Cancer diagnosis and treatment, including cancer stem cells

Abstract

This invention provides a method for diagnosing or treating cancerous diseases that contains cancer stem cells. [Solution] A method for measuring cancer stem cells is provided, comprising detecting cells expressing CLDN6. In particular, the present invention provides a method for measuring cancer stem cells, comprising detecting cells expressing CLDN6. Furthermore, the present invention provides a method for treating or preventing cancer, comprising inhibiting and / or eliminating cancer stem cells by administering an antibody having the ability to bind to CLDN6 to a cancer patient.

Description

[Technical Field]

[0001] Conventional cancer therapies have generally focused on selectively detecting and eradicating rapidly growing cancer cells (i.e., cells that form the tumor bulk) and generally attempting to toxicize cancer cells by disrupting cellular mechanisms involved in cell proliferation and DNA replication. Furthermore, standard oncology regimens are largely designed to administer the highest possible dose of radiation or chemotherapy agent, often referred to as the "maximum tolerated dose" (MTD), without causing excessive toxicity. [Background technology]

[0002] Chemotherapy protocols often include the combined administration of chemotherapeutic agents to enhance the therapeutic effect. Despite the wide variety of chemotherapeutic agents available, these therapies have many drawbacks. For example, chemotherapeutic agents can cause serious, and often dangerous, side effects due to nonspecific side effects on rapidly proliferating cells, whether normal or malignant.

[0003] Other types of cancer therapies include surgery to eradicate tumor cells in the patient, hormone therapy, immunotherapy, epigenetic therapy, anti-angiogenic therapy, targeted therapy, and radiation therapy. [Overview of the project] [Problems that the invention aims to solve]

[0004] However, all conventional approaches to cancer therapy have significant drawbacks for patients, including a lack of efficacy (particularly in terms of long-term outcomes) and toxicity. Therefore, new therapies are needed to treat cancer patients.

[0005] There is growing evidence that a subpopulation of cancer cells retaining stem-like characteristics exists within tumors. This subpopulation is called cancer stem cells (CSCs). Cancer stem cells have similar characteristics to normal stem cells and possess the ability to self-replicate and form all heterogeneous cell types in a tumor. A useful assay for analyzing the CSC-like characteristics of tumor cells is the colony formation assay. This assay allows for easy examination of the self-renewal capacity and tumorigenic potential of single tumor cells.

[0006] Cancer stem cells are thought to have the ability to initiate tumor formation, maintain tumor growth, and possibly lead to tumor dissemination to distant organ sites within the body. Cancer stem cells constitute a unique subpopulation of tumors that are more tumorigenic, grow relatively slowly or remain quiescent compared to the rest of the tumor cells (i.e., the tumor bulk), and are often relatively more chemotherapy-resistant than the tumor bulk. Because conventional cancer therapies target rapidly growing cells (i.e., cells that form the tumor bulk), these treatments are considered relatively ineffective in targeting and damaging cancer stem cells. Cancer stem cells can express other characteristics that make them relatively chemotherapy-resistant, such as multidrug resistance and anti-apoptotic pathways. The inability to properly target and eradicate cancer stem cells is a major reason why standard tumor treatment regimens fail to provide long-term benefits in many cancer patients. Therefore, cancer stem cells may be not only a major reason for cancer recurrence and drug ineffectiveness after treatment, but also a major reason for malignant cancer metastasis. Thus, one possibility for curing cancer is to eliminate cancer stem cells.

[0007] Claudins are endogenous membrane proteins located within the tight junctions between the epithelium and endothelium. Claudins possess four transmembrane segments and two extracellular loops, with their N-terminus and C-terminus expected to be localized within the cytoplasm. The CLDN family of transmembrane proteins plays a crucial role in maintaining tight junctions between the epithelium and endothelium, and is also thought to be involved in cytoskeleton maintenance and cellular signaling. CLDN6 is expressed in a range of different human cancer cells, but its expression in normal tissues is limited to the placenta.

[0008] In this specification, we present data demonstrating that CLDN6 expression is upregulated during the generation of pluripotent cells. Furthermore, CLDN6 is strongly associated with known markers of cancer stem cells, and CLDN6-positive tumor cells show enhanced colony formation. We also demonstrate that therapy using CLDN6-specific antibodies can overcome chemotherapy resistance in tumors such as ovarian cancer, and that the combination of chemotherapy and CLDN6 antibody therapy exhibits outstanding synergistic effects.

[0009] The findings presented herein indicate that CLDN6 is a novel marker for cancer stem cells and that targeting CLDN6 allows for the targeting of cancer stem cells for diagnostic and therapeutic purposes. [Means for solving the problem]

[0010] In one aspect, the present invention relates to a method for measuring cancer stem cells, which includes detecting cells expressing CLDN6.

[0011] In one embodiment, the presence of cells expressing CLDN6 indicates the presence of cancer stem cells, and / or the amount of cells expressing CLDN6 correlates with the amount of cancer stem cells. In one embodiment, cells expressing CLDN6 are detected in samples obtained from cancer patients before, during, and / or after treatment for cancer. In one embodiment, the method includes quantitative and / or qualitative measurement of cells expressing CLDN6. In one embodiment, the method includes comparing the amount of cells expressing CLDN6 to the amount of cells expressing CLDN6 in a reference sample or to a predetermined reference range. The reference sample may be a sample from a patient who has never been diagnosed with cancer. The predetermined reference range may be based on a population of patients who have never been diagnosed with cancer. In one embodiment, the method includes observing the amount of cancer stem cells in a cancer patient, where observing the amount of cancer stem cells in a cancer patient preferably includes comparing the amount of cancer stem cells in a sample obtained from the cancer patient to the amount of cancer stem cells in a sample obtained earlier from the cancer patient. In one embodiment, the sample obtained from a cancer patient is a sample taken from a cancer patient during or after the administration of cancer therapy.

[0012] In a further embodiment, the present invention relates to a method for observing the effect of cancer therapy in cancer patients, comprising (i) measuring the amount of cancer stem cells in a sample obtained from a cancer patient during or after administration of cancer therapy; and (ii) comparing the amount of cancer stem cells in a sample obtained from a cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient, wherein measuring the amount of cancer stem cells in a sample obtained from a cancer patient and / or measuring the amount of cancer stem cells in a sample obtained earlier from the cancer patient comprises measuring the amount of cells expressing CLDN6.

[0013] In one embodiment, the earlier samples obtained from cancer patients are samples taken from cancer patients before, during, or after the administration of cancer therapy.

[0014] In one embodiment of the method of all aspects of the present invention, stabilization or reduction of the amount of cancer stem cells indicates that the cancer therapy is effective. In one embodiment of the method of all aspects of the present invention, an increase in the amount of cancer stem cells indicates that the cancer therapy is ineffective. In one embodiment of the method of all aspects of the present invention, the cancer therapy is cancer therapy against cancer stem cells. In one embodiment of the method of all aspects of the present invention, the sample obtained from a cancer patient is a biological fluid or a tumor biopsy. In one embodiment of the method of all aspects of the present invention, the sample is subjected to one or more pretreatment steps. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 are detected or measured by detecting or measuring the amount of CLDN6 protein and / or CLDN6 mRNA. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 are detected or measured in quantity by an immunoassay, where the immunoassay is preferably selected from the group consisting of Western blotting, immunohistochemistry, radioimmunoassay, ELISA (solid-phase enzyme immunoassay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, immunofluorescence assay, protein A immunoassay, flow cytometry, and FACS analysis. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 are detected or measured in quantity by using an antibody having the ability to bind to CLDN6. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 are cancer cells and / or cells present in tumor sites that express CLDN6.

[0015] In a further embodiment, the present invention relates to a method for treating or preventing cancer, comprising inhibiting and / or eliminating cancer stem cells by administering an antibody having the ability to bind to CLDN6 to a cancer patient.

[0016] In one embodiment, cancer stem cells express CLDN6. In one embodiment, the method further comprises administering chemotherapy and / or radiotherapy. In one embodiment, inhibiting and / or eliminating cancer stem cells enhances the anticancer effect of chemotherapy and / or radiotherapy, where the enhancement of the anticancer effect of chemotherapy and / or radiotherapy preferably includes extending the survival time of cancer patients receiving chemotherapy and / or radiotherapy.

[0017] In a further embodiment, the present invention relates to a method for treating or preventing cancer, comprising administering to a cancer patient (i) an antibody having the ability to bind to CLDN6 and (ii) chemotherapy.

[0018] In one embodiment, the cancer comprises cancer stem cells expressing CLDN6. In one embodiment, administration of an antibody having the ability to bind to CLDN6 results in the inhibition or elimination of cancer stem cells expressing CLDN6. In one embodiment, administration of an antibody having the ability to bind to CLDN6 enhances the anticancer effect of chemotherapy, where the enhancement of the anticancer effect of chemotherapy preferably includes an extension of the survival time of cancer patients receiving chemotherapy.

[0019] In one embodiment of the methods of all aspects of the present invention, elimination of cancer stem cells results in a cure for cancer. In one embodiment of the methods of all aspects of the present invention, an antibody capable of binding to CLDN6 and chemotherapy are administered in synergistically effective amounts. In one embodiment of the methods of all aspects of the present invention, chemotherapy is administered at a dose lower than the maximum tolerated dose. In one embodiment of the methods of all aspects of the present invention, chemotherapy includes administering an active agent selected from the group consisting of taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, their prodrugs, their salts, and combinations thereof. In one embodiment of the methods of all aspects of the present invention, chemotherapy includes administering an active agent selected from the group consisting of paclitaxel, cisplatin, carboplatin, their prodrugs, their salts, and combinations thereof. In one embodiment of the methods of all aspects of the present invention, cancer stem cells are present in the tumor site of a cancer patient. In one embodiment of the methods of all aspects of the present invention, cancer is resistant to chemotherapy, particularly when administered as monotherapy. In one embodiment of the method of all aspects of the present invention, an antibody capable of binding to CLDN6 exerts an inhibitory and / or cytotoxic effect on cancer stem cells, where the antibody capable of binding to CLDN6 preferably imparts its inhibitory and / or cytotoxic effect to cancer stem cells by mediating one or more of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment of the method of all aspects of the present invention, the antibody capable of binding to CLDN6 is conjugated to a therapeutic component, which may be an antibody-drug conjugate as described herein. In one embodiment, the therapeutic component is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide. In one embodiment, the therapeutic component acts on slowly proliferating cells. In one embodiment of the method of all aspects of the present invention, the antibody capable of binding to CLDN6 binds to the first extracellular loop of CLDN6.In one embodiment of the method according to all aspects of the present invention, the antibody having the ability to bind to CLDN6 comprises a heavy chain variable region (VH) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 4.

[0020] In a further embodiment, the present invention relates to a method for treating or preventing cancer, comprising administering to a cancer patient an antibody-drug conjugate containing an antibody having the ability to bind to CLDN6, which is covalently bound to at least one toxic drug component by a linker.

[0021] In one embodiment, the toxic drug component is cell membrane permeable. In one embodiment, at least one of the toxic drug components acts on slowly proliferating cells. In one embodiment, the toxic drug component is a meitansinoid or auristatin. In one embodiment, the meitansinoid is selected from the group consisting of DM1 and DM4. In one embodiment, the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). In one embodiment, the linker is a cleavable linker, preferably a cathepsin-cleavable linker. In one embodiment, the antibody is linked to the linker via the antibody's cysteinethiol.

[0022] In one embodiment, the cancer comprises cancer stem cells expressing CLDN6. In one embodiment, administration of an antibody drug conjugate results in inhibition or elimination of the CLDN6-expressing cancer stem cells. In one embodiment, elimination of cancer stem cells results in a cure for the cancer. In one embodiment, the cancer stem cells are located in the tumor site of the cancer patient. In one embodiment, the antibody drug conjugate exerts an inhibitory and / or cytotoxic effect on the cancer stem cells, where the antibody drug conjugate preferably imparts its inhibitory and / or cytotoxic effect to the cancer stem cells by inducing apoptosis and / or inhibiting proliferation.

[0023] In one embodiment, the method further comprises administering chemotherapy and / or radiotherapy. In one embodiment, administering an antibody drug conjugate enhances the anticancer effect of chemotherapy and / or radiotherapy, where the enhancement of the anticancer effect of chemotherapy and / or radiotherapy preferably includes extending the survival time of cancer patients receiving chemotherapy and / or radiotherapy.

[0024] In one embodiment, an antibody drug conjugate and chemotherapy are administered in synergistically effective amounts. In one embodiment, chemotherapy is administered at a dose lower than the maximum tolerated dose. In one embodiment, chemotherapy includes administering an active agent selected from the group consisting of taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, their prodrugs, their salts, and combinations thereof. In one embodiment, chemotherapy includes administering an active agent selected from the group consisting of paclitaxel, cisplatin, carboplatin, their prodrugs, their salts, and combinations thereof. In one embodiment, cancer is resistant to chemotherapy, particularly when administered as monotherapy.

[0025] In one embodiment, an antibody capable of binding to CLDN6 has affinity and / or specificity to CLDN6 that is suitable for enabling endocytosis of the antibody and / or antibody-drug conjugate, particularly when present in an antibody-drug conjugate. In one embodiment, an antibody capable of binding to CLDN6 in an antibody-drug conjugate binds to the first extracellular loop of CLDN6. In one embodiment, an antibody capable of binding to CLDN6 in an antibody-drug conjugate contains a heavy chain variable region (VH) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 4.

[0026] In one embodiment of the method of all aspects of the present invention, CLDN6 has an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment of the method of all aspects of the present invention, cancer includes primary cancer, advanced cancer, metastatic cancer, recurrent cancer, or a combination thereof.

[0027] In a further embodiment, the present invention relates to a method for treating or preventing cancer, comprising (i) measuring cancer stem cells in a cancer patient by the method of the present invention and (ii) administering a cancer therapy targeting cancer stem cells to the cancer patient. In one embodiment, the cancer therapy targeting cancer stem cells comprises carrying out the method for treating or preventing cancer of the present invention.

[0028] In a further embodiment, the present invention relates to a method for preventing cancer chemotherapy resistance, cancer recurrence, or cancer metastasis, particularly during or after cancer treatment, which includes treating cancer by the method of the present invention.

[0029] In further embodiments, the present invention provides a medical preparation for treating or preventing cancer, comprising (i) an antibody capable of binding to CLDN6 and (ii) a chemotherapeutic agent. The antibody capable of binding to CLDN6 and the chemotherapeutic agent may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be in the form of a kit comprising a first container containing the antibody capable of binding to CLDN6 and a second container containing the chemotherapeutic agent. The medical preparation may further include printed instructions relating to the use of the preparation for treating or preventing cancer, particularly the use of the preparation in the method of the present invention. Various embodiments of the medical preparation, and in particular the antibody capable of binding to CLDN6 and the chemotherapeutic agent, are described herein.

[0030] In certain embodiments, the present invention provides a medical preparation comprising (i) an antibody capable of binding to CLDN6 and (ii) paclitaxel. The antibody capable of binding to CLDN6 and paclitaxel may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be intended to treat or prevent cancer, such as ovarian cancer. The medical preparation may be in the form of a kit comprising a first container containing the antibody capable of binding to CLDN6 and a second container containing paclitaxel. The medical preparation may further include printed instructions relating to the use of the preparation for the treatment or prevention of cancer, such as ovarian cancer, and in particular to the use of the preparation in the method of the present invention. Various embodiments of the medical preparation and, in particular, the antibody capable of binding to CLDN6, are described herein.

[0031] In a further embodiment, the present invention provides an antibody-drug conjugate containing an antibody having the ability to bind to CLDN6, which is covalently bound to at least one toxic drug component by a linker.

[0032] In one embodiment, the toxic drug component is cell membrane permeable. In one embodiment, at least one of the toxic drug components acts on slowly proliferating cells. In one embodiment, the toxic drug component is a meitansinoid or auristatin. In one embodiment, the meitansinoid is selected from the group consisting of DM1 and DM4. In one embodiment, the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). In one embodiment, the linker is a cleavable linker, preferably a cathepsin-cleavable linker. In one embodiment, the antibody is linked to the linker via the antibody's cysteinethiol.

[0033] In one embodiment, an antibody capable of binding to CLDN6 has affinity and / or specificity to CLDN6 that is suitable for enabling endocytosis of the antibody and / or antibody-drug conjugate, particularly when present in an antibody-drug conjugate. In one embodiment, an antibody capable of binding to CLDN6 in an antibody-drug conjugate binds to the first extracellular loop of CLDN6. In one embodiment, an antibody capable of binding to CLDN6 in an antibody-drug conjugate contains a heavy chain variable region (VH) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) containing an amino acid sequence or fragment thereof represented by SEQ ID NO: 4.

[0034] In a further embodiment, the present invention provides a pharmaceutical formulation containing the antibody drug conjugate of the present invention and a pharmaceutically acceptable diluent, carrier, or excipient.

[0035] In further embodiments, the present invention provides medical preparations containing the antibody-drug conjugate and chemotherapeutic agent of the present invention. Preferably, the medical preparations are intended to treat or prevent cancer. The antibody-drug conjugate and chemotherapeutic agent may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be in the form of a kit comprising a first container containing the antibody-drug conjugate and a second container containing the chemotherapeutic agent. The medical preparation may further include printed instructions relating to the use of the preparation for the treatment or prevention of cancer, in particular to the use of the preparation in the method of the present invention. Various embodiments of the medical preparations, and in particular the antibody-drug conjugate and chemotherapeutic agent, are described herein.

[0036] In certain embodiments, the present invention provides a medical preparation containing the antibody-drug conjugate and paclitaxel of the present invention. The antibody-drug conjugate and paclitaxel may be present in the medical preparation as a mixture or separately from each other. The medical preparation may be intended to treat or prevent cancer, such as ovarian cancer. The medical preparation may be in the form of a kit comprising a first container containing the antibody-drug conjugate and a second container containing paclitaxel. The medical preparation may further include printed instructions relating to the use of the preparation for the treatment or prevention of cancer, such as ovarian cancer, and in particular to the use of the preparation in the method of the present invention. Various embodiments of the medical preparation and, in particular, the antibody-drug conjugate, are described herein.

[0037] The present invention also provides active substances and compositions as described herein, such as antibody-drug conjugates, antibodies having the ability to bind to CLDN6, and / or chemotherapeutic agents for use in the methods described herein. For example, the present invention also provides antibody-drug conjugates or antibodies having the ability to bind to CLDN6 for administration together with chemotherapeutic agents such as paclitaxel.

[0038] In one embodiment, the antibody having the ability to bind to CLDN6 is a monoclonal, chimeric, or humanized antibody, or an antibody fragment. In one embodiment, the antibody mediates cell death when it binds to cell CLDN6, particularly CLDN6 expressed on the cell surface by a cell, where the cell is preferably a cancer stem cell, such as a cancer stem cell of cancer as described herein.

[0039] According to the present invention, cancers preferably include ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; lung cancer, particularly squamous cell carcinoma and adenocarcinoma of the lung, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC); large cell carcinoma (LCC); gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, particularly basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, particularly malignant pleomorphic adenoma; sarcoma, particularly synovial sarcoma and carcinosarcoma; bile duct cancer; bladder cancer, particularly metastatic cancer. The group is selected from epithelial carcinoma and papillary carcinoma, renal carcinoma, particularly renal cell carcinoma including clear cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestinal cancer including ileal cancer, particularly small intestinal adenocarcinoma and ileal adenocarcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and testicular embryonic cancer, germ cell tumors such as uterine cancer, teratomas or embryonic cancers, particularly germ cell tumors of the testes and ovaries, and their metastatic forms.

[0040] According to the present invention, cancer cells and / or cancer stem cells expressing CLDN6 are preferably cancer cells as described herein.

[0041] In one embodiment, the cancer described herein is CLDN6-positive. In one embodiment, the cancer cells of the cancer described herein are CLDN6-positive. In one embodiment, the cancer cells of the cancer described herein express CLDN6 on their cell surface.

[0042] In one embodiment, the cancer described herein includes primary cancer, advanced cancer, metastatic cancer, recurrent cancer, or a combination thereof, for example, a combination of primary cancer and metastatic cancer. In one embodiment, the cancer is partially or completely resistant to chemotherapy such as paclitaxel monotherapy. In one embodiment, the cancer is ovarian cancer, in particular ovarian cancer that is partially or completely resistant to chemotherapy such as paclitaxel monotherapy.

