2247 7C & 7G +++ (10%) ++ (25%) H/2011/12675 4B & 4L +++ (10%) ++ (5%) H/2010/15941 6SS & 8H +++ (90%) +++ (90%) H/2010/2986 2E & 2G +++ (5%) +++ (40%) H/2010/6709 9B & 9F +++ (70%) +++ (10%) H/2010/11569 5A & 5G +++ (80%) ++ (5%) H/2008/380 4A & 4G +++ (60%) +++ (90%) H/2009/13538 5B & 5E + (20%) + (10%) H/2009/11847 7C & 7F +++ (15%) ++ (15%) H/2009/23108 4A & 4J +++ (5%) +++ (70%) H/2009/4917 8E & 8S +++ (70%) +++ (25%) H/2009/214183C & 3L +++ (55%) ++ (70%) H/2009/20336 2D & 2F +++ (80%) +++ (90%) H/2009/17768 2C & 2E ++ (35%) ++ (20%) H/2008/13194 3A & 3J +++ (35%) +++ (10%) H/2008/13074 3B & 3C +++ (50%) +++ (50%) H/2008/12082 6D & 6G +++ (1%) - H/2008/11178 5B & 1SS +++ (80%) +++ (90%) H/2008/28150 4B & 4C ++ (15%) ++ (30%) H/2007/5478 4A & 4J +++ (90%) +++ (90%) H/2007/6216 3B & 3C +++ (50%) +++ (60%) H/2007/9047 1B & 1J +++ (70%) +++ (35%) H/2007/13983 6C & 6J ++ (15%) ++ (1%) H/2007/14400 6C & 6J - - H/2006/5616 3J & 3D +++ (35%) +++ (5%) H/2006/9779 3D & 3K + (5%) + (15%)

In 25 (92.5%) of the 27 cases analyzed, both the primary tumor and its associated lymph nodes were positive for CLDN18.2. In one case both tissues were negative and in one other case the primary tumor was positive for CLDN18.2 while the metastases were negative.

In 21 (80.7%) of 26 positive paired samples, the signal intensity of the primary tumor and metastatic tumor cells was the same. In 5 cases, the signal intensity decreased from +++ to ++. In 11 (44%) of 25 paired tissue samples, the number of positive tumor cells in metastases was less than in the primary tumor (Table 14).

Thus, the expression of CLDN18.2 is preserved during the transition of primary tumor cells to the metastasizing stage. The overall intensity and proportion of positive tumor cells in lymph node metastases was only slightly less than in the primary tumor (Fig. 10).

In a small number of patients, tissue samples were available from the primary tumor, lymph node metastases, and liver metastases. These triple pairs were subjected to staining to check the preservation of the expression of CLDN18.2 in distant metastases. Six of these triple pairs were analyzed with the 43-14A antibody.

Table 15 Expression of CLDN18.2 in paired samples of primary pancreatic tumors and their metastases. Paired samples of primary adenocarcinoma, liver metastases and lymph node metastases were analyzed for CLDN18.2 expression in tumor cells.

Patient primary tumor Metastases in
limf. nodes Metastases in the liver H/2011/17191-VA-VI-ISS +++ (60%) +++ (70%) +++ (1%) H/2010/14296XA +++ (5-10%) - - H/2010/4157 VIIA ++ (60%) ++ (30%) +++ (90%) H/2009/23598 VIIB +++ (40%) - - H/2011/3590-IIISS-VIIC-ISS ++ (20%) - - H/2008/10701 +++ (90%) +++ (100%) +++ (90%)

In 3 of the 6 triple pairs, all three tissue samples were comparable in terms of their positivity for CLDN18.2 (FIG. 11). In three cases, a proportion of tumor cells in the primary tumor were positive for CLDN18.2, while metastatic lesions did not stain for CLDN18.2 (Table 15).

Example 3 Target expression in human pancreatic cancer cell lines used for in vitro and in vivo models and pancreatic cancer models

Sources of cell lines

The main goal of this preclinical study was to analyze the inhibitory effects of IMAB362 in suitable model systems. To identify CLDN18.2 positive cell lines that can be used to characterize the effects of IMAB362 in vitro and in vivo, a set of 26 commercially available pancreatic cancer lines was screened for CLDN18.2 expression and characterized in detail. For each cell line, a cell bank was prepared immediately upon arrival for experimental use. They originated from primary pancreatic adenocarcinomas (10, of which 6 were mucosal adenocarcinomas), primary carcinomas (4), metastases of pancreatic adenocarcinomas in the liver (5) or spleen (1), or were isolated from ascitic fluid (5) (see .table 16). Some of these cell lines (8) were subjected to lentiviral transduction to express CLDN18.2.

Expression of the CLDN18.2 Transcript in Human Pancreatic Cancer Cell Lines

To identify CLDN18.2-expressing pancreatic cell lines, transcript levels were determined by quantitative real-time PCR (RT-PCR) using a forward primer that binds to CLDN18.2 exon-1 and a reverse primer that binds to CLDN18 exon-3. The endogenously expressing CLDN18.2 human gastric carcinoma cell line KATO-III and the CLDN18.2 negative breast cancer cell line SKBR-3 were included as positive and negative controls, respectively. Real-time PCR showed a clear endogenous expression of CLDN18.2 in pancreatic cancer cell lines DANG, Panc03.27, Panc05.04, Patu8988S and YAPC, with a relative level exceeding 1×10 ⁵ . Interestingly, Patu8988S cells showed a level of CLDN18.2 expression (˜1×10 ⁸ ) comparable to KATO-III gastric CA cells (FIG. 12A). Thus, we found strong expression of CLDN18.2 in 5 out of 22 pancreatic cancer cell lines.

Along with endogenous cell lines, LVT cell lines ectopically expressing CLDN18.2 were analyzed at the transcript level (FIG. 12A). In 6 out of 8 LVT cell lines, relative expression levels of CLDN18.2 were greater than 1×10 ⁸ . Only in HAPC-LVT and Suit2-LVT cells was the expression level higher than 1×10 ⁵ .

Next, it was investigated whether the expression of CLDN18.2 would be stable when cultured in vitro. Patu8988S, Panc05.04 cells and lentivirus-transduced Suit2-LVT, MiaPaCa2-LVT and Patu8902-LVT cell lines were passaged up to 15 times and analyzed for CLDN18.2 transcripts (Fig. 12B-D). With a large number of passages in both endogenous and transduced cells, a loss of CLDN18.2 expression was observed. The loss of expression was highest in the transduced cells. Therefore, for in vitro experiments, early passages were used where possible, and the following engraftment experiments tested the expression of CLDN18.2 in grafted tumors.