[0043] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0044] [Figure 1]CLDN6 mRNA is expressed in human iPS cells. Human foreskin fibroblasts (HFFs) were transfected with Lipofectamine RNAiMAX (Life Technologies) either without RNA (RNA-free control) or with a reprogrammed cocktail (unmodified OSKMNL + EBK + miR mix), and cells were harvested at days 5, 12, and 19 post-treatment. RNA was extracted, transcribed to cDNA, and then analyzed by quantitative real-time RT-PCR using the ABI PRISM 7300 sequence detection system and software (Applied Biosystems and QuantiTect SYBR green Kit (Qiagen)). The polyploid induction of CLDN6 expression in cells treated with the reprogrammed cocktail (black bars) compared to HFF cells (gray bars) from day 1 post-treatment is shown. CLDN6 mRNA expression was standardized to the mRNA expression of the housekeeping gene HPRT1. OSKMNL = transcription factors OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28; EBK = IFN escape proteins E3, K3, and B18R; miR mix = miRNA-302a / b / c / d, and 367. [Figure 2] CLDN6 is expressed on the surface of human iPS cells. HFF cells were transfected with RNA-free (RNA-free control) or a reprogrammed cocktail (unmodified OSKMNL + EBK + miR mix), and cells were harvested on day 5 (A), day 12 (B), and day 19 (C) post-treatment. Cells were stained with 1 μg / ml CLDN6-specific IMAB027-AF647 and SSEA-4-V450 antibodies (2.5 μl per test, purchased from BD) at 4°C for 30 minutes, and surface expression was analyzed by flow cytometry. Experiments were performed in duplicate, and representative dot plots are shown. OSKMNL = transcription factors OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28; EBK = IFN escape proteins E3, K3, and B18R; miR mix = miRNA-302a / b / c / d and 367. [Figure 3]CLDN6 surface expression in ovarian cancer cell lines. To analyze CLDN6 expression, 1E6 cells were stained with 1 μg / ml IMAB027-AF647 at 4°C for 30 minutes, and surface expression was analyzed by flow cytometry. (A) shows COV318 cells. The experiment was performed in three replicates, and one representative dot plot is presented. (B) shows PA-1 cells stably transfected with the control vector PA-1 76) or vectors expressing shRNA for CLDN6 (clones PA-1 50 and PA-1 54). The experiment was performed in three replicates, and one representative dot plot is presented. shRNA = small hairpin RNA. [Figure 4] CLDN6 is important for colony formation in ovarian cancer cells. To analyze colony formation behavior, COV318, PA-1 50, and PA-1 54 cells were stained with 1 μg / ml IMAB027-AF647 at 4°C for 30 minutes, and then 700 (COV318) or 500 (PA-1 50 / 54) CLDN6-positive or CLDN6-negative cells were sorted into 6-well plates. The cells were allowed to colonize for 14 days, and then stained with 0.5% crystal violet for 20 minutes. (A) Representative images of each cell line are shown. (B) Colony quantification was performed by manual counting. The mean and standard deviation of three independent experiments are shown. [Figure 5A] (Figure 5) CLDN6 is co-expressed with CSC markers CD24, CD90, and CD44 in the ovarian cancer cell line COV318. 1E6 COV318 cells were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triple replication. (Figure 5A) (A) shows representative dot plots of co-localization of various established CSC markers and CLDN6. [Figure 5B] In (B), the percentage of colocalization between CD44, CD24, CD90, and CLDN6-positive cells was calculated using various gating methods shown on the x-axis of the diagram. The mean and standard deviation of the three experiments are shown. [Figure 6A](Figure 6) Enrichment of CLDN6-expressing cells leads to accumulation of established CSC markers. COV318 cells were stained with 0.5 μg / ml IMAB027 and APC-conjugated goat anti-human IgG secondary antibody (1:300), and then CLDN6-positive and CLDN6-negative fractions were isolated by FACS sorting. Cells from both fractions were grown for 10 days. 1E6 cells from each fraction were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1. The experiment was performed in triple replication. (Figure 6A) (A) shows representative dot plots of the expression levels of various CSC markers in the CLDN6-positive and CLDN6-negative fractions, as well as their co-localization with CLDN6. [Figure 6B] In (B), the percentage of CSC marker expression levels is shown as a diagram, and the enrichment coefficients (multiple expression levels) for the related markers CD44, CD90, and CD24 were calculated by comparing the percentage of positive cells in the CLDN6-positive and CLDN6-negative fractions. [Figure 7A] (Figure 7) Cell lines with high CLDN6 expression show enrichment of CSC markers compared to cells with low CLDN6 expression. 1E6 cells of CLDN6-highly expressing ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triple replication, and representative dot plots are shown. [Figure 7B] 1E6 cells of CLDN6-highly expressing ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triple replication, and representative dot plots are shown. [Figure 7C]1E6 cells of CLDN6-highly expressing ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triple replication, and representative dot plots are shown. [Figure 7D] 1E6 cells of CLDN6-highly expressing ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) were stained with antibodies against various surface markers at 4°C for 30 minutes according to the FACS panel shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triple replication, and representative dot plots are shown. [Figure 8] Antitumor activity of IMAB027 in combination with paclitaxel in an early xenograft tumor model. Subcutaneous human ES-2 xenograft tumors ectopically expressing human CLDN6 were treated with 15 mg / kg paclitaxel via intravenous injection on days 3, 10, and 17 post-transplant. On day 4, antibody maintenance therapy was initiated with 35 mg / kg IMAB027 injections three times a week (alternating IV / IP / IP). (A) Mean tumor growth dynamics (±SEM) after treatment with IMAB027 (white squares), paclitaxel (gray circles), IMAB027 in combination with paclitaxel (black squares), or vehicle control (white circles). Arrows indicate the time of therapy initiation. (B) Survival curve of treated mice. Group size: n=12. [Figure 9]Antitumor effects of IMAB027 in combination with cisplatin in an advanced xenograft tumor model. Subcutaneous human NEC14 xenograft tumors were grown to an average size of approximately 100 mm3 before the start of treatment. Mice were treated with 1 mg / kg cisplatin by intravenous injection daily from day 6 to day 10 post-transplant, and with 35 mg / kg IMAB027 injections three times a week from day 6 as maintenance therapy (alternating IV / IP / IP). (A) Mean tumor growth dynamics (±SEM) after treatment with IMAB027 (black circles), cisplatin (white squares), IMAB027 in combination with cisplatin (black squares), or vehicle control (white circles). Arrows indicate the time of therapy initiation. (B) Individual tumor size (mean ± standard deviation) in mice at day 24 post-transplant. (C) Survival curve of treated mice. Group size: n=19. P-values: *, p<0.05; **, p<0.01 and ***, p<0.001. [Figure 10] Antitumor effects of IMAB027 in combination with carboplatin in advanced xenograft tumor models. Advanced human NEC14 xenograft tumors were treated with IMAB027 alone or in combination with a cell proliferation inhibitor, as described in Figure 9. Instead of cisplatin, mice were treated with 30 mg / kg carboplatin by bolus iP injection on days 6, 13, and 20. (A) Mean tumor growth dynamics (±SEM) after treatment with IMAB027 (black circles), carboplatin (white squares), IMAB027 in combination with carboplatin (black squares), or vehicle control (white circles). Arrows indicate the time of therapy initiation. (B) Individual tumor size (mean ± standard deviation) in mice at 24 days post-transplant. (C) Survival curve of treated mice. Group size: n=19. P-values: *, p<0.05; **, p<0.01 and ***, p<0.001. [Figure 11]CLDN6 is important for the sphere formation behavior of ovarian cancer cells. To analyze the effect of CLDN6 on sphere formation, CLDN6-positive and CLDN6-negative COV318 cells were isolated by fluorescence-activated cell sorting after staining with 0.5 μg / ml IMAB027. CLDN6-positive and CLDN6-negative COV318 cells were grown in ultra-low adhesion plates under sphere formation conditions (serum-free DMEM / F12 medium containing 0.4% bovine serum albumin, 20 ng / ml basic fibroblast growth factor, 10 ng / ml epidermal growth factor, and 5 μg / ml insulin). (A) Representative images of first-generation spheres of CLDN6-positive (CLDN6+) and CLDN6-negative (CLDN6-) COV318 cells at 3, 8, and 19 days post-sorting. (B) Representative images of second-generation spheres obtained from single cells of CLDN6+ first-generation spheres from (A) at 22 days post-sorting. [Figure 12] Enrichment of CLDN6-positive cells after treatment with a platinum derivative. COV318 cells were treated with 500 ng / ml cisplatin or 2,000 ng / ml carboplatin for 4 days. After treatment, cells were further grown for 3 days (white bars) and 6 days (black bars), respectively, in the absence of cell proliferation inhibitors. CLDN6 expression was analyzed by flow cytometry using the CLDN6-specific antibody IMAB027 and an isotype control antibody. Expression in treated COV318 cells is shown compared to untreated cells. For evaluation, isotype control values ​​were subtracted from CLDN6 staining. [Figure 13]Enrichment of CLDN6-positive cells after intraperitoneal transplantation. COV318 cells were injected intraperitoneally into athymic nude mice. Mice that developed ascites were euthanized, and both ascites and solid tumors were collected for further characterization. Isolated cells were analyzed for CLDN6 expression immediately after preparation and after being maintained for several passages during culture. (A) Flow cytometry analysis of CLDN6 expression in parental COV318 cells using CLDN6-specific antibody IMAB027 and isotype control antibody. (B) CLDN6 expression in cells derived from ascites and solid tumors from the ovary, liver, stomach, pancreas, and diaphragm at various time points after isolation (*: ascites at day 5 and day 35; **: solid tumors at day 12 and day 29). Fluorescence intensity is shown on the x-axis. The number of events shown on the y-axis is expressed as a percentage of the maximum number of events. [Figure 14] CLDN6 correlates with ovarian cancer stem cell markers in primary tumor samples. Forty-two ovarian cancer samples were analyzed for mRNA expression levels of CLDN6 and various described ovarian cancer stem cell markers by qRT-PCR using the Fluidigm detection system and software. Spearman correlation analysis was performed to analyze the correlation of CLDN6 with cancer stem cell-specific markers. (A) shows a scatter plot of significant correlations (P-value ≤ 0.05). (B) shows a summary of all correlations. [Figure 15A](Figure 15) IMAB027-mediated ADCC after treatment with carboplatin and paclitaxel. The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 copies) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15B] The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15C]The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15D] The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15E]The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15F] The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15G]The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15H] The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15I]The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 15J] The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Therefore, cells were treated for 4 days with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. After treatment, cells were grown for an additional 3 days (A-D) and 10 days (E-J), respectively, in the absence of cell growth inhibitors. Control cells were cultured without cell growth inhibitors. (A, C, E, G, I) ADCC experiments were performed using PBMCs from healthy donors at an effector (PBMC) to target cell ratio of approximately 40:1 with IMAB027 (black line) or isotype control antibody (gray line). Data points (n=4 replicas) are expressed as mean ± SD. (B, D, F, H, J) CLDN6 expression was analyzed by flow cytometry using IMAB027. The black dotted line represents CLDN6 expression in untreated cells, and the gray solid histogram represents CLDN6 expression after treatment. [Figure 16]Antitumor activity of IMAB027 in combination with PEB treatment in a highly advanced xenograft tumor model. Subcutaneous human NEC14 xenograft tumors were allowed to grow to a highly advanced stage in nude mice. Tumor treatment with PEB (cisplatin, etoposide, and bleomycin) and IMAB027 was initiated on day 13. Mice receiving the PEB regimen were treated with 1 mg / kg cisplatin and 5 mg / kg etoposide on days 13, 14, 15, 16, and 17, and with 10 mg / kg bleomycin by IV injection on days 13, 17, and 21. The antibody IMAB027 was administered three times a week from day 13 to day 101 post-transplant by alternating IV / IP / IP injections at a dose of 35 mg / kg. The vehicle control group was instead given 0.9% NaCl solution and drug substance buffer. Mice were observed for a total of 220 days. (A), (B) Mean tumor growth dynamics (±SEM) of untreated mice and mice treated with IMAB027, PEB, or PEB in combination with IMAB027. Arrows indicate the time of therapy initiation (Dunn's multiple comparison test: ***, p<0.001). (C) Survival curves of untreated mice and mice treated with IMAB027, PEB, or PEB in combination with IMAB027 (Mantel-Cox test: *, p<0.05; **, p<0.01). Group size: n=14. [Figure 17] Relative binding affinity and cytotoxicity of IMAB027, IMAB027-DM1, and IMAB027-vcMMAE. (A) Binding of IMAB027, IMAB027-DM1, and IMAB027-vcMMAE was measured by flow cytometry analysis in OV90 cells endogenously expressing CLDN6. (B) Dose-response curves of IMAB027-DM1 and IMAB027-vcMMAE-mediated reduction in OV90 cell viability. Tumor cells were incubated with IMAB027-DM1 or IMAB027-vcMMAE for 72 hours. Reduction in cell viability was measured using an XTT-based viability assay. Data points (n=3 replicas) are expressed as mean ± SD. MFI: Mean fluorescence intensity. [Figure 18]Antitumor effects of IMAB027-DM1 conjugate on advanced xenograft tumors. Nude mice carrying established subcutaneous human OV90 xenograft tumors were treated with a single intravenous injection of 1.78, 5.33, or 16 mg / kg of IMAB027-DM1 or a vehicle control at 10 days post-transplant. Subcutaneous tumor size was measured twice weekly (mean ± SEM). Group size: n=5, *: p<0.05; **: p<0.01. [Figure 19A] (Figure 19) Dose range setting of IMAB027-DM1 and IMAB027-vcMMAE conjugates for advanced OV90 xenograft tumors. Nude mice with established subcutaneous human OV90 xenograft tumors were treated with a single intravenous injection of IMAB027-DM1, IMAB027-vcMMAE, and vehicle or repeated injections of IMAB027 on day 10 post-transplant. (Figure 19A) Tumor growth in mice treated with 1.33, 2.67, or 5.33 mg / kg IMAB027-DM1 iv (top) or 4, 8, or 16 mg / kg IMAB027-vcMMAE iv (bottom) compared to vehicle control and IMAB027 (35 mg / kg, weekly iv / ip / ip). Subcutaneous tumor size was measured twice weekly (mean ± SEM). [Figure 19B] Kaplan-Meier survival curves of mice treated with vehicle or 4, 8, or 16 mg / kg IMAB027-vcMMAE. Mice were euthanized when the tumor reached a volume of 1400 mm3 or when the tumor became ulcerative. Group size: n=10, *: p<0.05; **: p<0.01, ***: p<0.001. [Figure 20]Dose range setting for IMAB027-vcMMAE conjugate in advanced PA-1 xenograft tumors. Nude mice with established subcutaneous human PA-1 xenograft tumors were treated at 15 days post-transplant with a single intravenous injection of IMAB027-vcMMAE, vehicle control, or repeated injections of IMAB027. (A) Mean tumor growth (mean ± SEM) and (B) Kaplan-Meier survival curves for mice treated with vehicle control, IMAB027 (35 mg / kg, weekly iv / ip / ip), or 4, 8, or 16 mg / kg IMAB027-vcMMAE. Mice were euthanized when the tumor reached a volume of 1400 mm3 or when the tumor became ulcerative. Group size: n=8, *: p<0.05; **: p<0.01. (C) Representative immunohistochemical staining for CLDN6 in PA-1 xenograft tumor sections at various post-transplant time points. [Figure 21] Antitumor activity of IMAB027-vcMMAE against advanced MKN74 xenograft tumors. Nude mice with established subcutaneous human MKN74 xenograft tumors were treated with 16 mg / kg IMAB027-vcMMAE or vehicle control intravenously on post-transplant day 7. (A) Mean tumor growth (mean ± SEM) and (B) Kaplan-Meier survival curves for mice treated with vehicle control or IMAB027-vcMMAE. Mice were euthanized when the tumor reached a volume of 1400 mm3 or when the tumor became ulcerative. Group size: n=10. (C) Flow cytometry analysis of CLDN6 expression in MKN74 tumor cells before transplantation and representative immunohistochemical staining of untreated MKN74 xenograft tumors on post-transplant day 31. **: p<0.01, ***: p<0.001. [Figure 22]Antitumor effects of IMAB027-DM1 and IMAB027-vcMMAE on advanced intraperitoneal metastatic human ovarian tumors. Nude mice were intraperitoneally transplanted with PA-1(Luc), a human ovarian cancer cell line ectopically expressing luciferase. After the formation of intraperitoneal metastatic xenograft tumors, the animals were treated by ip injection at 14 days post-transplantation with 16 mg / kg IMAB027-DM1, IMAB027-vcMMAE, or a vehicle control. After luciferin administration, metastatic growth was measured by luminescence activity using the IVIS Lumina Imaging System. (A) Quantification of metastatic volume in mice treated with IMAB027-DM1, IMAB027-vcMMAE, or vehicle. (B) In vivo whole-body luminescence images of nude mice at 28 days post-transplantation. Group size: n=8 (vehicle) or n=9 (IMAB027-DM1, IMAB027-vcMMAE), **: p<0.01, ****: p<0.0001. [Figure 23A] (Figure 23) Endocytosis of CLDN6-conjugated antibodies by human cancer cells. Endocytosis of CLDN6-conjugated IMAB027, chimAB5F2D2, or isotype control antibodies was measured using a cytotoxicity-based assay dependent on the co-internalization of the target-conjugated antibody with saporin-conjugated anti-human IgG Fab fragment (FabZap). PA-1, OV90, or NEC14 human cancer cells were incubated with IMAB027, chimAB5F2D2, or isotype control antibody and anti-human FabZap for 72 hours. (Figure 23A) Dose-response curves of IMAB027 / FabZap and chimAB5F2D2 / FabZap-mediated reduction in PA-1, OV90, and NEC14 cell viability, respectively. Data points (n=3 replicas) are expressed as mean ± SD. [Figure 23B] Comparison of IMAB027 standardized EC50 (rel EC50) and rel maximum values ​​for flow cytometry binding and endocytosis. [Modes for carrying out the invention]

[0045] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, and that the scope of the present invention is limited only by the accompanying claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0046] The elements of the present invention are described below. These elements are listed along with specific embodiments, but it should be understood that they may be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the expressly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the expressly described embodiments with many of the disclosed elements and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by this description unless specifically indicated in the context.

[0047] Preferably, the terms used herein are defined as those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0048] The implementation of this invention shall, unless otherwise specified, be based on the literature in the art (e.g., Molecular Cloning: A Laboratory Manual, 2 ndConventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology are used, as described in Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0049] Throughout this specification and the following claims, unless specifically required by context, the word “includes” and variations such as “includes” are understood to mean the inclusion of the member, integer or process or group of members, integers or processes described, but not the exclusion of any other member, integer or process or group of members, integers or processes; however, in some embodiments, such other members, integers or processes or groups of members, integers or processes may be excluded, i.e., the subject matter lies in the inclusion of the member, integer or process or group of members, integers or processes described. The terms “one” and “it” and similar references used in connection with describing the invention (particularly in connection with the claims) should be interpreted as including both singular and plural unless specifically indicated herein or unless it is clearly inconsistent with the context. Enumerations of ranges of values ​​in this specification are intended to be a simplified way of simply referring individually to each separate value belonging to that range. Unless specifically indicated herein, each individual value is incorporated herein as if it were individually enumerated. All methods described herein may be carried out in any suitable order, unless otherwise specifically indicated herein or if it is clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., "etc.") is intended solely to better illustrate the invention and not to limit the invention or the claims. No language herein should be construed as indicating that any non-claimed element is essential for carrying out the invention.

[0050] Throughout this specification, several sources are referenced. Each of the sources referenced herein (including all patents, patent applications, academic publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosures for the sake of prior art.

[0051] Claudins are a family of proteins that are the most important components of tight junctions, establishing a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that cross the membrane four times, with both their N-terminus and C-terminus located in the cytoplasm. The first extracellular loop, designated EC1 or ECL1, consists of an average of 53 amino acids, while the second extracellular loop, designated EC2 or ECL2, consists of approximately 24 amino acids. Cell surface proteins of the claudin family, such as CLDN6, are expressed in tumors of various origins and are particularly suitable as target structures for antibody-mediated cancer immunotherapy due to their selective expression (they are not expressed in normal tissues associated with toxicity) and localization to the plasma membrane.

[0052] CLDN6 has been identified as being specifically expressed in tumor tissue, with the placenta being the only normal tissue that expresses CLDN6, where low levels of CLDN6 are detected at the RNA level. CLDN6 has been shown to be expressed in, for example, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cell carcinoma, and bladder cancer.

[0053] In various embodiments of the present invention, cancers associated with CLDN6 expression include ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; lung cancer, particularly squamous cell carcinoma and adenocarcinoma, including small cell lung cancer (SCLS) and non-small cell lung cancer (NSCLC); gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, particularly basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, particularly malignant pleomorphic adenoma; sarcoma, particularly synovial sarcoma and carcinosarcoma; bile duct cancer; and bladder cancer. This includes cancers, particularly transitional cell carcinoma and papillary carcinoma; renal cancers, particularly renal cell carcinoma including clear cell carcinoma and papillary renal cell carcinoma; colon cancers; small intestinal cancers, particularly small intestinal adenocarcinoma and ileal adenocarcinoma; embryonic testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancers, particularly testicular seminoma, testicular teratoma and embryonic testicular cancer; germ cell tumors, particularly testicular germ cell tumors, such as uterine cancer, teratomas or embryonic cancers; and their metastatic forms. In one embodiment, cancers associated with CLDN6 expression are selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, ovarian cancer is carcinoma or adenocarcinoma. Preferably, lung cancer is carcinoma or adenocarcinoma, and preferably bronchiolar cancer such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0054] As used herein, the term "CLDN" means claudin and includes CLDN6. Preferably, the claudin is a human claudin.