Expression of CLDN18.2 protein in human pancreatic cancer cell lines

Detection of CLDN18.2 in cell lysates

In addition to transcript analysis, CLDN18.2 expression was analyzed at the protein level by Western blotting and IF. Cell lysates from 26 pancreatic cell lines were examined by Western blotting using Western blotting (WB) with CLDN18-specific anti-claudin 18 (C-terminus). Again, lysates of SKBR-3 cells were used as a negative control, and lysates of HEK293 cells stably transfected with CLDN18.2 (HEK293-p740) were used as a positive control. At the same time, a strong expression of the protein was noted in Patu8988S, DANG, and Panc05.04 cells, which confirms the data on RNA. Weak bands appeared in lysates of Panc03.27 and BxPC3 cells. YAPC cells that were RNA positive showed a faint smaller band on Western blots. All other cell lines were negative (FIG. 13).

Cellular expression of CLDN18 in pancreatic cancer cells

To obtain data confirming protein expression, pancreatic carcinoma cell lines were examined by immunofluorescence (IF) after cell fixation and permeabilization using the 35-22A antibody for detection. The IF analysis confirmed the previous RNA and protein data showing that the majority of pancreatic cancer cell lines were staining negative for CLDN18.2 (FIG. 14). In some cell lines (such as AsPC1, DANG, HUP-T3, T4-HUP, Panc01) spots were observed in the nuclei, which are most likely staining artifacts. DANG, Panc03.27 and BxPC3 cells, which showed low levels of CLDN18.2 at the RNA and/or protein level, were also negative in the IF assay, which is less sensitive. In contrast, control gastric carcinoma cells Panc05.04, Patu8988S and KATO-III had membranes and cytoplasm strongly positive for CLDN18.2. Staining intensity varied among cells, and negative cells were also observed in the population (FIGS. 14J and N). In LVT cell lines, strong membrane staining was observed in more than 80% of cells.

Confirmation of CLDN18.2 expression in pancreatic cancer cells

To confirm the expression of CLDN18.2 and assess the amount of this target on the cell surface, endogenous Panc05.04 and Patu8988S cell lines, as well as LVT cell lines, were stained with IMAB362 using the native staining method. Although IMAB362 staining of control gastric cancer cells Patu8988S, Panc05.04, and KATO-III was less intense and the percentage of positive cells decreased compared to staining of 35-22A cells (Fig. 16A-F), IF analysis confirmed that CLDN18.2 was expressed. on the surface of pancreatic cancer cells. The 8 LVT pancreatic cancer cell lines ectopically expressing CLDN18.2 showed clear membrane staining on almost all cells (as seen in the 6 LVT cell lines in FIG. 16G-L).

Thus, analysis of CLDN18.2 expression led to the identification of endogenously expressing pancreatic cancer cell lines Panc05.04 and Patu8988S and all 8 lentivirus-transduced cell lines BxPC3-LVT, CAPAN1-LVT, DANG-LVT, MiaPaCa-2-LVT, Suit2- LVT, Patu8902-LVT and YAPC-LVT as suitable for modeling CLDN18.2-positive cell systems.

Development of models of xenotransplantation and metastasis of pancreatic cancer

Engraftment studies to identify suitable subcutaneous tumor models in pancreatic cancer

A total of 37 engraftment studies were performed with various pancreatic cancer cell lines to identify suitable subcutaneous xenotransplantation models to test the efficacy of IMAB362 in vivo. From all the studied cell lines for subcutaneous models of xenotransplantation, BxPC3-LVT, CAPAN1-LVT, MiaPaCa-2-LVT, HPAC-LVT, DANG-LVT and YAPC-LVT cell lines with ectopic expression of CLDN18.2 were selected, showing a high degree of engraftment and homogeneous tumor growth. In addition, subcutaneous xenotransplantation models with endogenously expressing CLDN18.2 cell lines Patu8988S and DANG were selected to test the in vivo efficacy of IMAB362. Subcutaneous administration of Panc05.04 cells did not lead to the formation of subcutaneous tumors.

Table 17. Summary of Engraftment Testing for the Development of Subcutaneous Pancreatic Cancer Xenotransplantation Models

cell line Engraftment Check (EC) date Setting up an experiment results Notes BxPC3 ATCC EC1_BxPC3 ATCC 03.22. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 40%; average lifespan 56 days Not recommended BxPC3 ECACC EC2_BxPC3 ECACC 03.23. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 63 days suitable conditions for subcutaneous xenotransplantation BxPC3-LVT EC3_C179 03.25. 2011 1×10 ⁷ cells subcutaneously in the left side of 10 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 90%; average lifespan 64 days suitable conditions for subcutaneous xenotransplantation CAPAN1 EC1_CAPAN1 26.03. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 52 days suitable conditions for subcutaneous xenotransplantation CAPAN1-LVT EC1_C186 09.08. 2011 metastasis assay: 1-2×10 ⁶ cells in PBS i.v. 10 female mice HsdCpb: NMRI-Foxn1 ^nu no metastases in the lungs and liver; analysis 72 days after injection of cancer cells Not recommended CAPAN2 EC1_CAPAN2 06.04. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan of 100 days suitable conditions for subcutaneous xenotransplantation DANG EC3_C2 08.02. 2007 1×10 ⁷ cells subcutaneously in the left side of 3 nude mice survival rate 100%; very aggressive tumor growth; average lifespan is 18 days. inadequate conditions for xenotransplantation model DANG EC4_C2 10.10. 2007 5×10 ⁵ -1×10 ⁷ cells subcutaneously in the left side of 25 athymic mice survival rate 100%; very aggressive tumor growth; life span 16-23 days inadequate conditions for xenotransplantation model DANG subclone 1C5F2 EC5_C2 01.06. 2010 2.5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; cachexia in mice with tumors; average lifespan 17 days not recommended due to cancer cachexia DANG subclone 1C5F2 EC6_C2 10.02. 2011 5×10 ⁴ -2×10 ⁵ cells subcutaneously in the left side of 15 female mice HsdCpb: NMRI-Foxn1 ^nu 5 × 10 ⁴ \u003d 40% engraftment; 1×10 ⁵ , 2×10 ⁵ = 100% engraftment; life span 26-39 days not recommended due to cancer cachexia DANG subclone 1C5F2 EC7_C2 02.04. 2011 2×10 ⁵ cells subcutaneously in the left side of 5 female and 5 male mice HsdCpb: NMRI-Foxn1 ^nu ; weight kinetics survival rate 100%; cachexia in mice with tumors; average lifespan 29 days not recommended due to cancer cachexia DANG-LVT EC1_C180 21.03. 2011 5×10 ⁶ cells subcutaneously in the left side of 10 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; cachexia in mice with tumors; average lifespan 28 days not recommended due to cancer cachexia Patu8902 EC1_C197 25.07. 2011 1×10 ⁷ cells subcutaneously in the left side of 10 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; rapid growth of tumors; average lifespan is 21 days. suitable conditions for the xenotransplantation model Patu8902 EC2_C197 25.07. 2011 experimental analysis of metastases: i.v. administration of 1-2×10 ⁶ cells to 10 female mice HsdCpb: NMRI-Foxn1 ^nu no metastases in the tissues of the lungs and liver; 8 mice died after injection of cancer cells not recommended for metastasis analysis Patu8988S EC1_C178 15.03. 2011 1×10 ⁷ cells subcutaneously in the left side of 10 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; heterogeneous growth of tumors; ulceration of tumors; average lifespan 55 days Not recommended Patu8988S EC2_C178 05.04. 2011 experimental analysis of metastases: i.v. administration of 1-2×10 ⁶ cells to 10 female mice HsdCpb: NMRI-Foxn1 ^nu very slow metastasis model; analysis through
70 days suitable model for analysis of lung metastases Patu8988S EC3_C178 10.06. 2011 2.5×10 ⁶ -1.5×10 ⁷ cells subcutaneously in the left side of 15 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 95.5 days Not recommended Patu8988S (recult.) EC1_C202 11.08. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 88 days Not recommended Patu8988S subclone 17 EC1_C214 28.11. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 54 days Not recommended Patu8988S subclone 22 EC1_C215 28.11. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 47 days not recommended due to heterogeneity and ulceration of tumors; best growth curves of all Patu8988S subclones Patu8988S subclone 30 EC1_C216 28.11. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 35 days Not recommended Patu8988S subclone 34 EC1_C217 28.11. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 49 days Not recommended Patu8988S subclone 41 EC1_C218 28.11. 2011 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 108 days Not recommended Patu8988S subclone adM#13 EC1_C237 06.02. 2012 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu slow growth of tumors; bloody cysts, but no ulceration; average lifespan 73 days acceptable model for subcutaneous xenotransplantation Patu8988S subclone adM#19 EC1_C238 06.02. 2012 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 42 days Not recommended Patu8988S adM#1 subclone EC1_C239 13.02. 2012 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu heterogeneous growth of tumors; slow growth of tumors; ulceration of tumors; average lifespan 77 days Not recommended Patu8988S subclone adM#16 EC1_C240 13.02. 2012 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu slow growth of tumors; bloody cysts, but no ulceration; average lifespan is 66 days. acceptable model for subcutaneous xenotransplantation Patu8988S subclone adM#9 EC1_C241 13.02. 2012 5×10 ⁶ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu slow growth of tumors; bloody cysts, but no ulceration; average lifespan is 59 days. acceptable model for subcutaneous xenotransplantation Suit2 EC1_C196 25.07. 2011 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; rapid growth of tumors; ulceration of tumors; average lifespan 35 days Not recommended Suit2 EC2_C196 25.07. 2011 experimental analysis of metastases: iv administration of 2×10 ⁶ cells
10 HsdCpb female mice: NMRI-Foxn1 ^nu metastases in lungs, liver and muscles suitable model for analysis of lung metastases Panc02.03 EC1 Panc 02.03 12.05. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 54 days suitable conditions
for subcutaneous xenotransplantation model Panc03.27 EC2 Panc 03.27 12.05. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 91 days suitable conditions
for subcutaneous xenotransplantation model Panc04.03 EC3 Panc 04.03 17.06. 2010 1×10 ⁷ cells subcutaneously
to the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 39 days suitable conditions
for subcutaneous xenotransplantation model Panc05.04 EC4 Panc 05.04 18.06. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu no growth of subcutaneous tumors was noted Not recommended Panc05.04 EC5 Panc 05.04 09.05. 2011 2×10 ⁷ cells in RPMI subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu no growth of subcutaneous tumors was noted Not recommended MiaPaCa2 EC1_C195 25.07. 2011 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 42 days suitable conditions
for subcutaneous xenotransplantation model MiaPaCa2-LVT EC1_C219 18.11. 2011 5×10 ⁶ or 1×10 ⁷ cells subcutaneously in the left side of 10 female HsdCpb mice: NMRI-Foxn1 ^nu survival rate 100%; average lifespan of 40 days suitable conditions
for subcutaneous xenotransplantation model MiaPaCa2 EC2_C195 25.07. 2011 experimental analysis of metastases: iv administration of 2×10 ⁶ cells
10 HsdCpb female mice: NMRI-Foxn1 ^nu metastases in lungs, liver and lymph nodes suitable model for analysis of lung metastases HPAC EC1_HPAC 19.04. 2010 1.5×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; average lifespan 29 days suitable conditions
for subcutaneous xenotransplantation model YAPC EC1_YAPC 10.05. 2010 1×10 ⁷ cells subcutaneously in the left side of 5 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; very aggressive tumor growth; homogeneous growth of tumors; ulceration of tumors; average lifespan 28 days limited model of xenotransplantation YAPC-LVT EC2_YAPC 10.05. 2010 5×10 ⁵ -7.5×10 ⁶ cells subcutaneously in the left side of 20 female mice HsdCpb: NMRI-Foxn1 ^nu survival rate 100%; very aggressive tumor growth; homogeneous growth of tumors; ulceration of tumors; average lifespan 27 days limited model of xenotransplantation

Engraftment studies to identify suitable metastasis patterns

To study the effects of IMAB362 on the formation of metastases, models of cancer metastasis in nude mice were set up. Cells of pancreatic cancer lines were analyzed for their ability to metastasize after intravenous administration. CAPAN1-LVT, MiaPaCa-2, Patu8988S, Patu8902, and Suit-2 cells were injected into the tail vein of nude mice as described in Mohanty and Xu, 2010. To determine engraftment time and metastasis growth rate, mice were sacrificed at various time points (Table 18 ).

Table 18 Engraftment analysis of metastases from pancreatic cancer cell lines

cell line Number of cells Number of mice On which day mice were analyzed (number) What day did the mice die (number) What organs were isolated Metastases Patu8902 1×10 ⁶ 5 - 0(1), 1(3), 4(1) But No Patu8902 2×10 ⁶ 5 - 0(1), 1(3), 15(1) But No Patu8988S 1×10 ⁶ 7 34, 51, 59, 66, 70, 86, 108 - lungs and liver in the lungs Patu8988S 2×10 ⁶ 8 34, 41, 51, 59, 66, 70, 86, 108 - lungs and liver in the lungs suit-2 2×10 ⁶ 10 36(3), 45(3), 52(2), 59(2) - lungs and liver in lungs and liver CAPAN1-LVT 2×10 ⁶ 5 72(1) 0(1), 1(3) But But CAPAN1-LVT 1×10 ⁶ 5 72(4) 2(1) lungs and liver But MiaPaCa-2 2×10 ⁶ 10 32(2), 52(2), 59(2), 66(2), 73(2) - lungs and liver No

Engraftment analysis of Patu8902 and CAPAN1-LVT cells was not possible because most of the mice died almost immediately. In the lungs and liver of 5 surviving mice treated with CAPAN-LVT cells, no macroscopically visible metastases were found after 72 days. In contrast, the administration of Suit-2 and MiaPaCa-2 cells was well tolerated. The lung tissues of these mice were subjected to IHC analysis at various time points after injection. In mice treated with MiaPaCa-2 cells, no metastases were noted in the lungs after 73 days, so this cell line was not chosen as a model for treatment with IMAB362. Suit-2 cancer cells metastasized to the lungs of mice. There were multiple lesions throughout the tissue. That is why the Suit-2-LVT cell line transduced with lentiviral CLDN18.2 was chosen as a model system to analyze the effects of IMAB362 on the formation of metastases.