[0055] The term "CLDN6" preferably refers to human CLDN6, particularly a protein comprising the amino acid sequence of Sequence ID No. 1 or Sequence ID No. 2 in the sequence listing, or a variant thereof. The first extracellular loop of CLDN6 preferably contains amino acids 28-80, more preferably amino acids 28-76, of the amino acid sequence shown in Sequence ID No. 1 or Sequence ID No. 2. The second extracellular loop of CLDN6 preferably contains amino acids 138-160, more preferably amino acids 141-159, more preferably amino acids 145-157, of the amino acid sequence shown in Sequence ID No. 1 or Sequence ID No. 2. The first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0056] The term “mutant” in this invention refers, in particular, to mutants, splice mutants, conformational mutants, isoforms, allelic mutants, species mutants, and species homologs, especially those occurring in nature. Allelic mutants concern changes in the normal sequence of a gene, but their significance is often unclear. Complete gene sequencing often identifies numerous allelic mutants for a given gene. A species homolog is a nucleic acid sequence or amino acid sequence that originates from a different species than that of a given nucleic acid sequence or amino acid sequence. The term “mutant” encompasses any post-translational modification mutants and conformational mutants.

[0057] According to the present invention, the term "claudin-positive cancer" or similar terms means cancer comprising cancer cells that express claudin, preferably cancer cells that express claudin on the surface of said cancer cells. CLDN6 is expressed on the surface of cells if CLDN6 is located on the surface of the cells and is accessible by binding by a CLDN6-specific antibody added to the cells.

[0058] The term "cell surface" is used according to its usual meaning in this field and therefore includes the area outside the cell that is accessible for binding by proteins and other molecules. For example, transmembrane proteins that have one or more extracellular components are considered to be expressed on the cell surface.

[0059] In relation to the present invention, the term “extracellular component” refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is accessible from the outside of the cell by an antigen-binding molecule, such as an antibody, which is located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0060] The terms “part” or “fragment” are used interchangeably herein and refer to a contiguous element. For example, one part of a structure such as an amino acid sequence or a protein refers to one contiguous element of the structure. A part, part, or fragment of a structure preferably comprises one or more functional properties of the structure. For example, a part, part, or fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or peptide from which it originates. A part or fragment of a protein sequence preferably comprises at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 contiguous amino acid sequences of the protein sequence.

[0061] According to the present invention, CLDN6 is substantially not expressed in cells if its expression level is lower than that of placental cells or placental tissue. Preferably, the expression level is less than 10% of the expression level in placental cells or placental tissue, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower. Preferably, CLDN6 is substantially not expressed in cells if its expression level is at most twice, preferably 1.5 times, above the expression level in non-cancerous tissue other than the placenta, and preferably not above the expression level in said non-cancerous tissue. Preferably, CLDN6 is substantially not expressed in cells if its expression level is below the detection limit and / or if its expression level is so low that it does not allow binding by a CLDN6-specific antibody added to the cell.

[0062] According to the present invention, CLDN6 is expressed in cells if its expression level is at least twice, preferably 10 times, 100 times, 1000 times, or 10000 times higher than its expression level in non-cancerous tissues other than the placenta. Preferably, CLDN6 is expressed in cells if its expression level is above the detection limit and / or is high enough to allow binding by a CLDN6-specific antibody added to the cell. Preferably, CLDN6 expressed in cells is expressed or exposed on the surface of the cells.

[0063] CLDN6 expression is detectable as mRNA only in the placenta, but the protein is completely undetectable. Therefore, the descriptions made herein regarding CLDN6 expression in the placenta preferably relate to mRNA expression.

[0064] According to the present invention, the term “disease” refers to any pathological condition, including cancer, and in particular the forms of cancer described herein. References herein to cancer or specific forms of cancer also include its metastases. In preferred embodiments, the disease to be treated according to this application includes cells expressing CLDN6, and in particular cancer stem cells expressing CLDN6.

[0065] The phrase "disease associated with cells expressing CLDN6" or similar expressions means, according to the present invention, that CLDN6 is expressed in cells of diseased tissue or organ. In one embodiment, the expression of CLDN6 in cells of diseased tissue or organ is increased compared to its state in healthy tissue or organ. Increase means an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more. In one embodiment, the expression is observed only in diseased tissue, and the expression in the corresponding healthy tissue is suppressed. According to the present invention, diseases associated with cells expressing CLDN6 include cancer. Furthermore, according to the present invention, cancer is preferably characterized by cancer cells expressing CLDN6.

[0066] As used herein, “cancer disease” or “cancer” encompasses diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. “Cancer cells” means abnormal cells that grow by rapid, uncontrolled proliferation and continue to grow after the stimulus that initiated new growth has ceased. Preferably, “cancer disease” is characterized by cells expressing CLDN6, in particular cancer stem cells expressing CLDN6.

[0067] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as their metastases. Examples of these include lung carcinoma, breast carcinoma, prostate carcinoma, colon carcinoma, renal cell carcinoma, cervical carcinoma, or metastases of the above-mentioned cancer types or tumors. The term "cancer" according to the present invention also includes cancer metastases.

[0068] According to the present invention, "carcinoma" is a malignant tumor derived from epithelial cells. This group includes the most common cancers, such as breast cancer, prostate cancer, lung cancer, and colon cancer in their common forms.

[0069] Adenocarcinoma is a type of cancer that originates from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line the body's cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they possess secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated ones may not. By staining cells from a biopsy, pathologists determine whether a tumor is adenocarcinoma or some other type of cancer. Adenocarcinoma can occur in many tissues of the body due to the ubiquity of glands in the body. Not every gland secretes the same substance, but as long as there is exocrine function to the cells, it is considered a gland, and therefore its malignant form is named adenocarcinoma. Malignant adenocarcinoma invades other tissues and often metastasizes if there is enough time for it to do so. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. Ovarian adenocarcinoma includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0070] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process, depending on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then invasion of the target organ after being carried by the blood. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, because tumor cells or components may remain and exhibit metastatic potential. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis," which is a metastasis far from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present invention refers to lymph node metastasis.

[0071] Refractory cancer is a malignant disease in which specific treatments are ineffective, either from the outset or becoming refractory over time. The terms “refractory,” “refractory,” and “resistant” are used interchangeably herein.

[0072] As used herein, the term “cancer stem cells” refers to cells that may be precursors to highly proliferative cancer cells. Cancer stem cells have the ability to regrow tumors, as demonstrated by their ability to form tumors in immunocompromised mice. Cancer stem cells also typically proliferate slowly compared to the tumor bulk, i.e., cancer stem cells are generally quiescent. In some embodiments, but not all, cancer stem cells may constitute only a small fraction of the tumor, such as about 0.1–10%. Cancer stem cells may have one, more, or all of the following characteristics or properties: (i) they may have the ability to cause tumors and / or sustain tumor growth; (ii) they may generally be less mutated than the tumor bulk (e.g., due to slower growth and therefore fewer DNA replication-dependent errors, improved DNA repair, and / or epigenetic / non-mutagenic changes that contribute to their malignancy); (iii) they may have many characteristics of normal stem cells (e.g., similar cell surface antigens and / or intracellular expression profiles, self-renewal programs, multidrug resistance, immature phenotypes, etc., characteristic of normal stem cells), and may originate from normal stem cells (one or more); (iv) they may be a source of metastasis; (v) they may be slowly growing or quiescent; (vi) they may be tumorigenic (e.g., as measured by NOD / SCID transplantation experiments); (vii) they may be relatively resistant to conventional therapies (i.e., chemotherapy resistant); and (viii) they may contain a subpopulation of tumors (e.g., compared to the tumor bulk).

[0073] "To treat" means administering a treatment to a subject, such as a compound or composition or combination of compounds or compositions, to prevent or eliminate a disease, including reducing the size or number of tumors in the subject, to stop or slow the progression of a disease in the subject, to prevent or delay the onset of a new disease in the subject, to reduce the frequency or severity of symptoms and / or recurrences in a subject who currently has or has previously had the disease, and / or to extend, i.e., increase or extend the lifespan of the subject. In particular, the term "treatment of disease" includes curing, shortening the duration, improving, preventing, slowing or stopping the progression or worsening of a disease or its symptoms, or preventing or delaying its onset.

[0074] In relation to the present invention, terms such as “protect” or “prevent” relate to the prevention or treatment of the onset and / or transmission of disease in a subject, or both, and in particular to minimizing the likelihood of a subject developing a disease or delaying the onset of a disease. For example, a subject at risk of cancer is a candidate for a therapy to prevent cancer.

[0075] "At risk" refers to subjects identified as having a higher-than-usual likelihood of developing a disease, particularly cancer, compared to the general population. In addition, subjects who have had or currently have a disease, particularly cancer, are at high risk of developing the disease because they are still likely to develop it. Subjects who currently have or have had cancer are also at high risk of cancer metastasis.

[0076] The term "patient" means, according to the present invention, a subject for treatment, particularly a diseased subject, including humans, non-human primates, or other animals, especially mammals, such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a particularly preferred embodiment, the patient is human.

[0077] As used herein, the term “combined use” in relation to the administration of therapies refers to the use of more than one therapy or therapeutic agent. The use of the term “combined” does not limit the order in which therapies or therapeutic agents are administered to the subject. Therapies or therapeutic agents may be administered before, concurrently with, or after the administration of a second therapy or therapeutic agent to the subject. Preferably, therapies or therapeutic agents are administered to the subject in an order, amount, and / or time interval such that they can act together. In certain embodiments, therapies or therapeutic agents are administered to the subject in an order, amount, and / or time interval such that they provide a greater benefit than if they were administered in any other way, particularly independently of each other. Preferably, the greater benefit is a synergistic effect.

[0078] "Target cells" refers to any undesirable cells, such as cancer cells, particularly cancer stem cells. In a preferred embodiment, the target cells express CLDN6.

[0079] According to the present invention, the term "chemotherapy" refers to treatment with one or more chemotherapeutic agents or combinations of chemotherapeutic agents, such as cell proliferation inhibitory agents or cytotoxic agents. The chemotherapeutic agents according to the present invention include cell proliferation inhibitory compounds and cytotoxic compounds.

[0080] According to the present invention, the term "chemotherapeutic agent" includes taxanes such as paclitaxel and docetaxel, and platinum compounds such as cisplatin and carboplatin, as well as combinations thereof. Preferred combinations may include combinations of taxanes and platinum compounds, for example, paclitaxel and carboplatin, particularly in relation to the treatment of ovarian cancer. Further preferred combinations may include combinations of platinum compounds such as cisplatin and etoposide and / or bleomycin, particularly in relation to the treatment of ovarian cancer, particularly ovarian germ cell tumors, and / or germ cell tumors, particularly ovarian and testicular germ cell tumors. According to the present invention, reference to chemotherapeutic agents shall include derivatives such as any prodrugs such as esters, salts, or conjugates of the active substance. An example is a conjugate of the active substance with a carrier substance, for example, a protein-conjugated paclitaxel such as albumin-conjugated paclitaxel. Preferably, salts of the active substance are pharmaceutically acceptable.

[0081] Taxanes are a class of diterpene compounds initially derived from natural sources such as plants of the genus Taxus, although some are now artificially synthesized. The primary mechanism of action of taxane-class drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0082] According to the present invention, the term "docetaxel" is defined by the following formula: [ka] This refers to compounds that possess [a certain characteristic].

[0083] In particular, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-trityl-4-acetate-2-benzoate-13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropanoate}.

[0084] According to the present invention, the term "paclitaxel" is defined by the following formula: [ka] This refers to compounds that possess [a certain characteristic].

[0085] 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-epoxytaxic-11-en-2-ylbenzoate

[0086] According to the present invention, the term "platinum compound" refers to a compound that contains platinum in its structure, such as a platinum complex, and includes compounds such as cisplatin, carboplatin, and oxaliplatin.

[0087] The term "cisplatin" or "cisplatin" is derived from the following formula: [ka] This refers to the compound cis-diamminedichloroplatinum(II) (CDDP).

[0088] The term "carboplatin" is derived from the following formula: [ka] This refers to the compound cis-diammine(1,1-cyclobutanedicarboxylate)platinum(II).

[0089] The term "oxaliplatin" is derived from the following formula: [ka] This refers to a compound that is a platinum compound complexed with a diaminocyclohexane support ligand.

[0090] In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethandeoato-O,O')platinum(II). Injectable oxaliplatin is also commercially available under the trade name Eloxatine.

[0091] Further chemotherapeutic agents envisioned for use in the present invention, either alone or in combination with other chemotherapeutic agents such as taxanes or platinum compounds, include, but are not limited to, nucleoside analogs, camptothecin analogs, and anthracyclines.

[0092] The term "nucleoside analog" refers to structural analogs of nucleosides, a category that encompasses both purine analogs and pyrimidine analogs.

[0093] The term "gemcitabine" is derived from the following formula: [ka] It is a compound that is a nucleoside analog of [another compound].

[0094] In particular, this term refers to the compound 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythropentofuranosyl)pyrimidine-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidine-2-one.

[0095] The term “nucleoside analog” encompasses fluorouracil and its prodrugs, as well as fluoropyrimidine derivatives. The term “fluorouracil” or “5-fluorouracil” (5-FU or f5U) (marketed under the trademark names Adrucil, Carac, Efudix, Efudex, and Fluoroplex) is defined by the following formula: [ka] It is a compound that is a pyrimidine analog of [the compound in question].

[0096] In particular, this term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0097] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug converted to 5-FU in tissues. Orally administered capecitabine is given by the following formula: [ka] It has.

[0098] In particular, this term refers to the compound pentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidine-4-yl]carbamate.

[0099] The terms "folic acid" or "leucovorin" refer to compounds useful in synergistic combinations with the chemotherapeutic agent 5-fluorouracil. Therefore, where the administration of 5-fluorouracil or its prodrugs is referred to herein, in one embodiment, such administration may include administration in combination with folinic acid. Folinic acid is given by the following formula: [ka] It has.

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

[0101] According to the present invention, the term "camptothecin analog" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analog" refers to the following structure: [ka] This refers to compounds that contain [the specified element].

[0102] According to the present invention, preferred camptothecin analogs are inhibitors of the DNA enzyme topoisomerase I (Topo I). Preferred camptothecin analogs according to the present invention are irinotecan and topotecan.

[0103] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. In chemical terms, it is represented by the following formula: [ka] It is a semi-synthetic analog of the natural alkaloid camptothecin, which possesses [specific properties].

[0104] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4':6,7]-indolidino[1,2-b]quinoline-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0105] Topotecan is, formula: [ka] It is a topoisomerase inhibitor.

[0106] In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0107] Anthracyclines are a class of drugs commonly used in cancer chemotherapy, and are also antibiotics. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure, which typically requires glycosylation at a specific site.

[0108] Anthracyclines preferably induce one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by intercalating between base pairs in DNA / RNA strands, thus hindering the replication of rapidly proliferating cancer cells. 2. Inhibit topoisomerase II enzyme, preventing the relaxation of supercoiled DNA, thus blocking DNA transcription and replication. 3. Generate iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0109] According to the present invention, the term "anthracycline" preferably relates to a substance that induces apoptosis by inhibiting the recombination of DNA by topoisomerase II, and preferably to an anticancer agent.

[0110] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pirarubicin, barurubicin, N-trifluoro-acetyldoxorubicin-14-valerate, acrasinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and acrasinomycin A (acrarubicin). Mitoxantrone is a member of the anthracendione class of compounds, which are anthracycline analogs that lack the sugar moiety of anthracycline but retain a planar polycyclic aromatic ring structure that allows for intercalation into DNA.

[0111] In connection with the present invention, epirubicin is particularly intended as an anthracycline. Epirubicin is given by the following formula: [ka] It is an anthracycline drug that is marketed under the trade name Ellence in the United States and elsewhere under the trade names Pharmorubicin or Epirubicin Ebewe. In particular, the term "epirubicin" refers to the compound, (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. Epirubicin is considered to cause fewer side effects and is therefore preferred over doxorubicin, the most common anthracycline, in some chemotherapy regimens.

[0112] The term "etoposide" refers to a semi-synthetic derivative of podophyllotoxin that exhibits antitumor activity. Etoposide inhibits DNA synthesis by forming a complex with topoisomerase II and DNA. This complex induces breaks in double-stranded DNA and prevents repair by topoisomerase II binding. The accumulated DNA breaks prevent cells from entering the mitotic phase of cell division, leading to cell death. Etoposide is expressed by the following formula: [ka] It has.

[0113] In particular, this term refers to the compound 4'-demethyl-epipodophyllotoxin 9-[4,6-O-(R)-ethylidene-β-D-glucopyranoside],4'-(dihydrogen phosphate).

[0114] The term "bleomycin" refers to a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus. When used as an anticancer agent, it works by causing DNA breakage. Bleomycin is preferably formulated as follows: [ka] It contains compounds that have the following properties.

[0115] According to the present invention, when chemotherapy is administered in combination with an antibody having the ability to bind to CLDN6 (which may exist in a conjugate with at least one toxic drug component, i.e., as an antibody-drug conjugate), the chemotherapy is administered before and / or concurrently with the administration of the antibody (as a mixture or as separate compositions). Preferably, the administration of chemotherapy is started prior to the administration of the antibody. Preferably, the chemotherapy is started or administered before the administration of the antibody so as to increase CLDN6 expression in cancer cells such as cancer stem cells and enhance the antitumor activity of the antibody. Preferably, the administration of chemotherapy is started at least 2 days, at least 4 days, at least 6 days, at least 8 days, at least 10 days, at least 12 days, or at least 14 days before the first administration of the antibody. The administration of chemotherapy may continue during the administration of the antibody or may be stopped before or during the administration of the antibody, for example, 1 to 3 days, 1 to 7 days, 1 to 10 days, or 1 to 14 days before the administration of the antibody. Preferably, the chemotherapeutic agent includes taxanes such as paclitaxel or docetaxel and / or platinum compounds such as cisplatin or carboplatin.

[0116] The term “antigen” refers to an active substance, such as a protein or peptide, that contains an epitope to which an immune response is directed and / or should be directed. In preferred embodiments, the antigen is a tumor-associated antigen, such as CLDN6, i.e., a component of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus, and is preferably produced in large quantities as an intracellular or surface antigen on cancer cells.

[0117] In relation to the present invention, the terms “tumor-associated antigen” or “tumor antigen” preferably relate to proteins that are specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage under normal conditions, and that are expressed or abnormally expressed in one or more tumor or cancerous tissues. In relation to the present invention, tumor-associated antigens are preferably associated with the cell surface of cancer cells and preferably are not expressed at all or are expressed only rarely in normal tissues.

[0118] The term "epitope" refers to an antigenic determinant within a molecule, i.e., a part of a molecule recognized by the immune system, such as an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. The epitopes of proteins such as CLDN6 preferably comprise continuous or discontinuous portions of the protein and are preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acid lengths. For example, the epitopes may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths.

[0119] The term “antibody” encompasses glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and any molecule comprising an antigen-binding moiety of such a glycoprotein. The term “antibody” encompasses monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments or derivatives, for example, without limitation, single-chain antibodies, such as scFv and antigen-binding antibody fragments such as Fab and Fab' fragments, and also encompasses all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into more conserved, hypervariable regions called complementarity-determining regions (CDRs), interspersed with regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0120] As used herein, the term “monoclonal antibody” refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibodies exhibit single-binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma containing B cells obtained from a non-human animal, such as a mouse, fused to immortalized cells.

[0121] The term “recombinant antibody” as used herein includes all antibodies produced, expressed, created or isolated by recombinant means, for example (a) antibodies isolated from animals (e.g., mice) or hybridomas produced therefrom that are transgenic or transchromosomal with respect to an immunoglobulin gene, (b) antibodies isolated from host cells transformed to express the antibody, for example from a transfectoma, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies produced, expressed, created or isolated by any other means, including splicing an immunoglobulin gene sequence to another DNA sequence.

[0122] As used herein, the term "human antibody" is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation).

[0123] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, where the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may include a complete variable domain fused to a constant domain, or it may include only a complementation-determining region (CDR) transplanted into an appropriate framework region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble a human immunoglobulin. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more altered CDRs compared to the original antibody.

[0124] The term "chimeric antibody" refers to an antibody in which portions of the amino acid sequences of the heavy and light chains are homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another antibody. Typically, the variable regions of both the light and heavy chains mimic the variable region of an antibody derived from one species of mammal, while the constant region is homologous to the sequence of an antibody derived from another species. One obvious advantage of such a chimeric form is that the variable region can be conveniently induced from currently known sources using B cells or hybridomas from readily available non-human host organisms, for example, by combining it with a constant region derived from human cell preparations. The variable region has the advantage of ease of preparation and its specificity is not affected by the source, although the human constant region is less likely to elicit an immune response from human subjects than a constant region from a non-human source when the antibody is injected. However, the definition is not limited to this particular example.

[0125] Antibodies can originate from a variety of species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0126] The antibodies described herein include IgA antibodies such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more specifically IgG1, kappa, or IgG1, lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

[0127] As used herein, “heterogeneous antibody” is defined in relation to transgenic organisms that produce such antibodies. The term refers to antibodies that are not composed of transgenic organisms and have an amino acid sequence or coding nucleic acid sequence that corresponds to one found in organisms generally derived from non-transgenic species.

[0128] As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain bound to a mouse light chain is a heterohybrid antibody.

[0129] The antibodies described herein are preferably isolated. As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to CLDN6 substantially does not contain antibodies that specifically bind to antigens other than CLDN6). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN6 may, however, exhibit cross-reactivity to other related antigens, e.g., related antigens from other species (e.g., CLDN6 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, a combination of “isolated” monoclonal antibodies relates to antibodies having different specificities and being combined in a well-defined composition or mixture.

[0130] The terms “antigen-binding portion” (or simply “binding portion”) or “antigen-binding fragment” (or simply “binding fragment”) of an antibody, or similar terms, refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of a VH domain and a CH domain; (iv) Fv fragments consisting of a VL domain and a VH domain of one arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, which may be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination by synthetic linkers, which allow them to be constructed as single-chain proteins (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair up to form a monovalent molecule. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of the antibody. Further examples include binding-domain immunoglobulin fusion proteins, which contain (i) a binding-domain polypeptide fused to an immunoglobulin hinge-domain polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region.The binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Applications 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0131] In relation to the present invention, the term "binding domain" characterizes the structure of, for example, an antibody, which binds to / interacts with a given target structure / antigen / epitope. Therefore, the binding domain according to the present invention represents an "antigen interaction site."

[0132] For the purposes of this invention, all antibodies and antibody derivatives, such as antibody fragments, described herein are encompassed by the term “antibody.” The term “antibody derivative” refers to any modified form of antibody, such as a conjugate of an antibody with another active substance or another antibody, or an antibody fragment.

[0133] Naturally occurring antibodies are generally monospecific, meaning they bind to a single antigen. The present invention comprises antibodies that bind to target cells (by engaging with CLDN6) and to second entities such as cytotoxic cells (e.g., by engaging with the CD3 receptor). The antibodies of the present invention may be bispecific or multispecific, e.g., triplicate, quadruplicate, etc.

[0134] The term “bispecific molecule” is intended to encompass agents having two distinct binding specificities. For example, such a molecule may bind to or interact with receptors such as (a) cell surface antigens and (b) Fc receptors on the surface of effector cells. The term “multispecific molecule” is intended to encompass agents having more than two distinct binding specificities. For example, such a molecule may bind to or interact with (a) cell surface antigens, (b) receptors such as Fc receptors on the surface of effector cells and (c) at least one other component. Therefore, the term “antibody capable of binding to CLDN6” encompasses, but is not limited to, bispecific, triplicate, quadruplicate, and other multispecific molecules for CLDN6 and for other targets such as Fc receptors on effector cells. The term “bispecific antibody” also encompasses diabodies. Diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used to pair them with a complementary domain on another chain, thereby creating two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0135] In connection with the present invention, an antibody having the ability to bind to CLDN6 can preferably induce an immunoeffector function as described herein. Preferably, the immunoeffector function is directed toward cells such as cancer stem cells that carry the tumor-associated antigen CLDN6 on their surface.

[0136] In relation to the present invention, the term “immune effector function” encompasses any function mediated by components of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in the death of cancer cells, particularly cancer stem cells. Such functions include complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying tumor-associated antigens, cytolysis of cells carrying tumor-associated antigens, and / or inhibition of the proliferation of cells carrying tumor-associated antigens. Binding agents may also exert their effects simply by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies may block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of cancer cells.

[0137] According to the present invention, antibodies can be conjugated to therapeutic components or active substances such as toxic drug components, particularly cytotoxic substances, drugs (e.g., immunosuppressants), or radioisotopes. Cytotoxic or cytotoxic drugs include any active substances that are harmful to cells and, in particular, kill cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, amanitin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Suitable therapeutic agents for forming antibody conjugates include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), and antibiotics (e.g., dactinomycin (formerly acti) Examples of therapeutic agents include, but are not limited to, nomycin, bleomycin, mitramycin, and anthramycin (AMC), as well as antimitotic agents (e.g., vincristine and vinblastine). In preferred embodiments, the therapeutic agent is a cytotoxic or radiotoxic substance. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In preferred embodiments, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or lysine A. Particularly preferred toxic therapeutic components according to the present invention are compounds that inhibit microtubule aggregation and have antiproliferative and / or cytotoxic effects.

[0138] Particularly preferred according to the present invention are antibodies conjugated to therapeutic components or active substances, such as cytotoxics, that act on slowly proliferating or quiescent cells, such as cancer stem cells. Such therapeutic components include those that act on mRNA and / or protein synthesis. Several transcription inhibitors are known. For example, actinomycin D, which is both a transcription inhibitor and a DNA damaging agent, intercalates into DNA and thus inhibits the early stages of transcription. Flavopyridol targets the transcription elongation stage. α-amanitin directly binds to RNA polymerase, resulting in inhibition of both the initiation and elongation stages.

[0139] Antibodies can also bind to radioactive isotopes, such as iodine-131, yttrium-90, or indium-111, to produce cytotoxic radiopharmaceuticals.

[0140] The antibody conjugates of the present invention can be used to modulate a given biological response, and the drug components should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug components may be peptides, proteins, or polypeptides having the desired biological activity. Such proteins may include, for example, enzyme-active toxins or their active fragments, such as abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors. Further preferred drug components according to the present invention are curcumin, salinomycin, and sulforaphane.

[0141] Techniques for binding such therapeutic components to antibodies are well known, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody" See "In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0142] In one preferred embodiment, the antibody according to the present invention is conjugated to one or more maytansinoid molecules.

[0143] Maytansinoids are potent microtubule-targeting compounds that inhibit cell proliferation during mitosis. Maytansinoids are derivatives of maytansine, a 19-membered anthamacrollide structure bonded to a chlorinated benzene ring. Maytansine has the following formula: [ka] It has.

[0144] It has also been discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic maytansinol and maytansinol analogs are incorporated herein by reference, for example, U.S. Patents No. 4,137,230; No. 4,248,870; No. 4,256,746; No. 4,260,608; No. 4,265,814; No. 4,294,757; No. 4,307,016; No. 4,308,268; No. 4,30 This has been reported in issues 8,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,362,663 and 4,371,533, as well as in Kawai et al (1984) Chem. Pharm. Bull. 3441-3451.

[0145] Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Particularly preferred maytansinoids according to the present invention are thiol-containing derivatives of maytansine, such as DM1 and DM4. Such thiol-containing derivatives of maytansine include compounds in which the methyl group bonded to the carbonyl group is substituted with a group containing a free sulfhydryl group, such as a -R-SH group [wherein R represents an alkylene group or other carbon-containing atomic group].

[0146] DM1, also known as meltansine, is given by the following formula: [ka] It is a meitansinoid that possesses [a certain characteristic].

[0147] In particular, the terms "meltansine" or "DM1" refer to the compound, N 2' -deacetyl-N 2' This refers to -(3-mercapto-1-oxopropyl)-meytansine.

[0148] "DM4" is a compound, N 2' -deacetyl-N 2' This refers to -(4-methyl-4-mercapto-1-oxopentyl)-meytansine.

[0149] Anti-CLDN6 antibody-maytansinoid conjugates are prepared by chemically linking anti-CLDN6 antibodies to maytansinoid molecules without significantly reducing the biological activity of the antibody or maytansinoid molecule. While an average of 3-4 maytansinoid molecules can be conjugated per antibody molecule, even a single molecule of toxin / antibody is expected to enhance cytotoxicity compared to the use of a naked antibody.

[0150] In this regard, the term "antibody covalently bound to at least one toxic drug component" encompasses situations where one or more molecules of the same drug are covalently bound to the antibody molecule, as well as situations where different drugs are covalently bound to the antibody molecule. In the latter situation, one or more molecules of each of the different drugs may bind to the antibody molecule or a combination thereof (for example, one molecule of one drug bound to the antibody molecule and several molecules of another drug bound to it).

[0151] In some embodiments of the present invention, antibodies are conjugated to drastatin or auristatin, which is a drastatin peptide analog and derivative (U.S. Patents 5,635,483 and 5,780,588, incorporated herein by reference). Auristatin is a synthetic analog of drastatin 10, a natural product derived from the marine mollusk Dorabella auricularia. Like meitansinoids, auristatin is a microtubule disruptor. The drastatin or auristatin drug component can be conjugated to an antibody via the N (amino) or C (carboxyl) terminus of the peptide drug component.

[0152] Exemplary embodiments of auristatin preferably include monomethyl auristatin drug components such as N-terminally linked MMAE and MMAF.

[0153] MMAE, also known as monomethyl auristatin E, is given by the following formula: [ka] It has.

[0154] In particular, the term "MMAE" refers to the compound (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide. MMAE is actually desmethyl-aulistatin E, meaning the N-terminal amino group has only one methyl substituent instead of two, as in auristatin E itself.

[0155] Particularly preferred according to the present invention are antibody-vc auristatin conjugates, such as antibody-vcMMAE conjugates. According to the present invention, the terms "antibody-vc auristatin" or "vcMMAE" refer to antibody-drug conjugates (ADCs) containing auristatin, such as MMAE, linked to an antibody by a lysosome-cleavable dipeptide, valine-citrulline (vc).

[0156] MMAF, also known as monomethyl auristatin F, refers to the compound (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid.

[0157] Many linking groups known in this field exist for creating antibody-drug conjugates.

[0158] In one embodiment of the present invention, an antibody is linked to a drug by a bifunctional crosslinking reagent. As used herein, “bifunctional crosslinking reagent” refers to a reagent having two reactive groups, one of which can react with the antibody and the other with the drug, thereby forming a conjugate for linking an antibody to a drug. Any suitable bifunctional crosslinking reagent can be used in connection with the present invention, insofar as the linker reagent provides retention of the drug, e.g., cytotoxicity, and targeting properties of the antibody. Preferably, the linker molecule links the drug to the antibody via a chemical bond such that the drug and the antibody are chemically bonded to each other (e.g., covalently bonded).

[0159] In one embodiment, the bifunctional crosslinking reagent includes a non-cleavable linker. The non-cleavable linker is any chemical component that can link a drug, such as a meitansinoid, to an antibody via a stable covalent bond. Preferably, the non-cleavable linker is not cleaved under physiological conditions, particularly inside cells. Thus, the non-cleavable linker is substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions in which the drug or antibody remains active. Suitable crosslinking reagents for forming a non-cleavable linker between a drug and an antibody are well known in the art. In one embodiment, the drug is linked to the antibody via a thioether bond. Examples of non-cleavable linkers include linkers having a maleimide or haloacetyl-based component for reaction with drugs such as the sulfhydryl group of a meitansinoid. Such bifunctional crosslinkers are well known in the art and include, but are not limited to, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) and its "long-chain" analog, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidecaproate) (LC-SMCC). Preferably, the bifunctional crosslinker is SMCC. Using such a linker, a drug such as meltansine can be linked to an amino group, such as the free NH2 group of a lysine residue of an antibody, via 4-(3-mercapto-2,5-dioxo-1-pyrrolidinylmethyl)cyclohexanecarboxylic acid. Each antibody-drug conjugate molecule may contain one antibody molecule bound to several molecules of meltansine.

[0160] In one particularly preferred embodiment, the linking reagent is a cleavable linker. Preferably, the cleavable linker is cleavable under physiological conditions, particularly inside cells. Examples of suitable cleavable linkers include disulfide linkers, acid-unstable linkers, photo-unstable linkers, peptidase-unstable linkers, and esterase-unstable linkers. Disulfide-containing linkers are linkers that are cleavable via disulfide exchange, which can occur under physiological conditions. Acid-unstable linkers are linkers that are cleavable at acidic pH. For example, certain intracellular compartments such as endosomes and lysosomes have acidic pH (pH 4-5) and provide suitable conditions for cleaving acid-unstable linkers. Photo-unstable linkers are useful on the body surface and in many body coeloms where light is accessible. Furthermore, infrared light can penetrate tissues. Peptidase-unstable linkers can be used to cleave specific peptides inside or outside cells. In one embodiment, the cleavable linker is cleaved under mild conditions, i.e., intracellular conditions under which the activity of the cytotoxic agent is not affected.

[0161] In one particularly preferred embodiment, the linker is a linker comprising or consisting of a dipeptide, valine (Val)-citrulline (Cit)(vc), which is cleaved by cathepsin inside the tumor cell.

[0162] In this invention, the term “cancer therapy against cancer stem cells” refers to any therapy that can be used to target cancer stem cells, preferably to kill cancer stem cells and / or to reduce the proliferation or viability of cancer stem cells. Such therapies include i) naked or therapeutically conjugated antibodies, antibody fragments, and proteins (e.g., antibodies or antibody conjugates having the ability to bind to CLDN6 as described above) that target a specific cell surface target on cancer stem cells, e.g., CLDN6, or ii) small molecules that reduce the proliferation or viability of cancer stem cells. In certain embodiments, the active agent binds to an antigen that is expressed at a higher level on cancer stem cells than on normal stem cells. In certain embodiments, the active agent specifically binds to cancer stem cell antigens.

[0163] The term "binding" according to the present invention preferably relates to specific binding.

[0164] According to the present invention, when an antibody has significant affinity for a given target in a standard assay and binds to the given target, the antibody can bind to the given target. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant (K D ). Preferably, the term "significant affinity" means 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 of the dissociation constant (K D ) to bind to a given target.

[0165] If an antibody does not have significant affinity for a target in a standard assay and does not bind significantly, particularly not detectably, to the target, the antibody cannot (substantially) bind to the target. Preferably, when the antibody is present at a concentration of 2, preferably 10, more preferably 20, particularly 50 or 100 μg / ml or more, the antibody does not bind detectably to the target. Preferably, the K D for binding of the antibody to a given target to which it can bind is at least 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold or 10 6 -fold higher than the K D at which the antibody binds to the target, the antibody does not have significant affinity for the target. For example, if the K D for binding of the antibody to a target to which it can bind is 10 -7 M, the K Dat least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It is M.

[0166] An antibody is specific to a predetermined target if it can bind to a predetermined target but cannot bind to other targets, i.e., it does not have significant affinity for other targets in a standard assay and does not significantly bind to other targets. According to the present invention, an antibody is specific to CLDN6 if it can bind to CLDN6 but cannot (substantially) bind to other targets. Preferably, an antibody is specific to CLDN6 if its affinity and binding to such other targets do not significantly exceed its affinity or binding to proteins unrelated to CLDN6, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins, such as MHC molecules or transferrin receptors, or some other specific polypeptide. Preferably, an antibody has a K for binding to targets that it is not specific to. D At least 10 times, 100 times, 10 3 double, 10 4 double, 10 5 double or 10 6 K is twice as low D When the antibody binds to a predetermined target, the antibody is specific to that predetermined target. For example, regarding the binding of the antibody to a target to which the antibody is specific, K D 10 -7 If M, then K regarding the binding of the antibody to a non-specific target. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It is M.

[0167] The binding of antibodies to a target can be experimentally measured using any suitable method: see, for example, Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992); and the methods described herein. Affinity can be readily measured using conventional techniques, e.g., by equilibrium dialysis; by using the BIAcore 2000 instrument, using the general procedure outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigens; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NYAcad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may differ when measured under different conditions, e.g., different salt concentrations, pH. Therefore, affinity and other antigen-binding parameters, e.g., K D ,I C 50 The measurement is preferably performed using standard solutions and standard buffers of the antibody and antigen.

[0168] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0169] As used herein, “isotype switching” refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of other Ig classes.

[0170] As used herein, when applied to an object, the term “naturally occurring” refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by a human in a laboratory is considered naturally occurring.

[0171] As used herein, the term “reorganized” essentially refers to the configuration of a heavy-chain or light-chain immunoglobulin locus in which the V segment is directly adjacent to the DJ or J segment in the conformation encoding the complete VH or VL domain, respectively. Reorganized immunoglobulin (antibody) loci can be identified by comparison with germline DNA, and a reorganized locus has at least one recombinant heptameric / nocaper homologous element.

[0172] As used herein with respect to the V segment, the terms “unreorganized” or “germ cell configuration” refer to a configuration in which the V segment has not been rearranged to be directly adjacent to a D or J segment.

[0173] Preferably, binding of an antibody capable of binding to CLDN6 to cells expressing CLDN6 induces or mediates the death of the CLDN6-expressing cells. The cells expressing CLDN6 are preferably cancer stem cells, particularly those of cancerous diseases described herein, such as ovarian cancer stem cells. Preferably, the antibody induces or mediates cell death by inducing one or more of the following: complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation. Preferably, ADCC-mediated lysis of cells occurs in the presence of effector cells, which are selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs in certain embodiments. Inhibition of cell proliferation can be measured in vitro by quantifying cell proliferation in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside, an analog of thymidine, that can be incorporated into newly synthesized DNA in replicating cells (during the S phase of the cell cycle) and replace thymidine during DNA replication. For example, detecting the incorporated chemical using an antibody specific to BrdU indicates a cell that was actively replicating its DNA.

[0174] In preferred embodiments, the antibodies described herein have the following characteristics: a) Specificity for CLDN6; b) Binding affinity to CLDN6 of about 100 nM or less, preferably about 5 to 10 nM or less, more preferably about 1 to 10 nM or less; c) Ability to induce or mediate CDC on CLDN6-positive cells; d) Ability to induce or mediate ADCC on CLDN6-positive cells; e) Ability to inhibit the proliferation of CLDN6-positive cells; f) Ability to induce apoptosis in CLDN6-positive cells It may be characterized by one or more of the following.

[0175] In one embodiment, an antibody capable of binding to CLDN6 has the ability to bind to epitopes present in CLDN6, preferably within the extracellular domain of CLDN6, particularly within the first extracellular loop, preferably within amino acid positions 28-76 of CLDN6, or within the second extracellular loop, preferably within amino acid positions 141-159 of CLDN6. In a specific embodiment, an antibody capable of binding to CLDN6 binds to epitopes on CLDN6 that are not present on CLDN9. Preferably, an antibody capable of binding to CLDN6 binds to epitopes on CLDN6 that are not present on CLDN4 and / or CLDN3. Most preferably, an antibody capable of binding to CLDN6 binds to epitopes on CLDN6 that are not present on CLDN proteins other than CLDN6.

[0176] An antibody capable of binding to CLDN6 preferably binds to CLDN6 but not to CLDN9, and preferably not to CLDN4 and / or CLDN3. Preferably, the antibody capable of binding to CLDN6 is specific to CLDN6. Preferably, the antibody capable of binding to CLDN6 binds to CLDN6 expressed on the cell surface. In certain preferred embodiments, the antibody capable of binding to CLDN6 binds to a native epitope of CLDN6 present on the surface of living cells.

[0177] In a preferred embodiment, the antibody capable of binding to CLDN6 contains a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9 and their fragments.

[0178] In a preferred embodiment, the antibody capable of binding to CLDN6 contains a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 11, 12 and its fragments.

[0179] In certain preferred embodiments, the antibody having the ability to bind to CLDN6 comprises a combination of heavy chain variable region (VH) and light chain variable region (VL) selected from the following possibilities (i) to (vii): (i) VH includes the amino acid sequence or fragment represented by SEQ ID NO: 3, and VL includes the amino acid sequence or fragment represented by SEQ ID NO: 4. (ii) VH includes the amino acid sequence or fragment represented by SEQ ID NO: 5, and VL includes the amino acid sequence or fragment represented by SEQ ID NO: 6. (iii) VH includes the amino acid sequence or fragment represented by SEQ ID NO: 7, and VL includes the amino acid sequence or fragment represented by SEQ ID NO: 8. (iv) VH includes the amino acid sequence or fragment represented by SEQ ID NO: 9, and VL includes the amino acid sequence or fragment represented by SEQ ID NO: 10. (v)VH contains the amino acid sequence or fragment represented by SEQ ID NO: 5, and VL contains the amino acid sequence or fragment represented by SEQ ID NO: 4. (vi) VH contains the amino acid sequence or fragment represented by SEQ ID NO: 5, and VL contains the amino acid sequence or fragment represented by SEQ ID NO: 11. (vii)VH includes the amino acid sequence or fragment represented by SEQ ID NO: 5, and VL includes the amino acid sequence or fragment represented by SEQ ID NO: 12.

[0180] In a particularly preferred embodiment, the antibody having the ability to bind to CLDN6 includes the following combinations of heavy chain variable region (VH) and light chain variable region (VL): VH contains the amino acid sequence or fragment represented by SEQ ID NO: 5, and VL contains the amino acid sequence or fragment represented by SEQ ID NO: 4.

[0181] The term “fragment” refers in particular to one or more complementarity determination regions (CDRs) of the heavy chain variable region (VH) and / or light chain variable region (VL), preferably at least CDR3 variable region. In one embodiment, the one or more complementarity determination regions (CDRs) are selected from a set of complementarity determination regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term “fragment” refers to the complementarity determination regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL).

[0182] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises the CDRs together with their intervening framework regions. Preferably, the portion comprises at least about 50% of either or both of the first and fourth framework regions, wherein the 50% is 50% of the C-terminus of the first framework region and 50% of the N-terminus of the fourth framework region. The construction of the antibody by recombinant DNA technology may result in the introduction of N-terminal or C-terminal residues of the variable region encoded by a linker introduced to facilitate cloning or other manipulative steps, including the introduction of a linker to link the variable region of the present invention to an immunoglobulin heavy chain, other variable domains (e.g., in the construction of a diabody), or a further protein sequence containing a protein label.