Along with Suit-2, the ability to form metastases in Patu8988S cells endogenously expressing CLDN18.2 was also analyzed. An engraftment test was performed by intravenously injecting 2 different amounts of cells (1×10 ⁶ , 2×10 ⁶ ) into mice. Lungs and liver were isolated at various time points as indicated in Table 18. First, the various tissues obtained were analyzed by q-PCR. Lungs and liver obtained before day 70 were analyzed by amplification of α-satellite DNA from human chromosome 17. The lung results showed a clear increase in human DNA content in mouse lungs over time, which was independent of the number of injected cells. With intravenous administration of 1×10 ⁶ or 2×10 ⁶ cells, 5.8% and 3.7% of human DNA were observed after 70 days, respectively (Fig. 19). In the liver, human DNA was almost not amplified. After 70 days, its content increased slightly, but was still less than 0.005%.

To test for CLDN18.2 expression in Patu8988S metastases, lung tissues were subjected to immunohistochemical staining with anti-human MHC class I antibodies to detect human cells in mouse tissues, as well as anti-claudin 18 (Mid). Staining for MHC-I showed that mice had clear metastatic foci on lung tissue sections but not on liver sections (FIG. 20). Moreover, cell membranes in these lesions were stained with anti-claudin 18 (Mid), which clearly indicates the expression of the target protein IMAB362 in these cells. Therefore, this endogenous metastasis model was chosen to study the use of IMAB362 along with the Suit2-LVT model.

Example 4 IMAB362 Mediated Cell Killing Effects

IMAB362 cross-linking induces efficient apoptosis

Binding of antibodies to the target cell surface can trigger abnormal signaling leading directly to cell death. Such signaling events may depend on the epitope of the target, the valency of the binding, and whether the binding is associated with cross-linking of the target. For example, induction of apoptosis by rituximab in several CD20-positive lymphoma cell lines has been observed only under the condition of cross-linking. Such cross-linking can occur in vivo with a high-affinity interaction of Fc-positive immune cells with antibody-coated tumor cells.

Cross-linking of IMAB362 induces apoptosis directly within 18-42 hours in human gastric cancer cells NUGC-4 and KATO-III as determined by the TUNEL method. The degree of apoptosis correlates with the dose of antibody and the level of expression of the target in cancer cells. Treatment with gemcitabine results in cell cycle arrest of cancer cells, followed by cell apoptosis. Apoptosis of gemcitabine-treated pancreatic tumor cells is shown in FIG. 21.

IMAB362-mediated ADCC activity against pancreatic cancer cells

IMAB362 is very potent in inducing the recruitment and activation of Fcγ receptor positive immune effector cells such as normal killer cells. Binding of IMAB362 to target cells induces antibody dependent cellular cytotoxicity (ADCC) through the action of granzymes and perforins secreted by effector cells upon binding of their Fcγ receptors to the antibody. This mechanism of action has previously been shown to affect luciferase and CLDN18.2 positive gastric CA cells (NUGC-4 and KATO-III types) when incubated with IMAB362 for 24 hours in the presence of human peripheral blood mononuclear cells (PBMCs) (effector:target ratio = 40:1). Application up to 200 μg/ml IMAB362 gave a maximum degree of lysis of 80-100%.

Here, we determined the ADCC activity of IMAB362 against pancreatic cancer cell lines. IMAB362 at increasing concentrations was incubated with various cell lines at a ratio of E:T = 40:1. In each experiment, PBMCs from different donors were added. The results for all cell lines are shown in Table 19. Of the 5 initially identified CLDN18.2-positive pancreatic cell lines, only Patu8988S, Panc05.04 and DANG were effectively killed by the addition of IMAB362 and PBMCs (Fig. 22A). Although surface expression of CLDN18.2 in Panc05.04 and DANG was not detected by FACS, the level of expression was significant enough 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 concluded that only cells expressing RNA at a relative level of >5.5×10 ⁵ are efficiently lysed.

ADCC analysis was also performed with LVT pancreatic cancer cell lines and corresponding parental cell lines (FIGS. 22B-F). ADCC is strongly 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 killing and maximum killing of human pancreatic cancer cells by IMAB362 varied between PBMC donors and also depended on the number of cell passages affecting the level of CLDN18.2 expression.

The concentrations of IMAB362 that cause half-maximal killing of target cells as well as the maximum degree of killing are shown in FIG. 22G-H. LVT pancreatic CA cell lines were killed by the addition of small amounts of antibody to a large extent, while DANG and Panc05 required the highest concentrations of antibody to achieve a kill rate of ~50% of the maximum. The figure also shows the results for Panc05.04 obtained with subclone 15D3 (CLDN18.2-positive clone selected by Panc05.04 serial dilution and FACS), which exhibited a degree of ADCC-induced lysis comparable to LVT cell lines. Unfortunately, CLDN18.2 expression in this clone quickly subsided in vitro after cell passage, so this clone was not used for further experiments.

IMAB362-mediated CDC activity against pancreatic cancer cells

Pancreatic cancer cells that were killed by IMAB362 in the ADCC assay were analyzed for their sensitivity to the complement-dependent lytic activity of IMAB362. In addition, CDC and LVT cell lines and their parent strains were tested.

CDC activity is activated by antigen complexes with antibodies of the IgM or IgG type (classical pathway) or by microbial surfaces (alternative pathway). In the classical pathway, complement C5 is converted to C5b. Upon sequential binding of C5b, C7, C8, and C9, anaphylotoxins C3a, C4a, and C5b are released and the membrane attack complex (MAC) is formed. This pathway is suppressed by both soluble and membrane-bound proteins (eg, CR1, DAF, MCP, CD59, CD55, CD46) that protect their tissues.

CHO-K1 cells stably transfected with CLDN18.2 (p740) and luciferase were used as positive controls in each assay (FIG. 23A). The DANG, BxPC3, YAPC, Patu8988S, Panc05.04, CAPAN1, and Suit-2 cell lines did not undergo lysis when IMAB362 and pooled healthy human serum were added (Fig. 23B). Although DANG, Patu8988S and Panc05.04 cells are CLDN18.2 positive, as shown in all previous experiments, they do not lyse in a complement dependent manner. This is most likely due to the fact that cancer cells overexpress one or more proteins that inhibit membrane-bound complement (eg, CD46, CD55, and CD59) (Geis et al., Curr Cancer Drug Targets 2010, 10:922-931). However, it is still unclear whether the expression of these inhibitory proteins in tumor cells affects the clinical outcome of antibody therapy (Dzietczenia et al., Med. Oncol. 2010, 27:743-6; Weng and Levy et al., Blood 2001, 98: 1352-7).

Along with endogenous cell lines, CDC and all LVT cell lines were tested. As can be seen from FIG. 23, addition of IMAB362 and serum to MiaPaCa-2-LVT, Suit2-LVT and CAPAN1-LVT cells caused dose-dependent lysis with EC ₅₀ values from 0.3 to 2.6 μg/ml.

Example 5 Efficacy of IMAB362 in Pancreatic Cancer Xenotransplantation Models

10 out of 41 validated pancreatic cancer xenotransplantation models were selected to study the in vivo efficacy of IMAB362. In pancreatic xenograft models with high expression of CLDN18.2, treatment with IMAB362 showed a high antitumor effect. It was examined by treating mice carrying BxPC3-LVT or MiaPaCa-2-LVT subcutaneous xenografts in their left flank. Treatment was started 3 days after tumor inoculation with IMAB362 200 μg twice a week. IMAB362-treated mice exhibited significant tumor growth inhibition compared to mice treated with saline control. In addition, suppression of tumor growth in IMAB362-treated mice resulted in a prolongation of mean lifespan (FIG. 24 and FIG. 25). The effectiveness of IMAB362 correlates with the duration of treatment. If IMAB362 treatment is started at early time points, tumor growth inhibition is increased more than when treatment is started late to study the effect on established tumors. Moreover, the antitumor effect of IMAB362 depended on the degree of expression of CLDN18.2 in the target. In CLDN18.2 low-expressing tumors of the DANG and Patu8988S xenograft type, IMAB362-mediated growth inhibition was reduced compared to tumor growth inhibition when tumor xenografts with high CLDN18.2 expression were xenografted.

Example 6 Treatment in Mouse Models of Pancreatic Cancer Metastasis

Table 20. Processing Summary for Testing the Efficacy of IMAB362 on Pancreatic Cancer Metastases

cell line No. (date) of the experiment Experienced groups Setting up an experiment Suit2-LVT ET2_C220 (11/22/2011) 1.n=15; 200 mcg IMAB362 twice a week i/v/b
2. n=15; 200 mcg of isotype control 2 times a week iv-ip
3.n=15; PBS 2 times a week i/v/b - injection of 2×10 ⁶ cells in/in
into the tail vein of female nude mice Hsd: Foxn1 ^nu
- start of treatment 3 days after the injection of cancer cells Patu8988S ET1_C178 (05/24/2011) 1.n=10; 200 mcg IMAB362 twice a week i/v/b
2.n=10; PBS 2 times a week i/v/b - injection of 2×10 ⁶ cells in/in
into the tail vein of female nude mice Hsd: Foxn1 ^nu
- start of treatment 3 days after the injection of cancer cells Patu8988S ET1_C178b (06/03/2011) 1.n=15; 200 mcg IMAB362 twice a week i/v/b
2. n=15; isotype control 2 times a week i/v-i/b - injection of 2×10 ⁶ cells in/in
into the tail vein of female nude mice Hsd: Foxn1 ^nu
- start of treatment 3 days after the injection of cancer cells

Suit2-LVT metastasis model

Mice were intravenously injected with 2×10 ⁶ Suit2-LVT cells and treated with 200 μg of IMAB362, isotype control antibody (IMAB027) or PBS as indicated in Table 20. After 35 days, the first mouse (isotype control group) died. Thereafter, all mice were sacrificed on day 42 and the lungs and liver were removed for analysis by IHC and quantitative PCR.

Analysis of human DNA in the lungs of mice by quantitative PCR was repeated at least twice in triplicate. Calculation of human DNA content from the obtained Ct values showed a significant reduction (p<0.05) in Suit2-LVT metastases found in the lungs when mice were treated with IMAB362 (FIG. 26A) compared with PBS treatment and isotype control. To verify these results, lung tissue samples were sectioned and stained with anti-MHC-I antibodies. The surface area of positively stained cells was calculated on lung sections using the ImageJ program. Significant inhibition (p<0.05) was observed with IMAB362 treatment compared to PBS treatment, confirming the results obtained by q-PCR. However, differences were not significant for the isotype control antibody (FIG. 26B). This discrepancy is most likely due to differences in tissue processing: IHC processing of tissue sections only allows access to a very small area of the lung compared to q-PCR, which extracts genomic DNA from half of the tissue.

Along with tissue processing, it is possible that this result represents unexpected inhibitory effects of the anti-CLDN6 isotype control antibody. To explore this possibility, Suit2-LVT cells were analyzed for CLDN6 expression and IMAB027 binding by FACS. Addition of 200 μg/ml IMAB362 to Suit2-LVT cells showed strong binding to the cells, while addition of 200 μg/ml IMAB027 resulted in weak binding of the antibody to these target cells, indicating that CLDN6 is indeed weakly expressed in these cells. These results indicate that at least two factors (tissue processing and weak inhibition of IMAB027) caused the differences observed with the isotype control antibody.

Metastasis model Patu8988S

To analyze the effect of IMAB362 treatment on the development and growth of Patu8988S metastases in vivo, groups of 10 mice were injected with 2×10 ⁶ Patu8988S cells. The first experiment was carried out by comparing the treatment of IMAB362 with the treatment of mice with PBS. In each group, 1 mouse died immediately after cell injection. In the other 18 mice, metastases occurred very quickly compared to the engraftment experiments. After 63 days, the first 2 mice in the PBS group were sacrificed due to poor health. All other mice were sacrificed after 65 days. Optical analysis of the lungs showed large metastases throughout the lung tissue. The degree of metastasis was analyzed by quantitative PCR (Fig. 27). The results show that IMAB362 inhibits the growth of metastases in lung tissue.

A second experiment with 11 mice per group was performed by comparing IMAB362 treatment with isotype control (rituximab) treatment. In this experiment, metastases developed slowly, as was observed during engraftment. However, for data comparability, this second experiment was also terminated after 65 days. Again, lung tissue was analyzed by quantitative PCR, and again IMAB362 reduced the growth of metastases. One mouse from the IMAB362 group was set as an outlier, and excluding this outlier resulted in nearly significant (p=0.0588) inhibition. These data were verified by surface area using the IHC method as described for the Suit2-LVT metastasis experiment. This showed the same outlier, and when this value was omitted in the t-test, IMAB362 inhibition was also on the verge of significance (p = 0.0691), as were the values of this mouse.

Example 7 Primary Pharmacodynamics of IMAB362 in Combination with Chemotherapy

Sensitivity of pancreatic carcinoma cells to gemcitabine and oxaliplatin

To study the mechanisms of action of IMAB362 in combination with the chemotherapeutic agents oxaliplatin or gemcitabine, pancreatic cancer cells constitutively expressing CLDN18.2 (DANG, Patu8988S) and cells stably transducing CLDN18.2 (MiaPaCa-2-LVT, BxPC3-LVT) were used. .