[0183] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises the CDRs within a human antibody framework.

[0184] References herein to antibodies containing a specific chain, region, or sequence in relation to the heavy chain of the antibody preferably refer to a situation in which all of the heavy chains of the antibody contain the specific chain, region, or sequence. This also applies to the light chain of the antibody.

[0185] It should be understood that the antibodies described herein may be delivered to a patient by administering nucleic acids, such as RNA encoding the antibody, and / or by administering host cells containing nucleic acids, such as RNA encoding the antibody. Therefore, when administered to a patient, the nucleic acids encoding the antibody may be present in a naked form or in a suitable delivery vehicle, for example, in the form of liposomes or viral particles, or within host cells. The provided nucleic acids can produce antibodies over a long period in a sustained manner that mitigates the instability observed in at least some aspects of therapeutic antibodies. The nucleic acids to be delivered to the patient may be produced by recombinant means. If the nucleic acid is administered to a patient without being present in host cells, the nucleic acid is preferably taken up by the patient's cells for the expression of the antibody encoded by the nucleic acid. If the nucleic acid is present in host cells and administered to a patient, the nucleic acid is preferably expressed by the host cells within the patient to produce the antibody encoded by the nucleic acid.

[0186] As used herein, the term "nucleic acid" is intended to encompass DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcription RNA (IVT RNA) or synthetic RNA.

[0187] Nucleic acids may be contained in vectors. As used herein, the term “vector” encompasses any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as adenoviruses or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors encompass expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operablely linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0188] In connection with the present invention, the term "RNA" refers to a molecule comprising, and preferably entirely or substantially, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β,D-ribofuranosyl group. The term encompasses isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, for example, to or within the RNA, or at one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, such as nucleotides not found in nature or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0189] According to the present invention, the term "RNA" encompasses "mRNA," meaning "messenger RNA," and more preferably relates to "mRNA," and to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA typically comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0190] In one embodiment of the present invention, RNA is self-replicating RNA, for example, single-stranded self-replicating RNA. In one embodiment, self-replicating RNA is positive-sense single-stranded RNA. In one embodiment, self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, self-replicating RNA is alphaviral genomic RNA or derived from alphaviral genomic RNA. In one embodiment, self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is Semliki Forest virus. In one embodiment, self-replicating RNA comprises one or more transgenes, at least one of which encodes an antibody as described herein. In one embodiment, if RNA is viral RNA or derived from viral RNA, the transgene may partially or completely replace a viral sequence, for example, a viral sequence encoding a structural protein. In one embodiment, self-replicating RNA is in vitro transcription RNA.

[0191] The alphavirus genome is a positive-sense single-stranded RNA (ssRNA(+)) encoding two open reading frames (ORFs) for large polyproteins. The ORF at the 5' end of the genome encodes non-structural proteins nSP1~nSP4 (nsP1~4), which are translated and processed into RNA-dependent RNA polymerase (replicase); the ORF at the 3' end encodes structural proteins—the capsid and glycoprotein. Both ORFs are separated by a so-called subgenome promoter (SGP) that controls the transcription of the structural ORFs. When used as a gene vector, the structural proteins after the SGP are generally replaced by the transgene. To package such vectors into viral particles, the structural proteins are generally expressed in trans from helper constructs. Alphaviruses replicate primarily at the RNA level in the cytoplasm of infected cells. After infection, the ssRNA(+) genome acts as mRNA for the translation of the nsP1234 polyprotein precursor, which is in the early stages of the viral life cycle, processed into fragments nsP123 and nsP4 by autoproteolysis. Fragments nsP123 and nsP4 form a (-) strand replicase complex that transcribes (-) strand RNA from the genomic RNA template. In later stages, the nsP1234 polyprotein is completely cleaved into single proteins, which assemble into a (+) strand replicase complex that synthesizes a new (+) strand genome, as well as subgenomic transcripts encoding structural proteins or transgenes. The subgenomic RNA and the new genomic RNA are capped and polyadenylated, and are therefore recognized as mRNA after infection of target cells. Only the new genomic RNA contains a packaging signal that ensures exclusive packaging of the genomic RNA into budding virions. The advantage of alphaviral replicons for vectorics lies in the forward orientation of the capped and polyadenylated RNA genome. Translatable replicon RNA can be readily synthesized in vitro, thereby enabling capping by adding a cap analog to the in vitro transcription reaction, and allowing the poly-A tail to be encoded as a poly-T track on the plasmid template.In vitro transcribed (IVT) replicons are transfected using conventional transfection techniques and rapidly proliferate even with low amounts of starting IVT RNA. Within hours of transfection, the transgene positioned downstream of the SGP is transcribed into subgenomic RNA at a very high copy number of approximately 40,000 to 200,000 copies per cell, and therefore it is not surprising that the recombinant protein is strongly expressed. Depending on the specific purpose, IVT replicons can be directly transfected into target cells or packaged into alphavirus particles using a helper vector that provides the structural gene in trans. Transfection into the skin or muscle results in high and sustained local expression, in parallel with the potent induction of humoral and cellular immune responses.

[0192] To enhance the expression and / or stability of RNA used in accordance with the present invention, the RNA may be modified without altering the sequence of the expressed peptide or protein.

[0193] In relation to RNA used in accordance with the present invention, the term "modification" encompasses any modification of the RNA that is not present in the RNA in nature.

[0194] In one embodiment of the present invention, the RNA used according to the present invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0195] The RNA according to the present invention may have modified naturally occurring or synthetic ribonucleotides to enhance its stability and / or reduce its cytotoxicity. For example, in one embodiment, cytidine is partially or completely, preferably completely, substituted with 5-methylcytidine in the RNA used according to the present invention. Or, in addition, in one embodiment, uridine is partially or completely, preferably completely, substituted with pseudouridine in the RNA used according to the present invention.

[0196] In one embodiment, the term “modification” relates to providing RNA with a 5' cap or a 5' cap analogue. The term “5' cap” refers to a cap structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide linked to the mRNA by a distinctive 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at position 7. The term “conventional 5' cap” refers to a naturally occurring RNA 5' cap, preferably a 7-methylguanosine cap (m7G). In relation to the present invention, the term “5' cap” includes 5' cap analogues that are similar to the RNA cap structure and are modified, preferably in vivo and / or in cellular, to have the ability to stabilize RNA when bound to it.

[0197] Providing a 5' cap or 5' cap analogue to RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap or 5' cap analogue, thereby incorporating the 5' cap into the generated RNA strand by co-transcription, or by generating RNA by in vitro transcription, for example, and then attaching the 5' cap to the RNA after transcription using a capping enzyme, such as the capping enzyme of vaccinia virus.

[0198] The RNA may undergo further modifications. For example, further modifications of the RNA used in the present invention may include changes to the 5'UTR or 3'UTR, such as elongation or terminal cleavage of the naturally occurring poly(A) tail or introduction of an untranslated region (UTR) unrelated to the coding region of the RNA, or insertion of one or more, preferably two, copies of the 3'UTR derived from a globin gene, such as α2-globin, α1-globin, or β-globin, preferably β-globin, more preferably human β-globin.

[0199] Therefore, in order to increase the stability and / or expression of the RNA used according to the present invention, the RNA can be modified to be present with a polyA sequence having a length of preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the polyA sequence has a length of about 120 adenosine residues. In addition, the incorporation of two or more 3' untranslated regions (UTRs) into the 3' untranslated region of the RNA molecule can result in an increase in translation efficiency. In one particular embodiment, the 3' UTR is derived from the human β-globin gene.

[0200] Preferably, when the RNA is delivered to cells, particularly cells present in vivo, i.e., transfected, it expresses the protein or peptide it encodes.

[0201] The term "transfection" relates to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of the present invention, the term "transfection" also encompasses the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells can be present in a subject, e.g., a patient. Thus, according to the present invention, the cells for transfection of the nucleic acids described herein can be present in vitro or in vivo, e.g., the cells can form part of an organ, tissue, and / or organism of a patient. According to the present invention, the transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is expressed only transiently. Since the nucleic acids introduced in the transfection process are usually not integrated into the nuclear genome, the foreign nucleic acids are diluted or degraded via mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the rate of dilution. Stable transfection must occur if it is desired that the transfected nucleic acids actually remain within the genome of the cells and their daughter cells. The RNA can be transfected into cells to transiently express its encoded protein.

[0202] The term "stability" of RNA relates to the "half-life" of RNA. The "half-life" relates to the period required to remove half of the activity, amount or number of molecules. In the context of the present invention, the half-life of RNA is an indicator of the stability of said RNA. The half-life of RNA can affect the "duration of expression" of the RNA. RNA having a long half-life can be expected to be expressed over a long period of time.

[0203] In the context of the present invention, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", where the term "in vitro transcription" relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the production of the transcription product. These cloning vectors are generally referred to as transcription vectors and are included in the term "vector" according to the present invention.

[0204] The term "translation" according to the present invention relates to the process in the ribosome of a cell where the strand of messenger RNA instructs the assembly of a sequence of amino acids to produce a peptide or protein.

[0205] The term "expression" is used in its most general sense according to the present invention and includes, for example, the production of RNA and / or peptide or protein by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" relate particularly to the production of peptide or protein. It also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable. According to the present invention, the term "expression" also includes "ectopic expression" or "abnormal expression".

[0206] "Ectopic expression" or "abnormal expression," according to the present invention, means that the expression is altered, preferably increased, compared to the state in a subject without a disease associated with ectopic or abnormal expression of a reference, e.g., a specific protein, e.g., a tumor antigen. Increased expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100% or more. In one embodiment, expression is observed only in affected tissue, while expression in healthy tissue is suppressed.

[0207] The term "specifically expressed" means that a protein is expressed primarily in a specific tissue or organ. For example, a tumor antigen specifically expressed in the placenta means that the protein is primarily expressed in the placenta and not expressed in other tissues or to a significant degree in other tissue or organ types. Therefore, a protein that is exclusively expressed in placental cells and expressed to a significantly lower degree in other tissues is specifically expressed in placental cells. In some embodiments, a tumor antigen may also be specifically expressed in more than one tissue type or organ under normal conditions, for example, two or three tissue types or organs, but preferably three or fewer different tissue or organ types. In this case, the tumor antigen is specifically expressed in these organs.

[0208] According to the present invention, the term "RNA encoding" means that when RNA is present in a suitable environment, preferably within a cell, it can be expressed to produce the protein or peptide it encodes.

[0209] Some aspects of the present invention are based on adoptive transfer of host cells, which are transfected in vitro with nucleic acids such as RNA encoding the antibodies described herein, after ex vivo proliferation from a low precursor frequency to a clinically appropriate cell number, and then transferred to a recipient such as a patient. The host cells used in the treatment according to the present invention may be autologous, allogeneic, or syngeneic to the recipient being treated.

[0210] The term "autologous" is used to describe anything that originates from the same subject. For example, "autotransplantation" refers to the transplantation of tissue or organs from the same subject. Such a procedure is advantageous because it overcomes immunological barriers that would otherwise lead to rejection.

[0211] The term "homogenetic" is used to describe anything that originates from different individuals of the same species. Two or more individuals are said to be homogeneous if they do not have identical genes at one or more loci.

[0212] The term "related" is used to describe individuals or tissues that have the same genotype, i.e., identical twins or animals of the same inbred lineage, or any tissues derived from them.

[0213] The term "xenotransplant" is used to describe something consisting of multiple different elements. For example, the transfer of bone marrow from one individual to another constitutes xenotransplantation. Xenogenes are genes that originate from a source other than the subject.

[0214] The term "peptide" according to the present invention includes oligopeptides and polypeptides and refers to a substance containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably 21 or more, and preferably up to eight, ten, twenty, thirty, forty or fifty, and particularly up to 100 amino acids, which are covalently linked by peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having more than 100 amino acid residues, but generally the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

[0215] Any teachings given herein with respect to a particular amino acid sequence, for example, those shown in the sequence listing, should be interpreted as relating to a variant of the particular sequence that is functionally equivalent to the particular sequence, for example, a variant of the particular sequence that produces an amino acid sequence exhibiting the same or similar properties as the particular amino acid sequence. One important property is to retain the antibody's binding to its target or to maintain the antibody's effector function. Preferably, a variant of a particular sequence, when it substitutes the particular sequence in the antibody, retains the antibody's binding to CLDN6 and, preferably, the antibody's function as described herein, such as CDC-mediated lysis or ADCC-mediated lysis.

[0216] For example, the sequences shown in the sequence listing can be modified to remove one or more, preferably all, free cysteine ​​residues by substituting them with amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine, or methionine, most preferably alanine or serine.

[0217] Those skilled in the art will recognize that the sequences of the CDR, hypervariable region, and variable region can be modified without losing their ability to bind to CLDN6. For example, the CDR region is identical or highly homologous to the region of the antibody specified herein. "Highly homologous" means that 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2 substitutions may be made within the CDR. In addition, the hypervariable region and variable region can be modified to exhibit substantial homology to the region of the antibody specifically disclosed herein.

[0218] For the purposes of this invention, the "mutants" of amino acid sequences include amino acid insertion mutants, amino acid addition mutants, amino acid deletion mutants, and / or amino acid substitution mutants. Amino acid deletion mutants, which include deletions at the N-terminus and / or C-terminus of a protein, are also called N-terminal and / or C-terminal cleavage mutants.

[0219] Amino acid insertion mutants contain the insertion of one, two, or more amino acids within a specific amino acid sequence. In amino acid sequence mutants with insertions, one or more amino acid residues are inserted at specific sites within the amino acid sequence, but random insertions are also possible, provided that the resulting products are properly screened.

[0220] Amino acid addition mutants include amino-terminal and / or carboxyl-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0221] Amino acid deletion mutants are characterized by the removal of one or more amino acids from a sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be located at any position in the protein.

[0222] Amino acid substitution mutants are characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. It is preferable that the modification is located at a non-conserved position in the amino acid sequence among homologous proteins or peptides and / or that an amino acid is substituted with another amino acid having similar properties. Preferably, the amino acid changes in protein mutants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of the families of amino acids related to the side chain. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

[0223] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably given with respect to an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given with respect to at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably contiguous amino acids. In a preferred embodiment, the degree of similarity or identity is given with respect to the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, for example using Align, with standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap open 10.0, gap extend 0.5.

[0224] "Sequence similarity" indicates the percentage of amino acids that are identical or are conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between these sequences.

[0225] The term "identity percentage" is intended to represent the percentage of amino acid residues that are identical between two sequences being compared, obtained after best alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are conventionally performed by comparing them after optimal alignment, and such comparisons are performed segment by segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be achieved manually, by local homology algorithms (Smith and Waterman, 1981, Ads App.Math.2, 482; Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85, 2444; or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0226] The identity percentage is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared to obtain the identity percentage between the two sequences, and multiplying the result by 100.

[0227] The terms “cell” or “host cell” preferably refer to an intact cell, i.e., a cell having an intact membrane from which its normal intracellular components, such as enzymes, organelles, or genetic material, have not been released. An intact cell is preferably a viable cell, i.e., a living cell capable of performing its normal metabolic functions. Preferably, the terms refer to any cell that can be transfected with exogenous nucleic acids according to the present invention. Preferably, a cell, when transfected with exogenous nucleic acids and transferred to a recipient, can express said nucleic acids within the recipient. The term “cell” includes bacterial cells; other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include Gram-negative strains, such as Escherichia coli, Proteus, and Pseudomonas, as well as Gram-positive bacterial strains, such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from Trichoderma, Neurospora, and Aspergillus species. Suitable yeast cells include those from the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK cells, etc. However, amphibian cells, insect cells, plant cells, and any other cells used in this field for heterologous protein expression can also be used.Mammalian cells, such as those from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred for adoptive transfer. Cells may originate from many histological types, including primary cells and cell lines, such as immune system cells, particularly antigen-presenting cells like dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells, and other cell types. Antigen-presenting cells are cells that present antigens on their surface in association with the major histocompatibility complex. T cells can recognize this complex using their T cell receptor (TCR).

[0228] The term “transgenic animal” refers to an animal having a genome that includes one or more transgenes, preferably heavy chain and / or light chain transgenes, or a transchromosome (which may or may not be integrated into the animal’s natural genomic DNA), and which is preferably capable of expressing the transgenes. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, so that the mouse produces human anti-CLDN6 antibodies when immunized with cells expressing the CLDN6 antigen and / or CLDN6. The human heavy chain transgene may be integrated into the mouse’s chromosomal DNA, as in transgenic mice such as HCo7 or HCol2 mice, e.g., HuMAb mice, or the human heavy chain transgene may be maintained extrachromosomally, as in transchromosomal (e.g., KM) mice described in International Publication No. 02 / 43478. Such transgenic and transchromosomal mice may produce multiple isotypes (e.g., IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN6 by undergoing VDJ recombination and isotype switching.

[0229] As used herein, “reduce,” “decrease,” or “inhibit” means the ability to cause an overall reduction or overall decrease of a level, for example, a level of cell expression or proliferation, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, most preferably 75% or more.

[0230] Terms such as “increase” or “enhance” preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even greater.

[0231] The following provides a consideration of the underlying mechanisms of the therapeutic effect of antibodies, but this should not be considered a limitation to the present invention in any sense.

[0232] The antibodies described herein preferably interact with components of the immune system, preferably via ADCC or CDC. The antibodies described herein can also be used to directly kill tumor cells by targeting a payload (e.g., a radioisotope, drug, or toxin), or to attack tumors via complementary mechanisms of action, which may include an antitumor immune response that may be impaired due to the cytotoxic side effects of chemotherapeutic agents on T lymphocytes, in synergistic action with traditional chemotherapeutic agents. However, the antibodies described herein can also exert their effects simply by binding to CLDN6 on the cell surface, and thus, for example, by blocking cell proliferation.

[0233] Antibody-dependent cell-mediated cytotoxicity ADCC represents the ability to kill effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells be marked with an antibody.

[0234] ADCC preferably occurs when an antibody binds to an antigen on tumor cells and the antibody's Fc domain engages with an Fc receptor (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and certain cell populations characteristically express defined Fc receptors. ADCC can be considered a mechanism that directly induces varying degrees of immediate tumor destruction, resulting in antigen presentation and induction of a T-cell response against the tumor. Preferably, in vivo induction of ADCC results in a T-cell response and a host-derived antibody response against the tumor.

[0235] Complement-dependent cell injury CDC is another method of cell death that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex involves the C of an antibody molecule, such as an IgG molecule. H This results in the exposure of multiple very close C1q binding sites on the 2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-avidity interaction, which initiates a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of effector cell chemotactic / activators C3a and C5a. Preferably, the complement cascade terminates with the formation of membrane-damaging complexes, which create pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0236] The antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, can also be used.

[0237] The preferred animal system for producing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma production in mice is a very widely established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are well known in this field. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also well known.

[0238] Other preferred animal strains for producing hybridomas that secrete monoclonal antibodies are rat and rabbit strains (see, for example, Spieker-Polet et al., Proc.Natl.Acad.Sci.USA92:9348 (1995); also see Rossi et al., Am.J.Clin.Pathol.124:295 (2005)).

[0239] In yet another preferred embodiment, human monoclonal antibodies can be produced using transgenic or transchromosomal mice that carry a part of the human immune system rather than a mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.” Production of human antibodies in such transgenic mice can be carried out as detailed with respect to CD20 in International Publication No. 2004 / 035607.

[0240] Another method for producing monoclonal antibodies is to directly isolate the gene encoding the antibody from lymphocytes that produce antibodies with defined specificities, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For further details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodydes for cancer therapy ISBN-0-89603-918-8 and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0241] To produce antibodies, mice can be immunized with a carrier-bound peptide derived from an antigen sequence, i.e., the sequence against which antibodies should be directed; an enriched preparation of a recombinantly expressed antigen or its fragment; and / or, as described above, with cells expressing the antigen. Alternatively, mice can be immunized with DNA encoding the antigen or its fragment. If immunization using purified or enriched antigen preparations does not yield antibodies, mice can also be immunized with cells expressing the antigen, such as a cell line, to promote an immune response.

[0242] During the course of the immunization protocol, the immune response can be observed using plasma and serum samples obtained by tail vein or posterior orbital blood collection. Mice with sufficient immunoglobulin titers can be used for fusion. To increase the proportion of hybridomas that secrete specific antibodies, mice can be additionally immunized with antigen-expressing cells via intraperitoneal or intravenous routes three days prior to sacrificial death and splenectomy.

[0243] To produce hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells can be isolated from immunized mice and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can then be screened for antigen-specific antibody production. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies specific to the antigen can be identified by immunofluorescence and FACS analysis using antigen-expressing cells. The antibody-secreting hybridomas can be re-plated and screened again, and if still positive for monoclonal antibodies, they can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro, and antibodies can be produced in tissue culture medium for characterization.

[0244] Antibodies can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods, as is well known in this field (Morrison, S. (1985) Science 229:1202).

[0245] For example, in one embodiment, a gene of interest, such as an antibody gene, can be ligated to an expression vector, such as a eukaryotic expression plasmid, as used in the GS gene expression system disclosed in International Publication No. 87 / 04462, 89 / 01036, and European Patent No. 338841, or other expression systems well known in the art. A purified plasmid containing the cloned antibody gene can be introduced into eukaryotic host cells such as CHO cells, NS / O cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells such as plant-derived cells, fungal or yeast cells. Methods used to introduce these genes may be those described in the art, such as electroporation, lipofectin, lipofectamine, or others. After the introduction of these antibody genes into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomas, which can then be amplified in terms of expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0246] Alternatively, the cloned antibody gene can be expressed in other expression systems, including microorganisms, such as prokaryotic cells like Escherichia coli. Furthermore, antibodies can be produced in transgenic non-human animals such as milk from sheep and rabbits or eggs from chickens, or in transgenic plants; see, for example, Verma, R., et al. (1998) J.Immunol.Meth.216:165-181; Pollock, et al. (1999) J.Immunol.Meth.231:147-157; and Fischer, R., et al. (1999) Biol.Chem.380:825-839.

[0247] Chimera transformation Mouse antibodies are highly immunogenic in humans when repeatedly applied, leading to reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimerizing or humanizing the respective antibodies. A chimeric antibody is one in which different parts originate from different animal species, for example, a variable region derived from a mouse antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by ligating the variable regions of the heavy and light chains of a mouse antibody with the constant regions of the heavy and light chains of a human antibody (as described, for example, in Kraus et al., Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by ligating the human κ light chain constant region with the mouse light chain variable region. Similarly, in a preferred embodiment, a chimeric antibody can be produced by ligating the human λ light chain constant region with the mouse light chain variable region. The preferred heavy chain constant regions for the production of chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for the production of chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0248] Humanization Antibodies primarily interact with target antigens via amino acid residues located within six heavy-chain and light-chain complementarity-determining regions (CDRs). Therefore, the amino acid sequences within the CDRs are more diverse among individual antibodies than the sequences outside the CDRs. Since CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors containing CDR sequences from specific naturally occurring antibodies, transplanted into framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully constructed variable genes formed by V(D)J ligation during B cell maturation. Germline gene sequences also differ from the sequences of high-affinity secondary repertoire antibodies, which are uniformly distributed throughout the variable region.

[0249] The ability of an antibody to bind to an antigen can be determined using standard binding assays (e.g., ELISA, Western blotting, immunofluorescence, and flow cytometry).

[0250] To purify the antibody, the selected hybridoma can be grown in a 2-liter spinner flask for monoclonal antibody purification. Alternatively, the antibody can be produced in a dialysis bioreactor. The supernatant can be filtered and concentrated as needed, then subjected to affinity chromatography with protein G-Sepharose or protein A-Sepharose. To ensure purity, the eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography. The buffer can be replaced with PBS, and the concentration can be measured by OD280 using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80°C.

[0251] Site-specific or multi-site mutagenesis can be used to determine whether the selected monoclonal antibody binds to a unique epitope.

[0252] To determine the antibody isotype, isotyping ELISA can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics). Wells on a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with a monoclonal antibody or purified isotyping control at ambient temperature for 2 hours. The wells can then be reacted with either mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or a mouse IgM-specific peroxidase-binding probe. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at an OD of 405–650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, catalog no. 1493027) can be used as described by the manufacturer.

[0253] Flow cytometry can be used to determine the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing the antigen. Cell lines that express the antigen naturally or after transfection, and negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, APC-labeled or Alexa647-labeled anti-IgG antibodies can be bound to antigen-binding monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument that utilizes the side-light scattering properties to gate single living cells. To distinguish antigen-specific monoclonal antibodies from non-specific binding antibodies in a single measurement, a simultaneous transfection method can be used. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected using different fluorescence channels than antibody-stained cells. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies selectively bind to cells expressing the fluorescent marker, while non-specific antibodies bind to non-transfected cells at a similar rate. A selective assay using fluorescence microscopy can be added to or replaced by the flow cytometry assay. Cells can be stained precisely as described above and examined by fluorescence microscopy.

[0254] Immunofluorescence microscopy can be used to determine the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing antigens. For example, cell lines expressing antigens spontaneously or after transfection, along with negative controls lacking antigen expression, are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen at 25°C for 30 minutes. After washing, the cells can be reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0255] Cell extracts from antigen-expressing cells and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed on an ECL substrate.

[0256] Antibodies can be further tested for reactivity with the antigen by immunohistochemistry using methods well known to those skilled in the art, for example, using paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgery, or from mice carrying xenograft tumors inoculated with cell lines expressing the antigen spontaneously or after transfection. For immunostaining, antibodies reactive to the antigen can be incubated and then incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (DAKO) according to the supplier's instructions.

[0257] Antibodies can be tested for their ability to mediate phagocytosis and death of cells expressing CLDN6. Testing monoclonal antibody activity in vitro provides an initial screening prior to testing in vivo models.

[0258] Antibody-dependent cell-mediated cytotoxicity (ADCC) In short, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells, or other effector cells from healthy donors can be purified by Ficoll Hypaque density gradient centrifugation, followed by lysis of contaminating red blood cells. The washed effector cells are suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum, and CLDN6 expression is performed. 51 Cr-labeled target cells can be mixed with effector cells in various ratios to target cells. Alternatively, target cells may be labeled with a fluorescence-enhancing ligand (BATDA). Highly fluorescent chelates of europium containing the enhancing ligand released from dead cells can be measured by fluoroscopy. Another selective technique can utilize the transfection of target cells with luciferase. The added Lucifer Yellow can then be oxidized only by living cells. Purified anti-CLDN6 IgG can then be added at various concentrations. Unrelated human IgG can be used as a negative control. The assay can be carried out at 37°C for 4–20 hours, depending on the effector cell type used. 51 Samples can be assessed for cell lysis by measuring Cr release or the presence of EuTDA chelates in the culture supernatant. Alternatively, luminescence resulting from the oxidation of Lucifer Yellow can be a measure of viability.

[0259] Anti-CLDN6 monoclonal antibodies can also be tested in various combinations to determine whether cell lysis is enhanced by multiple monoclonal antibodies.

[0260] Complement-dependent cell injury (CDC) Monoclonal anti-CLDN6 antibodies can be tested for their ability to mediate CDC using various known techniques. For example, serum for complement can be obtained from blood by methods known to those skilled in the art. Various methods can be used to measure the CDC activity of mAbs. For example, 51 Cr release can be measured, or increased membrane permeability can be evaluated using a propidium iodide (PI) exclusion assay. Briefly, target cells are washed and 5 × 10⁻⁶ 5 The mAbs / ml can be incubated with various concentrations of mAbs at room temperature or 37°C for 10-30 minutes. Then, serum or plasma can be added to a final concentration of 20% (v / v), and the cells can be incubated at 37°C for 20-30 minutes. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be immediately analyzed by flow cytometry using a FACS array.

[0261] In a selective assay, the induction of CDC can be measured in adherent cells. In one embodiment of this assay, cells are prepared 24 hours before the assay by measuring 3 × 10⁶ cells. 4 Inoculate tissue culture cells into flat-bottom microtiter plates at a density of / well. The following day, remove the growth medium and incubate the cells in triplicates with the antibody. Incubate control cells with growth medium or growth medium containing 0.2% saponin, respectively, for background lysis and maximal lysis measurements. After incubation at room temperature for 20 minutes, take the supernatant and add 20% (v / v) human plasma or serum in DMEM (preheated to 37°C) to the cells, then incubate at 37°C for a further 20 minutes. Add all cells from each sample to propidium iodide solution (10 μg / ml). Next, replace the supernatant with PBS containing 2.5 μg / ml ethidium bromide and measure the fluorescence emission at 600 nm after excitation at 520 nm using a Tecan Safire. Calculate the percentage of specific lysis as follows: Specific lysis % = (Sample fluorescence - Background fluorescence) / (Maximal lysis fluorescence - Background fluorescence) × 100.

[0262] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies To test the ability to initiate apoptosis, monoclonal anti-CLDN6 antibodies can be incubated with, for example, CLDN6-positive tumor cells or CLDN6-transfected tumor cells at 37°C for approximately 20 hours. Cells can be harvested, washed in Annexin V-conjugated buffer (BD biosciences), and incubated with Annexin V (BD biosciences) conjugated with FITC or APC for 15 minutes in the dark. All cells from each sample can be added to PI solution (10 μg / ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, catalog no. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) integration during DNA synthesis in proliferating cells in a microplate. The integrated BrdU is detected using a europium-labeled monoclonal antibody. To enable antibody detection, cells are fixed using a fix solution to denature their DNA. Unbound antibodies are washed away, and a DELFIA inducer is added to dissociate europium ions from the labeled antibody into the solution. In the solution, these ions form highly fluorescent chelates with the components of the DELFIA inducer. The fluorescence measured using time-resolved fluorescence spectroscopy in the detection is proportional to the DNA synthesis in the cells of each well.

[0263] Preclinical trials The binding agents described herein can also be tested in vivo models (for example, in immunodeficient mice carrying xenograft tumors inoculated with CLDN6-expressing cell lines) to measure their effects in controlling the proliferation of tumor cells expressing CLDN6.

[0264] In vivo studies after xenotransplantation of tumor cells expressing CLDN6 into immunocompromised mice or other animals can be performed using the antibodies described herein. Antibodies can be administered to tumor-free mice, followed by injection of tumor cells to measure the antibody's effect in preventing tumor formation or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice to measure the therapeutic effect of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibody application can be combined with the application of other substances, such as cell proliferation inhibitors, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies, to measure synergistic effects and potential toxicity. To analyze antibody-mediated toxic side effects, animals can be inoculated with the antibody or a control reagent and thoroughly examined for symptoms that may be associated with CLDN6 antibody treatment. Possible side effects of in vivo application of CLDN6 antibodies include toxicity in CLDN6-expressing tissues, particularly the placenta. Antibodies that recognize CLDN6 in humans and other species, such as mice, are particularly useful for predicting potential side effects mediated by the application of monoclonal CLDN6 antibodies in humans.

[0265] The mapping of antibody-recognized epitopes can be performed as detailed in "Epitope Mapping Protocols (Methods in Molecular Biology)" ISBN-089603-375-9 by Glenn E. Morris and "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 by Olwyn MrWestwood and Frank C. Hay.

[0266] The compounds and active ingredients described herein may be administered in the form of any suitable pharmaceutical composition.

[0267] The pharmaceutical composition of the present invention is preferably sterile and contains an effective amount of the antibody and optionally further active substances discussed herein to produce a desired reaction or effect.

[0268] Pharmaceutical compositions are usually provided in unit dose form and can be prepared by methods known to the public. Pharmaceutical compositions may be in the form of solutions or suspensions, for example.

[0269] The pharmaceutical composition may contain salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" means that the substance is non-toxic and does not interact with the action of the active ingredient of the pharmaceutical composition.

[0270] Medicinally unacceptable salts can be used to prepare medicinally acceptable salts and are included in the present invention. Such medicinally acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Medicinally acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0271] Suitable buffering agents for use in pharmaceutical compositions include acetic acid in salts, citric acid in salts, boric acid in salts, and phosphoric acid in salts.

[0272] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0273] Injectable formulations may contain pharmaceutically acceptable excipients such as Ringer's lactate solution.

[0274] The term “carrier” refers to a natural or synthetic organic or inorganic component that is combined with an active ingredient to facilitate, enhance, or enable its application. According to the present invention, the term “carrier” also includes one or more suitable solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to a patient.

[0275] Suitable carriers for parenteral administration include, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0276] As used herein, the term “excipient” is intended to refer to all substances that may be present in a pharmaceutical composition and are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings, or colorants.

[0277] The active substances and compositions described herein may be administered by any conventional route, for example, by parenteral administration including injection or infusion. Administration is preferably parenteral, for example, by intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular routes.

[0278] Compositions suitable for parenteral administration typically contain sterile aqueous or non-aqueous preparations of the active compound, preferably isotonic with the recipient's blood. Examples of suitable carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, a fixative oil, usually sterile, is used as the medium for the solution or suspension.

[0279] The active substances and compositions described herein are administered in effective doses. “Effective dose” refers to the amount that, alone or in combination with further doses, achieves the desired response or effect. In the case of treating a particular disease or condition, the desired response preferably relates to inhibiting disease progression. This includes slowing disease progression and, in particular, preventing or reversing disease progression. The desired response in the treatment of a disease or condition may also be delaying or preventing the onset of the disease or condition. In particular, the term “effective dose” refers to an amount of treatment sufficient to result in the prevention of the onset, recurrence, or development of cancer and one or more symptoms of cancer; to reduce the severity or duration of cancer; to improve one or more symptoms of cancer; to prevent cancer progression; to induce cancer regression; and / or to prevent cancer metastasis. In one embodiment of the present invention, the therapeutic dose is effective in achieving stabilization, reduction, or elimination of a cancer stem cell population and / or eradication, removal, or suppression of primary cancer, metastatic cancer, and / or recurrent cancer.

[0280] The effective amount of the active substance or composition described herein depends on the condition being treated, the severity of the disease, the patient's age, physiological state, size and weight, and other individual patient parameters, the duration of treatment, the type of accompanying treatment (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the active substance described herein may depend on these various parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0281] The active substances and compositions described herein may be administered to patients to treat or prevent cancerous diseases, such as those described herein, characterized by the presence of cancer stem cells expressing CLDN6.

[0282] The active ingredients and compositions provided herein may be used alone or in combination with conventional treatment regimens such as surgery, radiation therapy, chemotherapy and / or bone marrow transplantation (autologous, allogeneic, allogeneic, or unrelated).

[0283] In cancer treatment, combination strategies are particularly desirable because the combined effects of two, three, four, or even more anticancer drugs / therapies often produce significantly stronger synergistic effects than monotherapy approaches. Therefore, in another embodiment of the present invention, cancer treatment can be effectively combined with various other agents. These include, for example, combinations with conventional oncology, multiepitope strategies, additive immunotherapy, and therapeutic approaches targeting angiogenesis or apoptosis (see, for example, Andersen et al. 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57(11):1735-1743). Sequential administration of different agents can inhibit cancer cell proliferation at different checkpoints, while other agents can inhibit, for example, neovascularization, malignant cell survival, or metastasis, potentially transforming cancer into a chronic disease.

[0284] The following list provides some non-limiting examples of anticancer agents and therapies that can be used in combination with the present invention:

[0285] 1.Chemotherapy Chemotherapy is the standard treatment for many types of cancer. The most common chemotherapeutic agents work by killing rapidly dividing cells, which is one of the main characteristics of cancer cells. Therefore, the combination of conventional chemotherapeutic agents such as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents that affect either cell division or DNA synthesis may significantly improve the therapeutic effect of the present invention by making tumor cells more susceptible to immune-mediated death by eliminating suppressor cells and restarting the immune system, or by additional activation of immune system cells. The synergistic anticancer effects of chemotherapy drugs and immunotherapy drugs based on vaccination have been demonstrated in many studies (see, for example, Quoix et al. 2011: Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial. Lancet Oncol. 12(12):1125-33; also see Liseth et al. 2010: Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: the hematological experience. J Biomed Biotechnol. 2010:6920979; and also see Hirooka et al 2009: A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38(3):e69-74). There are hundreds of chemotherapy drugs available that are basically suitable for combination therapy.Some (non-limiting) examples of chemotherapy drugs that can be used in combination with the present invention include carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), crizotinib (Xalkori), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), and gemcitabine (Gem These include zar, imatinib mesylate (Gleevec), irinotecan (Camptosar), liposomal-encapsulated doxorubicin (Doxil), methotrexate (Folex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), trabectedin (Yondelis), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0286] 2.Surgery Cancer surgery—the procedure to remove tumors—remains the foundation of cancer treatment. Surgery can be combined with other cancer treatments to remove any remaining tumor cells. Combining surgical methods with subsequent immunotherapy is a promising approach that has been demonstrated repeatedly.

[0287] 3. Radiation Radiation therapy remains a crucial component of cancer treatment, with approximately 50% of all cancer patients receiving it during the course of their disease. The primary goal of radiation therapy is to incapacitate cancer cells by causing them to lose their ability to proliferate (divide). The types of radiation used to treat cancer are photon radiation (X-rays and gamma rays) and particle radiation (electron beams, proton beams, and neutron beams). There are two methods for delivering radiation to the site of cancer. External radiation is delivered from outside the body by directing high-energy beams (photons, protons, or particle radiation) to the tumor site. Internal radiation, or brachytherapy, is delivered directly to the tumor site from inside the body by a sealed source in a catheter or seed. Radiotherapy techniques that can be applied in combination with the present invention include, for example, fractionation therapy (radiotherapy delivered in fractions, e.g., daily fractional doses of 1.5-3 Gy given over several weeks), three-dimensional conformal radiotherapy (3DCRT; delivering radiation to a macroscopic tumor volume), intensity-modulated radiotherapy (IMRT; computer-controlled intensity modulation of multiple radiation beams), image-guided radiotherapy (IGRT; a technique including pre-radiotherapy imaging that takes corrections into account), and stereotactic radiotherapy (SRBT, delivering very high individual doses to only a few treatment fractions). For a review of radiotherapy, see Baskar et al. 2012: Cancer and radiation therapy: current advances and future directions. Int. J Med Sci. 9(3): 193-199.

[0288] 4. Antibodies Antibodies (preferably monoclonal antibodies) achieve their therapeutic effects against cancer cells through various mechanisms. Antibodies can directly induce apoptosis or programmed cell death. They can effectively halt tumor cell proliferation by blocking components of signaling pathways, such as growth factor receptors. In cells expressing monoclonal antibodies, they can lead to the formation of anti-idiotype antibodies. Indirect effects include recruiting cytotoxic cells such as monocytes and macrophages. This type of antibody-mediated cell death is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies can also bind to complement, resulting in direct cytotoxicity known as complement-dependent cell-mediated cytotoxicity (CDC). Combining surgical methods with immunotherapy drugs or methods is a successful approach, as demonstrated, for example, in Gadri et al. 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 32(4):333-40. The following list provides some non-limiting examples of anticancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present invention: avagovomab (CA-125), absiximab (CD41), adecatumumab (EpCAM), aftuzumab (CD20), aracizumab pegol (VEGFR2), artumomab pentetate (CEA), amatsuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), alsitumomab (CEA), bavituximab (phosphatidylserine), vectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), vibatuzumab meltansine (CD44) v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab meltansine (mucin CanAg), cantuzumab labtansine (MUC1), capromab pendetide (prostate cancer cells), carrumab (CNTO888), catumakisomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab vogatox (EpCAM), thixutumumab (IGF-1 receptor), claudiximab (claudin), cribatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), darotuzumab (insulin Phosphate-like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), droditumab (DR5), eclomeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enabatuzumab (PDL192), encituximab (NPC-1C), epratuzumab (CD22), ertzumaxomab (HER2 / neu, CD3), etalacizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficratuzumab (SCH900105), Figitumumab (IGF-1 receptor), Frambotumab (glycoprotein 75), Fresolimmubab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab / Ozogamicin (CD33), Gevokizumab (IL-1β), Dilentuximab (carbonic anhydrase 9 (CA-IX)), Grembatumumab / Vedotin ( GPNMB), ibritumomab / tiuxetan (CD20), iclucumab (VEGFR-1), igovomab (CA-125), indatuximab / labtansine (SDC1), intetumumab (CD51), inotuzumab / ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), rabetsumab (CEA), lexatumumab Br (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorbotuzumab meltansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matsuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitsumomab (GD3 ganglio) SID), mogamulizumab (CCR4), moxetumomab / pasdotox (CD22), nacolomab / tafenatox (C242 antigen), naptumomab / estafenatox (5T4), nalnatumumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumumab (PDGF-R)α), Onartuzumab (human scattering factor receptor kinase), Oportuzumab monatox (EpCAM), Olegobomab (CA-125), Oxerumab (OX-40), Panitumumab (EGFR), Patritumumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pintumomab (adenocarcinoma antigen), Pritumumab (vimentin), Lacothomomab (N-glycolylneuraminic acid), Radretumumab (fibronectin extradomain B), Rafibirumab (rabies virus glycoprotein) (Primary), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Lobatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrotuzumab (FAP), Siltuximab (IL-6), Tabalumab (BAFF), Takatuzumab Tetraxetan (α-fetoprotein), Tapritumomab Paptox (CD19), Tenatumomab (Tenascin C), Teprotumumab (CD221), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotzumab / selmoleukin (EpCAM), ubrituximab (MS4A1), urerumab (4-1BB), borosiximab (integrin α5β1), botumumab (tumor antigen CTAA16.88), saltumumab (EGFR), zanorimumab (CD4).