Chemically, gemcitabine (Gemzar, manufactured by Eli Lilly & Co) is a nucleoside analogue. Like 5-fluorouracil (5-FU) and other pyrimidine analogs, the triphosphate analog of gemcitabine replaces one of the building blocks of nucleic acids in DNA replication. This process stops the growth of tumors, since only one more nucleoside can attach to the “wrong” nucleoside, which leads to apoptosis.

Oxaliplatin functions by forming interstrand and intrastrand crosslinks in DNA. Crosslinks in DNA prevent DNA replication and transcription leading to cell death (Graham J., Mushin M., Kirkpatrick P. (2004) “Oxaliplatin” Nature Reviews Drug Discovery 3(1): 11-2).

Dose-response curves for gemcitabine and oxaliplatin indicate different sensitivity of the studied pancreatic cancer cell lines (Fig. 28 and Fig. 29).

Table 21. IC ₅₀ values of gemcitabine and oxaliplatin for pancreatic cancer cell lines

cell line IC ₅₀ for proliferation inhibition gemcitabine oxaliplatin BxPC3-LVT ~2 ng/ml 500-1000 ng/ml Capan1-LVT ~1 ng/ml ~100 ng/ml DANG ~1 ng/ml ~500 ng/ml Patu8988S-luci#6 >100 ng/ml >500 ng/ml MiaPaCa-2-LVT 50 ng/ml 50-100 ng/ml

High concentrations of gemcitabine (IC ₅₀ >100 ng/mL) or oxaliplatin (IC ₅₀ >500 ng/mL) are required to inhibit proliferation of Patu8988S cells. DANG and BxPC3-LVT cells are highly sensitive to gemcitabine, but not to oxaliplatin. MiaPaCa-2-LVT cells are most sensitive to oxaliplatin but less sensitive to gemcitabine treatment (FIG. 28, FIG. 29 and Table 21).

Effect of Chemotherapeutic Agents on CLDN18.2 Expression in Pancreatic Carcinoma Cell Lines

The mode of action triggered by IMAB362 binding strongly depends on the presence and density of its target, CLDN18.2, on the cell surface. Pretreatment of DANG and Patu8988S cells with gemcitabine (GEM), as well as gemcitabine in combination with oxaliplatin (GemOx), resulted in increased levels of CLDN18.2 mRNA and protein as measured by RT-PCR analysis (Fig. 30) and Western blotting (Fig. .31) cells without treatment and cells with prior chemotherapy. Consequently, the amount of CLDN18.2 protein available to IMAB362 on the surface of pretreated Gem or GemOx cells of pancreatic cancer lines increased as shown by flow cytometry (FIG. 32).

Treatment of DANG and Patu8988S with gemcitabine results in an increase in CLDN18.2 levels. Patu8988S shows strong upregulation of CLDN18.2 when exposed to Gem and less when exposed to GemOx.

Effects of Chemotherapeutic Drugs on the Cell Cycle and CLDN18.2 Expression

Cell cycle progression refers to the sequence of events from one mitotic division to the next in a cell. The resting phase (G0/G1) is followed by the phase of DNA synthesis (S), and then the phase of cell growth (G2) and DNA replication (M), followed by cell division into two daughter cells. Any intervention in the cell machine can prevent the passage of the entire cycle in any phase of the cell cycle. For example, certain chemotherapeutic agents can block it in the G2 or M phase, or both in G2 and M (G2/M).

Gemcitabine treatment of DANG or Patu8988S cells resulted in S-phase cell cycle arrest (Fig. 33, Fig. 34). Patu8988S was analyzed when cultured with Gem. Gemcitabine treatment not only resulted in cell cycle arrest, but also altered CLDN18.2 expression (FIG. 34B). The change in CLDN18.2 density after gemcitabine treatment was even greater when comparing proliferating cells in S phase and resting cells in G0/G1 phase (Fig. 34C). In Patu8988S cells, CLDN18.2 is expressed at all phases of the cell cycle. After treatment with gemcitabine, its expression is still increased, and the highest levels of CLDN18.2 in cells are observed in the S-phase cell population.

This distortion of the tumor cell phenotype has a significant impact on the biological effectiveness of therapeutic antibodies. ADCC and CDC are dose dependent, so increasing the target CLDN18.2 provides a synergistic benefit with standard chemotherapy regimens.

KATO-III cells, a human gastric cancer cell line, were cultured in RPMI 1640 medium (Invitrogen) containing 20% FCS (Perbio) and 2 mM Glutamax (Invitrogen) at 37°C and 5% CO ₂ , with or without cytostatic compounds . 5-FU (Neofluor from NeoCorp AG) was tested at 10 or 100 ng/mL and oxaliplatin (Hospira) was tested at 50 or 500 ng/mL. Cultured 8×10 ⁵ KATO-III cells for 96 hours without medium change or 72 hours followed by cultivation for another 24 hours in standard medium to remove cell cycle arrest in a 6-well tissue culture plate at 37°C, 5% _CO2 . Cells were harvested with trypsin/EDTA, washed and analyzed.

For extracellular detection of CLDN18.2, cells were stained with anti-CLDN18.2 monoclonal antibody IMAB362 (Ganymed) or a control antibody of the same isotype (Ganymed). A goat anti-huIgG-APC antibody from Dianova was used as a secondary reagent.

Cell cycle stages were determined by measuring the DNA content of the cells. This makes it possible to distinguish between cells in the G1, S, or G2 phase of the cell cycle. In the S phase, DNA duplication occurs, while in the G2 phase, cells grow and prepare for mitosis. Cell cycle analysis was performed using the CycleTest Plus DNA Reagent Kit from BD Biosciences according to the manufacturer's method. Flow cytometry data were acquired and analyzed on a BD FACS CantoII setup (BD Biosciences) using the FlowJo program (Tree Star).

In FIG. 35a and b, the bars show the percentage of cells in the G1, S, or G2 phase of the cell cycle, respectively. When KATO-III cells are cultivated in the medium, the cell cycle stops, mainly in the G1 phase. When cells are treated with 5-FU, they are blocked mainly in the S phase. When cells are treated with KATO-III with oxaliplatin, cells are enriched mainly in the G1 and G2 phases. As can be seen from FIG. 35c, cell cycle arrest in S phase or G2 phase results in stabilization or elevation of CLDN18.2 levels. Once the cells exit any phase of the cell cycle (Fig. 35b), CLDN18.2 expression on the surface of KATO-III cells is upregulated (Fig. 35d).

KATO-III cells were pretreated with irinotecan or docetaxel for 4 days and analyzed for CLDN18.2 expression and cell cycle arrest. Treatment of cells with irinotecan resulted in dose-dependent inhibition of cell growth and cell cycle arrest in the S/G2 phase (FIG. 36). Treatment of cells with docetaxel resulted in dose-dependent inhibition of cell growth and cell cycle arrest in the G2 phase (Fig. 36).