[0289] 5. Cytokines, chemokines, costimulatory molecules, and fusion proteins The combined use of cytokines, chemokines, costimulatory molecules, and / or fusion proteins thereof with pharmaceutical compositions encoding the antigens of the present invention to induce beneficial immunomodulatory or tumor inhibitory effects is another embodiment of the present invention. Various chemokines having C, CC, CXC, and CX3C structures may be used to increase the infiltration of immune cells into tumors and to promote the migration of antigen-presenting cells to lymph nodes that drain tumors. Some of the most promising chemokines are, for example, CCR7 and its ligands CCL19 and CCL21, as well as CCL2, CCL3, CCL5, and CCL16. Other examples are CXCR4, CXCR7, and CXCL12. Furthermore, costimulatory or modulating molecules such as B7 ligands (B7.1 and B7.2) may be useful. Other cytokines, such as interleukins (e.g., IL-1 to IL-17), interferons (e.g., IFNα1 to IFNα8, IFNα10, IFNα13, IFNα14, IFNα16, IFNα17, IFNα21, IFNβ1, IFNW, IFNE1, and IFNK), hematopoietic factors, TGF (e.g., TGF-α, TGF-β, and other members of the TGF family), and finally members of the tumor necrosis factor family of receptors and their ligands, as well as 4-1BB, 4-1BB-L, CD137, CD137L, CTLA-4GITR, GITRL, Fas, Fas-L, T Other stimulating molecules, including but not limited to NFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LTβR, RANK, EDAR1, XEDAR, Fn114, Troy / Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT, and CD95 (Fas / APO-1), glucocorticoid-inducible TNFR-related proteins, TNF receptor-associated apoptosis-mediated protein (TRAMP), and cell death receptor 6 (DR6), are also useful. CD40 / CD40L and OX40 / OX40L, in particular, are important targets for combination immunotherapy due to their direct effects on T cell survival and proliferation.For a review article, see Lechner et al. 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3(11), 1317-1340.

[0290] 6. Bacterial treatment Researchers have used anaerobic bacteria such as Clostridium novyi to deplete the inside of hypoxic tumors. These should then die upon contact with the oxygenated side of the tumor, meaning they are harmless to the rest of the body. Another strategy is to use anaerobic bacteria transformed with an enzyme that can convert non-toxic prodrugs into toxic drugs. Along with tumor necrosis and bacterial growth in hypoxic areas, the enzyme is expressed only within the tumor. Thus, a systemically applied prodrug is metabolized into a toxic drug only within the tumor. This has been demonstrated to be effective with the non-pathogenic anaerobic bacterium Clostridium sporogenes.

[0291] 7. Kinase inhibitors Another large group of potential targets for complementary cancer therapies are kinase inhibitors, because cancer cell proliferation and survival are closely linked to the dysregulation of kinase activity. A wide range of inhibitors have been used to restore normal kinase activity and thus reduce tumor growth. The group of target kinases includes receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-α, PDGFR-β, c-Kit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK; cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, JAK-2; serine / threonine kinases, e.g., ATM, Aurora A & B, CDK, mTOR, PKCi, PLK, b-Raf, S6K, STK11 / LKB1; and lipid kinases, e.g., PI3K, SK1. Examples of small molecule kinase inhibitors include PHA-739358, nilotinib, dasatinib, and PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), batalanib (PTK787 / ZK222584), sutent (SU11248), sorafenib (BAY 43-9006), and leflunomide (SU101). For further information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0292] 8. Toll-like receptors Members of the Toll-like receptor (TLR) family are a crucial link between innate and adaptive immunity, and the action of many adjuvants depends on TLR activation. Many established vaccines against cancer incorporate TLR ligands to enhance the vaccine response. In addition to TLR2, TLR3, and TLR4, TLR7 and TLR8 in particular have been investigated for cancer therapy in passive immunotherapy approaches. Closely related TLR7 and TLR8 contribute to the antitumor response by influencing immune cells, tumor cells, and the tumor microenvironment and can be activated by nucleoside analog structures. All TLRs have been used as monoimmunotherapies or as cancer vaccine adjuvants and may be used synergistically with the formulations and methods of the present invention. For further information, see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

[0293] 9. Angiogenesis inhibitors In addition to therapies targeting immunomodulatory receptors affected by tumor-mediated escape mechanisms and immunosuppression, there are therapies that target the tumor environment. Angiogenesis inhibitors prevent the widespread growth of blood vessels (angiogenesis) necessary for tumor survival. For example, angiogenesis, which is promoted by tumor cells to meet their increased nutrient and oxygen demands, can be blocked by targeting various molecules. Non-limiting examples of angiogenesis-mediated molecules or angiogenesis inhibitors that can be combined with the present invention include soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors neuropilin 1 and neuropilin 2) 1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatins and related molecules, endostatins, vasostatins, calreticulin, platelet factor 4, TIMP and CDAI, Meth-1 and Meth-2, IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18, prothrombin (cringle domain 2), These include antithrombin III fragments, prolactin, VEGI, SPARC, osteopontin, Maspin, canstatin, proliferin-related proteins, lestin, and drugs such as bevacizumab, itraconazole, carboxamide triazole, TNP-470, CM101, IFN-α, platelet factor IV, suramin, SU5416, thrombospondin, VEGFR antagonists, angiogenesis-inhibiting steroids + heparin, cartilage-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin α Vβ3 inhibitors, linamide, and tascinimod. For a review, see Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9): 976-81.

[0294] 10. Small molecule targeted therapy drugs Small molecule targeted therapies are generally inhibitors of mutated, overexpressed, or otherwise important enzyme domains on proteins within cancer cells. Notable and non-limiting examples include the tyrosine kinase inhibitors imatinib (Gleevec / Glivec) and gefitinib (Iressa). The use of small molecules targeting certain kinases in combination with vaccines for cancer therapy, such as sunitinib malate and / or sorafenib tosylate, is also described in a previous patent application, U.S. Patent Application No. 2009004213.

[0295] 11. Virus-based vaccines Many virus-based cancer vaccines are available or under development that can be used in combination therapy approaches with the formulations of the present invention. One advantage of using such viral vectors is their endogenous ability to initiate an immune response, with an inflammatory response occurring as a result of viral infection that creates the danger signals necessary for immune activation. Ideally, viral vectors should be safe and should not introduce an anti-vector immune response that would enhance the antitumor-specific response. Recombinant viruses, such as vaccinia virus, herpes simplex virus, adenovirus, adeno-associated virus, retrovirus, and avipoxvirus, have been used in animal tumor models, and based on their promising results, human clinical trials have been initiated. Of particular importance are virus-like particles (VLPs), which are small particles containing specific proteins derived from the viral coat. Virus-like particles do not contain any genetic material from the virus and cannot cause infection, but can be constructed to present tumor antigens on their coat. VLPs can originate from a variety of viruses, including hepatitis B virus or other viral families such as Parvoviridae (e.g., adeno-associated viruses), Retroviridae (e.g., HIV), and Flaviviridae (e.g., hepatitis C virus).For a review, see Sorensen and Thompsen 2007: Virus-based immunotherapy of cancer: what do we know and where are we going? APMIS 115(11):1177-93; for virus-like particles against cancer, see Buonaguro et al. 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert Rev Vaccines 10(11):1569-83; and Guillen et al. 2010: Virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies. Procedia in Vaccinology 2(2), 128-133.

[0296] 12. Multi-epitope strategy The use of multiepitopes shows promising results for vaccination. Rapid sequencing technology combined with intelligent algorithmic systems enables the development of tumor mutanomes and may provide multiepitopes for personalized vaccines that can be used in conjunction with the present invention. For further information, see 2007:Vaccination of metastatic colorectal cancer patients with matured dendritic cells loaded with multiple major histocompatibility complex class I peptides. J Immunother 30:762-772; and also Castle et al. 2012:Exploiting the mutanome for tumor vaccination. Cancer Res 72(5):1081-91.

[0297] 13. Adoptive T cell transfer For example, the combination of tumor antigen vaccination and T cell transfer is described in Rapoport et al. 2011: Combination immunotherapy using adoptive T-cell transfer and tumor antigen vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood 117(3):788-97.

[0298] 14. Peptide-based targeted therapy Peptides can bind to cell surface receptors or to the affected extracellular matrix surrounding tumors. Radionuclides linked to these peptides (e.g., RGD) ultimately kill cancer cells if the nuclides decay in the vicinity of the cells. Oligomers or multimers of these binding motifs are particularly interesting because they can lead to enhanced tumor specificity and avidity. For a non-limiting example, see Yamada 2011: Peptide-based cancer vaccine therapy for prostate cancer, bladder cancer, and malignant glioma. Nihon Rinsho 69(9):1657-61.

[0299] 15. Other therapies Numerous other cancer therapies exist that can be combined with the present invention to produce synergistic effects. Non-limiting examples include apoptosis-targeted therapies, hyperthermia, hormone therapy, telomerase therapy, insulin enhancement therapy, gene therapy, and photodynamic therapy.

[0300] Various methods known in this field can be used to detect cells expressing CLDN6 and / or measure the quantity of such cells.

[0301] For example, immunoassays can be used to detect CLDN6 protein expression in or on the cell surface. According to the present invention, immunoassays include, but are not limited to, Western blotting, immunohistochemistry, radioimmunoassays, ELISA (solid-phase enzyme immunoassay), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, immunofluorescence assays, protein A immunoassays, flow cytometry, or FACS analysis.

[0302] In one embodiment, cells are bound to one or more labeled antibodies having the ability to bind to CLDN6 before detection and / or measurement of quantity.

[0303] Alternatively, to detect cells expressing CLDN6 and / or measure the quantity of such cells, the expression of CLDN6 mRNA may be detected or the quantity of CLDN6 mRNA may be measured.

[0304] In certain embodiments of the present invention, the sample obtained from a patient to detect and / or measure the quantity of cells expressing CLDN6 is a biological fluid, including but not limited to blood, bone marrow, serum, urine, or interstitial fluid. In other embodiments, the sample from the patient is a tissue sample (e.g., a biopsy from a subject having or suspected of having cancerous tissue). Most preferably, the sample is a tumor biopsy.

[0305] According to the method of the present invention, the sample may be a biological sample subjected to one or more pretreatment steps before detection of cells expressing CLDN6 and / or measurement of the quantity of cells. In certain embodiments, the biological fluid is pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps. In other embodiments, the tissue sample is pretreated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization, or homogenization, and then pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or a combination of such pretreatment steps.

[0306] The amount of cancer stem cells in a sample can be expressed, for example, as a percentage of the total cells or total cancer cells in the sample, or quantified in terms of area (e.g., cells per field of view), volume (e.g., cells per ml), or weight (e.g., cells per ml).

[0307] The amount of cancer stem cells in a test sample can be compared to the amount of cancer stem cells in a reference sample(s). In one embodiment, the reference sample is a sample obtained from a subject receiving therapy at an earlier point in time (e.g., before therapy as a baseline reference sample, or at an earlier point in time during therapy). In this embodiment, the therapy preferably results in a decrease in the amount of cancer stem cells in the test sample compared to the reference sample. In another embodiment, the reference sample is obtained from a healthy subject without detectable cancer or from a patient in remission for the same type of cancer. In this embodiment, the therapy preferably results in a test sample having an amount of cancer stem cells equal to or less than the amount of cancer stem cells detected in the reference sample. In certain embodiments, a stabilization or decrease in the amount of cancer stem cells compared to an earlier (previously detected) amount of cancer stem cells measured for the subject indicates an improved prognosis or a beneficial response to the therapy, while an increase compared to an earlier amount of cancer stem cells indicates the same prognosis or a worsening prognosis and / or poor response to the therapy.

[0308] In some embodiments, a combination of a cell surface marker, such as CLDN6, and other markers typical of cancer stem cells is used to measure the amount of cancer stem cells in a sample.

[0309] The present invention also provides a kit comprising one or more containers filled with reagents for detecting, measuring, or observing cells expressing CLDN6. In one embodiment, the kit optionally includes instructions for the use of the reagents, particularly for the use of the reagents in the method of the present invention, for quantifying cancer stem cells or observing the effect of cancer therapy by detecting and / or measuring cells expressing CLDN6. In one embodiment, the kit comprises an activator that specifically binds to the CLDN6 protein or CLDN6 mRNA. In some embodiments, the activator is an antibody or antibody fragment. In other embodiments, the activator is a nucleic acid. For nucleic acid detection, the kit generally comprises (but is not limited to) a probe specific to CLDN6 mRNA. For quantitative PCR, the kit generally comprises pre-selected primers specific to the CLDN6 nucleic acid sequence. Quantitative PCR kits may also comprise an enzyme suitable for amplifying the nucleic acid (e.g., a polymerase such as Taq), and deoxyribonucleotides and buffers required for the reaction mixture for amplification. Quantitative PCR kits may also comprise a probe specific to the CLDN6 nucleic acid sequence. In some embodiments, the quantitative PCR kit also includes components suitable for reverse transcribing RNA, such as an enzyme (e.g., reverse transcriptase) and primers for reverse transcription, along with the deoxynucleotides and buffers necessary for the reverse transcription reaction.

[0310] In certain embodiments, the active substance is labeled for detection. Furthermore, the kit may include instructions for performing the assay and methods for interpreting and analyzing data resulting from the performance of the assay.

[0311] Based on the results obtained (i.e., whether cancer stem cells are present or whether the amount of cancer stem cells has stabilized or decreased), the physician may choose to select a specific cancer therapy, such as a cancer therapy targeting cancer stem cells, or to continue therapy. Alternatively, based on the results that cancer stem cells are absent or the amount of cancer stem cells has increased, the physician may choose to administer a cancer therapy that does not target cancer stem cells, or to continue, change, or discontinue therapy.

[0312] In certain embodiments of the present invention, if the reduction of the cancer stem cell population in a sample obtained from a patient undergoing cancer therapy is determined to be insufficient when compared to a patient-derived sample taken earlier from the same patient, the physician has numerous options for adjusting the therapy.

[0313] For example, the physician may then increase the dose, frequency, duration, or any combination thereof of the cancer therapy. In certain embodiments, after a decision has been made, the patient may be given additional cancer therapy in lieu of or in combination with the initial therapy.

[0314] In other specific embodiments, if a reduction in the cancer stem cell population in a sample obtained from a patient undergoing cancer therapy is determined to be acceptable when compared to a patient-derived sample taken earlier from the same patient, the physician may choose not to adjust the cancer therapy. For example, the physician may choose not to increase the dose, frequency, duration, or any combination thereof of the cancer therapy. Furthermore, the physician may choose to add or combine additional therapies.

[0315] The present invention will be further illustrated by the following embodiments, but these should not be construed as limiting the scope of the invention. [Examples]

[0316] (Example 1) CLDN6 is expressed on the surface of human induced pluripotent stem cells. To analyze whether CLDN6 is expressed in human induced pluripotent stem cells (iPSCs), CLDN6 transcript expression was treated at several time points in neonatal HFF (human precipitous fibroblasts, System Bioscience) with a reprogrammed cocktail (unmodified OSKMNL + EBK + miR mix; OSKMNL = in vitro transcription (IVT) RNA of transcription factors OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28; EBK = IVT-RNA of IFN escape proteins E3, K3, and B18R, and a miRNA mix consisting of miR-302a / b / c / d and 367; following the protocol described in International Publication No. PCT / EP2012 / 04673) or mock-transfected HFF (RNA-free control). The ABI PRISM 7300 sequence detection system and software (Applied Biosystems and QuantiTect SYBR green) were used. Quantitative real-time RT-PCR (qRT-PCR) was performed using Kit (Qiagen). Cells were cultured in Nutristom serum-free medium (Stemgent, Cambridge (MA)) supplemented with 10 ng / ml bFGF and 0.5 μM thiazovibin. The reprogrammed cocktail was transfected with Lipofectamine RNAiMAX (Life Technologies) on days 1, 2, 3, 4, 8, 9, 10, and 11 of the experiment. As a control, cells were treated with Lipofectamine RNAiMAX alone (RNA-free control). A clear upregulation of CLDN6 of nearly 6000-fold was detected on day 19 of treatment compared to untreated HFF cells, and an upregulation of approximately 2000-fold was observed on day 12 of treatment (Figure 1). Therefore, CLDN6 is expressed in human induced pluripotent stem cells (iPSCs).

[0317] Flow cytometry was used to investigate whether CLDN6 is also expressed on the surface of iPSCs. Since iPSCs proliferate on HFF-supported cells, this analysis was combined with staining for the widely accepted stem cell marker, SSEA-4, to ensure specific detection of iPSCs. For this purpose, HFF cells treated with a reprogrammed cocktail or mock control (without RNA) were collected on days 5, 12, and 19 of treatment, stained with 1 μg / ml CLDN6-specific IMAB027-AF647 and 2 μl SSEA-4 antibody at 4°C for 30 minutes, and surface expression was analyzed by flow cytometry. Viability Dye 7-AAD was also included in the staining protocol to exclude dead cells from the analysis. The experiment was performed in duplicate, recording 50,000 events from each sample using a BD Canto II flow cytometer. Analysis of the recorded cells was performed using FlowJo software, and representative dot plots are shown (Figure 2).

[0318] On day 5, CLDN6 is undetectable on surface HFFs, whether treated with the reprogramming cocktail or not. Surprisingly, SSEA-4 expression is observed in 15% of HFFs, regardless of whether they were treated with the reprogramming cocktail. This can be explained by the fact that the HFFs used are neonatal fibroblasts, and these cells are capable of maintaining a certain positivity rate for SSEA-4. On day 12 of treatment, approximately 63% of the treated HFFs are positive for SSEA-4, and approximately 15% show a CLDN6-SSEA-4 bipositive fraction. On day 19 of treatment, 15% of the treated HFFs are positive for both CLDN6 and SSEA-4, showing distinct subpopulations. The CLDN6-SSEA-4 positive subpopulation represents only iPSCs, while the CLDN6-SSEA-4 negative subpopulation is considered to be either HFF-supporting cells or not reprogrammed cells, and it is presumed that the SSEA-4 single-positive cells represent cells at the start of reprogramming.

[0319] The inventors observed that 15% of HFF cells were positive for SSEA-4 but not for CLDN6, suggesting that CLDN6 is a more specific marker for human iPSCs than SSEA-4. While SSEA-4 is expressed in neonatal HFF cells, CLDN6 appears to be specifically expressed only in fully reprogrammed HFF cells, which are part of the iPSC fraction.

[0320] Therefore, CLDN6 is specifically expressed on the surface of human iPSCs.

[0321] (Example 2) CLDN6 is important for colony formation of ovarian cancer cells. A colony formation assay is an effective assay for analyzing the CSC-like properties of tumor cells. This assay allows for easy examination of the self-renewal capacity and tumorigenic potential of single tumor cells. To analyze whether CLDN6 plays a role in tumorigenesis, we selected, on the one hand, the ovarian tumor cell line COV318, which exhibits only a subpopulation of CLDN6-positive cells, and on the other hand, the uniform CLDN6-expressing cell line PA-1 (clones PA-1 50, PA-1 54), which carries a stable lentiviral small hairpin RNA (shRNA)-mediated CLDN6 knockdown; see Figure 3.

[0322] Cells were stained for CLDN6 with 1 μg / ml IMAB027-AF647 at 4°C for 30 minutes, and then sorted by FACS (fluorescence-activated cell sorting) using a BD FACSAria cell sorter for CLDN6 expression. 500 (PA-1 50, PA-1 54) or 700 (COV318) cells from CLDN6-positive and CLDN6-negative subpopulations were directly sorted into the wells of a 6-well plate and grown for up to 14 days until sufficient colonies were formed. The medium was changed twice a week. Colonies were stained with 0.5% crystal violet in 10% ethanol for 20 minutes, fixed, washed three times with distilled water, and air-dried. Photographs were taken, and colonies were manually counted. At least 50 cells were considered a colony. Figure 4 shows representative colony formation assays of COV318 and CLDN6 knockdown cell lines PA-1 50 and 54. Interestingly, in both cell lines, CLDN6-negative cells showed significantly lower colony formation compared to CLDN6-positive cells. From these results, we conclude that CLDN6 plays a crucial role in the colony-forming ability, which is an essential characteristic of cancer stem cells.

[0323] (Example 3) CLDN6 is co-expressed with CSC markers CD24, CD90, and CD44 in ovarian cancer cell lines. The use of specific surface marker expression profiles is a common strategy for the identification and isolation of cytoplasmic sperm cells (CSCs) from solid tumors and cell lines. Surface markers used in the literature for the isolation of CSCs from ovarian cancer include CD44, CD24, CD90, CD34, CD117, and CD133. To analyze whether a CSC subpopulation can be identified in ovarian cancer cell lines containing a small subpopulation of CLDN6-positive cells, we constructed a FACS panel containing antibodies against these surface markers (Table 1). Furthermore, to examine the proportion of co-localization between the widely established CSC markers and CLDN6, we also included an antibody for the detection of CLDN6 in the panel, thus determining the potential of CLDN6 to act as a marker for CSCs. For this purpose, 1E6 cells of cell line COV318 were stained with an indicator of antibody (see Table 1) at 4°C for 30 minutes, and the cells were then analyzed for their surface marker expression profiles by flow cytometry. The inventors also included Viability Dye eFluor® 506 in their staining protocol to exclude dead cells from their analysis. The experiment was performed in three replicates, recording 50,000 events from each sample using a BD Canto II flow cytometer. The recorded cells were analyzed using FlowJo software.

[0324] [Table 1] Table 1: CSC FACS Panel. This table shows the FACS panels used for the analysis of CSC markers and CLDN6 expression in ovarian cancer cell lines. The amount of antibody and conjugating fluorescent dye used for the corresponding markers are listed.