Effect of Chemotherapy on IMAB362-Induced Antibody-Dependent Cellular Cytotoxicity (ADCC)

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

Dose-response curves of Gem (1 ng/mL) or GemOx (1 ng/mL Gem + 10 ng/mL Ox) pretreated DANG cells (2 days) shifted upward and to the left compared to untreated target cells (Fig. 37A) . Treatment of cancer cells with Gem or GemOx resulted in increased levels of CLDN18.2 and greater susceptibility to IMAB362-mediated ADCC. After treatment with chemotherapeutic agents, DANG cells showed a decrease in EC ₅₀ values and an increase in maximum cell lysis in IMAB362-mediated ADCC (FIG. 37B).

Peripheral blood mononuclear cells (PBMCs), including NK cells, monocytes, mononuclear cells or other effector cells from healthy donors, were purified by Ficoll Hypaque density gradient centrifugation. The washed effector cells were plated in X-Vivo medium. In this case, KATO-III cells endogenously expressing CLDN18.2 and originating from the stomach were used as target cells. Target cells stably expressed luciferase, and Lucifer yellow is only oxidized by viable cells. Purified anti-CLDN18.2 antibody IMAB362 was added at various concentrations, and a foreign chimeric anti-huIgG1 antibody was used as isotype control antibody. Samples were analyzed for cytolysis by measuring luminescence from oxidation of Lucifer yellow, which is a measure of the number of viable cells remaining after IMAB362-induced cytotoxicity. KATO-III pretreated for 3 days with irinotecan (1000 ng/mL), docetaxel (5 ng/mL), or cisplatin (2000 ng/mL) were compared to untreated target cells cultured in medium and IMAB362- induced ADCC.

When pretreated for 3 days with irinotecan, docetaxel, or cisplatin, KATO-III cells showed a decrease in viable cells compared to target cells cultured in medium (Fig. 38a), and claudin 18.2 expression in cells pretreated with irinotecan, docetaxel, or cisplatin increased compared to cells cultured in the medium (Fig. 38b).

In addition, pretreatment of KATO-III cells with irinotecan, docetaxel, or cisplatin enhanced the ability of IMAB362 to induce ADCC (Fig. 38c, d).

Effect of chemotherapy on IMAB362-induced CDC

The effect of IMAB362 on CDC was examined by incubation with target cells in the presence of human serum as a source of complement.

Media cultured MiaPaCa-2-LVT cells exhibited EC ₅₀ values for IMAB362-specific lysis of 7665 ng/ml. Gem treatment resulted in a decrease in EC ₅₀ to 4677 ng/ml along with an increase in the maximum degree of lysis (Fig. 39).

The effect of chemotherapeutic agents on IMAB362-induced CDC was analyzed by pretreatment of gastric cancer cells with KATO-III with 10 ng/ml 5-FU and 500 ng/ml oxaliplatin (5-FU + OX) for 48 hours. Representative dose-response curves for IMAB362-induced CDC during chemotherapeutic pretreatment of KATO-III cells are shown in FIG. 40. Pretreatment of cancer cells for 48 hours enhanced the ability of IMAB362 to induce ADCC, which resulted in an increase in the maximum degree of lysis in pretreated cancer cells compared to untreated cells.

Example 8 Efficacy of IMAB362 in Combination with Chemotherapy in Mouse Tumor Models

Investigated the antitumor activity of IMAB362 in combination with Gem or GemOx in subcutaneous models of pancreatic carcinoma xenotransplantation, which were previously used to test the effectiveness of IMAB362 as a single agent.

IMAB362-treated nude mice with BxPC3-LVT or MiaPaCa-2-LVT tumors showed a significant reduction in tumor growth compared to control mice treated with control saline. Chemotherapy with gemcitabine up to 100 mg/kg without additional treatment with IMAB362 had no significant therapeutic effect on BxPC3-LVT or MiaPaCa-2-LVT xenografts. In contrast, combined treatment with 50-100mg/kg of gemcitabine plus IMAB362 resulted in a significant increase in tumor growth inhibition and prolongation of lifespan in mice with tumors compared to mice treated with chemotherapy alone (Fig. 41, Fig. 42, Fig. 43). These observations indicate the existence of synergistic therapeutic effects when gemcitabine is combined with IMAB362 immunotherapy.

When using high doses of gemcitabine 2x150 mg/kg per week, tumor growth was strongly inhibited in grafted MiaPaCa-2-LVT tumor xenografts regardless of IMAB362 treatment (Fig. 44A). However, mice treated with combination therapy of IMAB362 and gemcitabine exhibited significantly longer survival compared to mice treated with gemcitabine alone (FIG. 44B).

Example 9 Treatment with ZA/IL-2 leads to the expansion of a large number of Vγ9Vδ2 T cells

PBMCs were cultured for 14 days in RPMI medium with or without 300 U/ml IL-2 and 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.

Survival and growth of lymphocytes require the addition of IL-2 to cultures of PBMCs. They effectively expand in cultures when 300 U/ml IL-2 is added. FACS using Vγ9 and Vδ2 specific antibodies showed that the addition of ZA/IL-2 specifically induces the accumulation of Vγ9Vδ2 T cells. After 14 days, the CD3+ lymphocyte population may contain up to 80% Vγ9Vδ2 T cells. Some of the Vγ9Vδ2 T cells express CD16, and the enrichment of these cells in the population of CD3+ lymphocytes is 10-700 times, depending on the donor. The enrichment of CD16+ Vγ9+ Vδ2+ T cells in cultures is 10-600 times compared to cultures growing without ZA. We conclude that in vitro treatment of PBMCs with ZA/IL-2 results in upregulation of the ADCC-mediated FcγIII receptor CD16 in a significant proportion of γδ T cells.

Like NK cells, when ZA/IL-2 induced expansion, Vγ9Vδ2 T cells will be positive for the CD16 FcγRIII receptor, through which cell-bound antibody triggers ADCC. To determine whether Vγ9Vδ2 T cells would be able to induce strong ADCC in combination with IMAB362, a number of experiments were performed.

PBMCs obtained from 2 different donors (#1 and #2) were cultured in medium with or without 300 U/ml IL-2 and 1 μM ZA. After 14 days, cells were harvested and added at increasing concentrations (0.26 ng/ml - 200 μg/ml) of IMAB362 to NUGC-4 cells expressing CLDN18.2. Specific cell death was determined by the luciferase method. An ADCC assay was performed with 27 donors cultured in the presence of 300 U/ml IL-2 with or without ZA, with NUGC-4 cells serving as the target. For each donor, EC ₅₀ values were calculated from dose-response curves and the maximum extent of specific death at a dose of 200 μg/ml IMAB362 was estimated on dot plots.

Using PBMCs cultured for 14 days with ZA/IL-2, strong IMAB362-dependent ADCC activity was observed against CLDN18.2-positive NUGC-4 cells. When using ZA/IL-2-treated cultures of PBMCs, ADCC activity is dependent on the presence of Vγ9Vδ2 T cells. When cells are cultured without ZA, ADCC activity is reduced in most donors. In these cultures, residual ADCC activity depends on NK cells. When testing more than 20 donors, ADCC analysis shows that treatment of PBMCs with ZA/IL-2 improves EC ₅₀ values and maximum specific death rate compared to culturing PBMCs with IL-2 alone.

Example 10 Efficacy of IMAB362 in combination with gemcitabine in a mouse metastasis model

To analyze the effect of treatment with IMAB362 in combination with gemcitabine on Patu8988S lung metastases in vivo, 12 nude Hsd: Foxn1 ^nu nude mice per group received an intravenous injection of 2×10 ⁶ Patu8988S cells into the tail vein. 14 days after tumor cell injection, mice received 200 μg of IMAB362 or PBS as controls (i.v.-i.p.) twice a week plus a weekly dose of 100 mg/kg of gemcitabine ip for 4 weeks. Treatment with IMAB362 or PBS was continued until mice were sacrificed on day 70 after tumor cell injection. The load of the lungs with xenotransplanted tumors was analyzed by quantitative PCR on human DNA in lung preparations and by optical immunohistological staining with an antibody against human MHC-I (clone EPR1394Y). The results show that mice treated with IMAB362 plus gemcitabine significantly reduced the amount of human DNA in the lungs (Fig. 45A), and the surface area stained with anti-human MHC-I complex on lung sections was significantly less than in the lungs of mice. treated with foreign antibody plus gemcitabine (FIG. 45B). Both methods showed a reduction in lung xenograft loading of Patu8988s tumors in IMAB362 plus gemcitabine-treated mice, indicating that the combination with IMAB362 is significantly superior to gemcitabine alone.

--->

SEQUENCE LIST

<110> Ganymed Pharmaceuticals AG

<120> COMBINATION THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2

FOR TREATMENT OF CANCER

<130> 342-73 PCT

<150> PCT/EP2013/000505

<151> 2013-02-20

<160> 54

<170>PatentIn version 3.5

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Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

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Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

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Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

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Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

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Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

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Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

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Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

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Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

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Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

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Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

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Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

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Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

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Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu

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Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly

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Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg

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Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

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Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

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Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

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Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

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Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

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Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

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Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

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Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

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Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

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Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

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Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

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Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

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Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

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Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

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Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

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Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

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Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

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Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

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Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

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Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

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Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

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Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

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Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

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Ser Leu Ser Pro Gly Lys

325

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Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu

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Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn

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Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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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

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Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

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Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

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Gly Lys

465

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<223> Description of artificial sequence: chimeric monoclonal antibody

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Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

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Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

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Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

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Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

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Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

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Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

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Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

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Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

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Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

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Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

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Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

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Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

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Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

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Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

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Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

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Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

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Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

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Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

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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

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His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

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Pro Gly Lys

465

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<223> Description of artificial sequence: chimeric monoclonal antibody

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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

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Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

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Thr Asp Tyr Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu

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Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn

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Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

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Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

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Tyr Phe Cys Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly

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Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

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Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

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Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

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Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

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Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

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Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

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Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

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Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

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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

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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

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

Lys

465

<210> 17

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<212> PRT

<213> Artificial

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<223> Description of artificial sequence: chimeric monoclonal antibody

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

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Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

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Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

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Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

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Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

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Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

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Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

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Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

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Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

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Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

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Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

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Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

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Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

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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

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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

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His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

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Pro Gly Lys

465

<210> 18

<211> 466

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

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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

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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 sequence: chimeric monoclonal antibody

<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 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 sequence: chimeric monoclonal antibody

<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> 21

<211> 235

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 21

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> 22

<211> 234

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 22

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> 23

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 23

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> 24

<211> 240

<212> PRT

<213> Artificial

<220>

<223> Description of artificial sequence: chimeric monoclonal antibody

<400> 24

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 sequence: chimeric monoclonal antibody

<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 sequence: chimeric monoclonal antibody

<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 Glyn 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 sequence: chimeric monoclonal antibody

<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 sequence: chimeric monoclonal antibody

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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 sequence: Translation of PCR product

<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> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 51

agagagctct ggcttcaccg agtg 24

<210> 52

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 52

ccagaagtta gtcaccagca tgttgg 26

<210> 53

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 53

gggataattt cagctgacta aacag 25

<210> 54

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 54

ttccgtttag ttaggtgcag ttatc 25

<---

Claims (18)

1. A method of treating a precancerous lesion of the pancreas characterized by pancreatic cells expressing claudin 18 (CLDN18.2) splicing variant 2, comprising administering to a patient (i) an antibody capable of binding to CLDN18.2; and (ii) an agent that stabilizes or increases the expression of CLDN18.2, where (a) the antibody binds to CLDN18.2 and kills cells expressing CLDN18.2, wherein the antibody comprises an antibody heavy chain containing the amino acid sequence shown in SEQ ID NO: 32 and an antibody light chain containing the amino acid sequence shown in SEQ ID NO: 39, and (b) the agent is a nucleoside analog selected from the group consisting of gemcitabine and an ester or salt thereof. 2. The method of claim 1, wherein the expression of CLDN18.2 occurs on the cell surface of the cancer cells. 3. The method according to claim. 1 or 2, characterized in that it includes the introduction of an agent selected from the group consisting of platinum compounds, taxanes, their salts and combinations thereof. 4. The method of claim 1 or 2, which comprises administering an agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, albumin-bound paclitaxel, salts thereof, and combinations thereof. 5. The method of claim 1 or 2, which comprises administering a combination of gemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin, a combination of gemcitabine and carboplatin, or a combination of paclitaxel or albumin-bound paclitaxel and gemcitabine. 6. A method for treating a precancerous lesion of the pancreas characterized by pancreatic cells expressing CLDN18.2, comprising administering to a patient (i) an antibody capable of binding to CLDN18.2; And (ii) gemcitabine, wherein the antibody mediates the killing of cells expressing CLDN18.2, wherein the antibody comprises an antibody heavy chain comprising the amino acid sequence represented by SEQ ID NO: 32 and an antibody light chain comprising the amino acid sequence represented by SEQ ID NO: 39, and wherein the method comprises administering bisphosphonate to the patient. 7. The method of claim 6 wherein the bisphosphonate is a nitrogen-containing bisphosphonate (aminobisphosphonate). 8. The method of claim 6 wherein the bisphosphonate is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid and salts thereof. 9. The method of claim 6 wherein the bisphosphonate is administered in combination with interleukin-2. 10. The method according to any one of claims 1 to 9, wherein the method comprises administering an antibody capable of binding to CLDN18.2 at a dose of up to 1000 mg/m ² . 11. The method according to any one of claims 1 to 9, wherein the method comprises administering an antibody having the ability to bind to CLDN18.2 repeatedly at a dose of 300 to 600 mg/m ² .

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