[0325] FACS analysis revealed that COV318 cells express subpopulations of the CSC markers CD44, CD90, and CD24, and that CLDN6 colocalizes at least partially with all three markers (Figure 5A). The inventors then used various gating strategies to calculate the percentage of colocalization of all four markers. First, they calculated the percentage of CD44, CD24, CD90, and CLDN6-positive cells in the entire viable cell population. They found that 0.18% of viable cells were positive for all four markers. Next, they calculated the percentage of CD44, CD24, and CD90-positive cells in the viable cell population that could represent the CSC fraction. Compared to the entire viable cell population, 0.23% of cells were positive for all three markers, while compared to the CLDN6-positive subpopulation, 20.1% of the cell fraction was triple-positive, showing an 87-fold increase in the concentrations of the three markers in the CLDN6-positive subfraction. In the final stage, we calculated the percentage of CLDN6-positive cells in the entire viable cell population, and the percentage of CLDN6-positive cells in the CD44 / CD24 / CD90-positive subpopulation. We observed a 74-fold increase in the concentration of CLDN6-expressing cells, from 0.91% in the entire cell population to 66.87% in the CSC fraction (Figure 5B).

[0326] Considering these findings together, these data indicate that CLDN6 accumulates in the CSC fraction, and conversely, CSC markers are enriched in the CLDN6-positive subpopulation. These findings suggest that CLDN6 is a marker for CSC.

[0327] (Example 4) Enrichment of CLDN6-expressing cells leads to accumulation of established CSC markers CD44, CD24, and CD90. CSC fractions isolated from cell lines and tumors have often been shown to be enriched with CSC markers such as CD44 and CD24. To analyze the potential of CLDN6 as a novel CSC marker, we investigated whether cell isolation of CLDN6-positive fractions from bulk cells resulted in accumulation of established ovarian CSC markers.

[0328] For this purpose, COV318 cells were stained with 0.5 μg / ml IMAB027 at 4°C for 30 minutes, then incubated with goat anti-human IgG secondary antibody (1:300) at 4°C for 10 minutes. Subsequently, CLDN6-positive and CLDN6-negative cell fractions were isolated from COV318 cells by FACS sorting using a BD FACSAria cell sorter. The selected cells were then grown under standard growth conditions for 10 days. 1E6 cells from both subpopulations were stained for CSC markers CD44, CD24, CD90, CD34, CD117, and CD133 at 4°C for 30 minutes (see Table 1 for details), and their surface marker expression profiles were analyzed by flow cytometry. 50,000 events were recorded from each sample using a BD Canto II flow cytometer, and the recorded cells were analyzed using FlowJo software.

[0329] FACS analysis showed that approximately 50% of the cells in the fraction classified as CLDN6-positive remained positive for CLDN6 after 10 days of culture under standard conditions, while the cells classified as CLDN6-negative were completely negative for CLDN6. Importantly, we observed that the CLDN6-positive fraction showed accumulation of CSC markers CD44, CD24, and CD90 compared to the CLDN6-negative fraction of COV318 cells. Representative dot plots for various samples are shown in Figure 6A. Further quantification of the expression levels of these markers revealed a 99-fold enrichment of CD44, an 8-fold enrichment of CD90, and a 33-fold enrichment of CD24 when comparing the CLDN6-positive subpopulation with the CLDN6-negative subpopulation (Figure 6B).

[0330] These findings demonstrate that CLDN6 can be used as a selective marker for separating the CSC fraction from bulk cell lines, and indicate that CLDN6 is a novel CSC marker.

[0331] (Example 5) Cell lines that express high levels of CLDN6 show increased enrichment of CSC markers compared to cells that express low levels of CLDN6. CLDN6 has been shown to be highly expressed in germ cell tumors, ovarian adenocarcinoma, and some cancers with primitive phenotypes. If CLDN6 is a CSC marker, then accumulation of cells with CSC-like characteristics, and therefore accumulation of CSC marker-positive cells, is expected in such cell lines or tumors.

[0332] The inventors investigated four CLDN6-overexpressing cell lines, ovarian cancer cell lines OV90 and PA-1, and testicular cancer cell lines NEC-8 and NEC-14, in relation to their established CSC marker expression levels. For this purpose, 1E6 cells of each cell line were stained at 4°C for 30 minutes for surface markers CD44, CD24, CD90, CD34, CD117, and CD133, as well as CLDN6 (see Table 1 for details). The cells were then analyzed for their expression profiles by flow cytometry. The experiment was performed in triplicate, recording 50,000 events from each sample using a BD Canto II flow cytometer, and the recorded cells were analyzed using FlowJo software. Dead cells were excluded from the analysis by counterstaining with viability dye eFluor® 506. Representative dot plots from each sample are shown in Figure 7.

[0333] FACS analysis revealed that approximately 95% of the cell lines examined were positive for CLDN6. As expected, these CLDN6-high-expression cell lines also showed accumulation of established CSC markers in addition to CLDN6; OV90 cells showed high expression of CD44, CD133, CD24, and CD117; PA-1 cells showed high expression of CD44, CD133, CD90, and CD117; and NEC-8 and NEC-14 cells showed elevated expression levels of the markers CD133, CD90, CD24, and CD117.

[0334] These findings indicate that CLDN6-highly expressing cell lines are enriched with respect to CSC-like cells, further supporting the idea that CLDN6 is a CSC marker.

[0335] (Example 6) Treatment of advanced human xenograft tumors with CLDN6 antibodies in combination with chemotherapy drugs synergistically inhibits tumor cell proliferation and extends survival time. HSD: Athymia nude Foxn1 nu Human cancer cell lines were transplanted into mice. After tumors were established, tumor-bearing mice were divided into groups and administered CLDN6-specific monoclonal antibody (IMAB027), chemotherapy drugs, or a combination of both. The control group was given antibody buffer (vehicle control).

[0336] Specifically, for the treatment of human ES-2 (CLDN6) xenograft tumors, human ovarian cancer cell line ES-2 stably transfected with human CLDN6 was cultured at 37°C in a humidified incubator containing 5% CO2 in minimal essential medium (Life Technologies) containing 1× non-essential amino acid solution (Life Technologies), 700 μg / ml G418 (Life Technologies), and 10% FCS (Life Technologies). For transplantation, 6-week-old female Hsd: athymoid nude Foxn1 nu 5 × 10 in 200 μl of PBS for mice 6 ES-2(CLDN6) cells were subcutaneously inoculated into the flank. Three days after subcutaneous tumor inoculation, mice were treated with one of the following: saline control, antibody or chemotherapy monotherapy, or antibody / cell proliferation inhibitor combination therapy (n=12 in each group). 15 mg / kg paclitaxel or saline control was administered on days 3, 10, and 17 post-transplant. Antibody maintenance therapy was initiated on day 4 with bolus injections of 35 mg / kg IMAB027 or vehicle control (IMAB027 buffer) three times a week (alternating IV / IP / IP). Tumor volume and animal health status were monitored twice a week. The tumor reached 1400 mm. 3 Mice were euthanized when the tumor reached its maximum volume or when it became ulcerated. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the Posthoch-Dunn multiple comparison test.

[0337] For the treatment of advanced human NEC14 xenograft tumors, the human testicular germ cell tumor cell line NEC14 was cultured in RPMI 1640 medium GlutaMAX (Life Technologies) containing 10% FCS (Life Technologies) at 37°C in a humidified incubator with 5% CO2, according to the supplier's instructions. For transplantation, 6-8 week old female Hsd: athymoid nude Foxn1 nu 2 × 10⁶ mice in 200 μl of PBS 7 NEC14 cells were subcutaneously inoculated into the flank. In the post-progression treatment study, the tumor was 50-150 mm. 3 The tumors were grown to a certain volume, and before treatment, the mice were divided into control, antibody or cell growth inhibitor monotherapy, and antibody / cell growth inhibitor combination therapy groups (n=19 per group). On day 6 post-transplant, the drugs alone, in combination, or with a vehicle control (saline) were administered as follows: bolus IP injection of 1 mg / kg cisplatin on days 6, 7, 8, 9, and 10; bolus IP injection of 30 mg / kg carboplatin on days 6, 13, and 20; and antibody maintenance therapy with bolus injections of 35 mg / kg IMAB027 or vehicle control (IMAB027 buffer) three times a week (alternating IV / IP / IP). Tumor volume was observed twice a week. The tumor reached 1400 mm. 3 Mice were euthanized when the tumor reached its maximum volume or when it became ulcerated. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the Posthoch-Dunn multiple comparison test.

[0338] Compared to the control group, treatment with paclitaxel was ineffective and did not show antitumor activity in human ES-2 (CLDN6) xenograft tumors ectopically expressing human CLDN6. In contrast, IMAB027 inhibited tumor growth and extended survival in mice. Treatment with IMAB027 and paclitaxel in combination synergistically inhibited tumor growth (Figure 8).

[0339] Furthermore, both cisplatin and IMAB027, as monotherapies, can significantly reduce tumor growth in NEC14 tumor-bearing animals. However, after initial inhibition of tumor growth, the inventors observed recurrent tumor growth in most animals. In the combination therapy approach, cisplatin and IMAB027 act synergistically, not only inhibiting tumor growth but also inducing complete NEC14 tumor remission. Survival data most impressively demonstrate the therapeutic effect of IMAB027 in combination with cisplatin. Compared to the monotherapy approach, almost all mice treated with IMAB027 together with cisplatin were still alive at 90 days post-tumor transplantation (Figure 9).

[0340] In post-stage treatment of NEC14 xenograft tumors carried by mice, other platin derivatives such as carboplatin exhibit only very limited antitumor effects. However, the combination of IMAB027 and carboplatin results in a synergistic tumor inhibitory effect through highly efficient inhibition of tumor growth and extension of survival (Figure 10).

[0341] Therefore, the combination of chemotherapy drugs and CLDN6-specific antibodies enhances the inhibition of tumor growth and extends survival in mice transplanted with human tumor cells. The combination of chemotherapy drugs and antibodies produces a synergistic effect in inhibiting tumor cell growth and extending survival.

[0342] (Example 7) CLDN6 is a CSC marker. Example 7.1: CLDN6 is important for the sphere formation behavior of ovarian cancer cells. Another effective assay for analyzing the CSC-like properties of tumor cells is the sphere formation assay. This assay allows for easy examination of the cell's ability to grow in an anchorage-independent manner, a typical characteristic of CSCs. To analyze whether CLDN6 plays a role in the anchorage-independent growth of tumor cells, we selected the ovarian tumor cell line, COV318, which contains only a subpopulation of CLDN6-positive cells. COV318 cells were screened for their CLDN6 expression, and sphere formation was allowed in CLDN6-positive and CLDN6-negative cell populations under stem cell-specific conditions for 21 days. The sphere formation assay revealed that CLDN6-positive COV318 cells showed the ability to form spheres when cultured under stem cell-specific conditions, while CLDN6-negative cells were almost completely killed (Figure 11A). These findings indicate that the CLDN6-positive fraction is a stem cell-enriched population exhibiting the ability to grow in an anchorage-independent manner.

[0343] To define the ability of CLDN6-positive COV318 cells to undergo numerous cell division cycles while maintaining an undifferentiated state, we analyzed the ability of spheres to generate second-generation spheres. For this purpose, first-generation spheres (22 days after seeding) of CLDN6-positive COV318 cells were dissociated into single cells and then re-plated. On day 23 after re-platering, second-generation spheres were formed, and it was clearly observed that these newly formed spheres were morphologically more regular than the initial first-generation spheres (Figure 11B). These observations further confirm that the CLDN6-positive fraction of COV318 cells is the stem cell fraction of this cell line.

[0344] Considering these results together, it is clear that CLDN6 plays a significant role in anchorage-independent proliferation of ovarian cancer cells, which is an essential characteristic of cancer stem cells.

[0345] Example 7.2: Enrichment of CLDN6-positive COV318 cells after in vitro treatment with chemotherapy drugs The ovarian cancer cell line COV318 exhibits heterogeneous expression of CLDN6, with a very small subpopulation of cells (approximately 0.3–0.5%) expressing CLDN6. In vitro treatment of COV318 with a platinum derivative generates a residual cell population with a higher percentage (>2%) of CLDN6-positive cells in each case (Figure 12). The specific accumulation of CLDN6-positive cells after treatment suggests that these cells may have a selective survival or proliferation advantage during chemotherapy. Resistance to conventional chemotherapy is a characteristic feature of CSCs.

[0346] Example 7.3: In vivo enrichment of CLDN6-positive COV318 cells In previous studies, the inventors observed that COV318 cells injected subcutaneously into the flanks of athymic nude mice exhibited weak tumorigenicity. In contrast, mice ingested COV318 cells via intraperitoneal injection developed malignant ascites and peritoneal tumors within 100 days. Unlike parental COV318 cells (Figure 13A), the majority of cells isolated from the ascites and tumors were CLDN6-positive (Figure 13B, upper panel). After culturing under standard conditions, COV318 cells again lost CLDN6 expression in vitro (Figure 13B, lower panel). This suggests that CLDN6-positive COV318 cells exhibit greater tumorigenic potential and that a small CLDN6-positive subpopulation contains CSCs.

[0347] Example 7.4: CLDN6 correlates with ovarian cancer stem cell markers in primary tumor samples. Our previous results showed that CLDN6 co-localizes with some CSC markers in ovarian cancer cell lines. Therefore, we next investigated whether CLDN6 expression correlates with CSC markers in primary tumor samples from ovarian cancer patients.

[0348] For this purpose, mRNA expression of CLDN6 and selected markers described in the literature as specific to ovarian cancer stem cells (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1A1, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch 1, Pou5F1, CD133, Snail, Sox 2) was analyzed by qRT-PCR in 42 human ovarian cancer samples. Subsequent correlation analysis of these markers with CLDN6 was performed using Spearman r. Scatter plots of significant correlations and summaries of all correlations are shown in Figure 14.

[0349] Positive correlations were observed between CTCFL, LIN28B, CD24, GNL3, and EPCAM and CLDN6, while CD44 was negatively correlated with CLDN6 in the analyzed ovarian cancer samples (Figure 14A). No significant correlations were found for any of the other markers examined (Figure 14B).

[0350] These findings further support the idea that CLDN6 is a CSC marker.

[0351] (Example 8) The antitumor activity of anti-CLDN6 antibodies is enhanced when used in combination with chemotherapy drugs. Example 8.1: Effects of chemotherapeutic agents on IMAB027-mediated ADCC The effects of chemotherapeutic agents on IMAB027-mediated ADCC were analyzed using COV362(Luc) cells pre-treated with carboplatin, gemcitabine, paclitaxel, doxorubicin, and topotecan, respectively. As shown by flow cytometry, pre-treatment of target cells resulted in increased CLDN6 protein levels on the cell surface (Figure 15B, D, F, H, and J). Compared to untreated target cells, the maximum lysis of cells treated with chemotherapeutic agents increased up to threefold (Figure 15A, C, F, G, and I). In summary, the antitumor activity of IMAB027 may be enhanced in combination with chemotherapeutic agents.

[0352] Example 8.2: The combination of IMAB027 and multi-agent chemotherapy PEB (cisplatin, etoposide, and bleomycin) highly effectively inhibits tumor growth and extends survival in mice transplanted with the human tumor cell line NEC14. HSD: Athymia nude Foxn1 nu Mice were subcutaneously transplanted with the human testicular germ cell tumor cell line NEC14. Mice with very advanced tumors were randomized and treated with the antibody IMAB027, multi-agent chemotherapy PEB (cisplatin, etoposide, and bleomycin), or a combination of IMAB027 and PEB.

[0353] Compared to untreated and IMAB027-treated mice, multi-agent chemotherapy significantly reduced tumor growth (Figure 16A). However, after the tumor initially responded to PEB treatment, tumor growth began in most animals by day 30. Sustained inhibition of tumor growth was achieved only with a combination approach in which PEB and IMAB027 acted synergistically (Figure 16B). Untreated mice had a mean survival time of 30 days, while mice treated with IMAB027 and PEB had mean survival times of 34 days and 97 days, respectively. In the PEB-treated group, 3 out of 14 mice (21%) showed complete tumor remission, and 1 out of 14 mice (7%) had approximately 30 mm of tumor size at the end of the study. 3 The mice presented with residual tumor mass. Surprisingly, complete tumor regression was observed in 12 out of 14 mice (86%) treated with PEB in combination with IMAB027. Eleven mice were cured without any recurrence for more than 6 months, and one mouse without a tumor was euthanized on day 93 due to poor overall health (Figure 16C). The study showed that animals with very advanced human NEC14 testicular tumors treated with PEB in combination with IMAB027 had significantly longer survival times and significantly higher response rates compared to animals treated with multi-agent chemotherapy alone.

[0354] (Example 9) Anti-CLDN6 antibody-drug conjugates are highly effective in treating CLDN6-expressing tumors. Example 9.1: In vitro binding and antitumor activity of toxin-binding IMAB027 The relative binding affinity of the IMAB027-drug conjugates IMAB027-DM1 and IMAB027-vcMMAE was tested in ovarian cancer cell line OV90 using flow cytometry. In saturation binding experiments, antibody concentration was plotted against median fluorescence intensity (MFI), and EC50 (antibody concentration binding to half of the binding sites at equilibrium) and maximum binding were calculated by nonlinear regression. Compared to unconjugated IMAB027, both IMAB027-drug conjugates showed similarly low EC50 values, and binding saturation was achieved at low concentrations (Figure 17A). The cytotoxic activity of the IMAB027-drug conjugates was measured in OV90 cells using the XTT proliferation assay. Dose-response curves showed similar inhibition of tumor cell proliferation in vitro for IMAB027-DM1 and IMAB027-vcMMAE (Figure 17B). In summary, IMAB027 bound to DM1 or vcMMAE binds to CLDN6-positive target cells with similar relative affinity and highly effectively induces tumor cell death.

[0355] Example 9.2: Treatment of advanced human xenograft tumors with toxin-conjugated IMAB027 anti-CLDN6 antibody inhibits tumor cell proliferation, prolongs survival, and mediates complete tumor regression. The antitumor activity of IMAB027, a CLDN6-specific antibody conjugated to a cytotoxic agent, was tested in mice transplanted with CLDN6-positive human cancer cell lines. In this targeted approach, IMAB027 was conjugated to either the meitansinoid DM1 or auristatin E (MMAE) and then targeted in athymic nude Foxn1 cells carrying advanced xenograft tumors. nu Tumor growth was observed in mice.

[0356] Treatment of advanced human OV90 ovarian subcutaneous xenograft tumor models: The antitumor activity of toxin-binding IMAB027 was tested in mice carrying advanced human xenograft tumors with uniform CLDN6 expression. Treatment was initiated 10 days post-tumor cell transplantation. Following a single IV bolus injection, IMAB027-DM1 and IMAB027-vcMMAE significantly inhibited tumor growth in OV90 ovarian cancer cell xenograft tumors uniformly expressing CLDN6 (Figures 18 and 19A). More importantly, a single application of 16 mg / kg IMAB027-vcMMAE resulted in complete tumor remission in 60% of treated mice (Figure 19B).

[0357] Treatment of advanced human PA-1 ovarian subcutaneous xenograft tumor models: The antitumor activity of toxin-bound IMAB027 was also tested in an advanced xenograft tumor model with heterogeneous CLDN6 expression. After subcutaneous transplantation, PA-1 xenograft tumors lost CLDN6 expression after a certain period (Figure 20C). Treatment was initiated on day 15. At that point, PA-1 xenograft tumors began to lose CLDN6 expression. Animals were administered 4, 8, or 16 mg / kg of IMAB027-vcMMAE by a single IV bolus injection. Control animals were instead given unconjugated IMAB027 or vehicle control buffer.

[0358] Treatment with IMAB027-vcMMAE significantly inhibited tumor growth in PA-1 xenograft tumors and extended the survival time of tumor-bearing mice, while IMAB027 or IMAB027-DM1 (data not shown) did not affect PA-1 tumor growth (Figures 20A and B).

[0359] The reduced level of CLDN6-positive tumor cells in the tumor bulk is likely the reason for the weak antitumor activity of IMAB027 and IMAB027-DM1 in this in vivo tumor model. IMAB027-DM1 is bound to the non-membrane-permeable toxin DM1 by a non-cleavable linker. In contrast, IMAB027-vcMMAE is bound to the cell membrane-permeable toxin MMAE by a cathepsin-cleavable linker. The release of the membrane-permeable form of MMAE after cell processing promotes the death of tumor cells lacking specific epitopes (bystander effect). Therefore, treatment with IMAB027-vcMMAE is highly effective in eradicating PA-1 tumors containing both CLDN6-positive and CLDN6-negative cells.

[0360] In summary, IMAB027-vcMMAE is highly efficient at killing human xenograft tumors with heterogeneous CLDN6 expression through target cell activation and death by bystander cells.

[0361] Treatment of advanced human MKN74 subcutaneous xenograft tumor models: Unlike PA-1 xenograft tumors, which lose CLDN6 expression in advanced tumors, MKN74 xenograft tumors acquire CLDN6 expression. As shown by flow cytometry using the CLDN6-specific antibody IMAB027, <0.3% of cells in the gastric cancer cell line MKN74 are CLDN6-positive in vitro. Interestingly, a significant number of tumor cells show CLDN6 expression after subcutaneous transplantation in athymic nude mice (Figure 21C). Treatment of established MKN74 xenograft tumors with 16 mg / kg IMAB027-vcMMAE results in a very significant inhibition of tumor growth and extension of survival (Figures 21A and B).

[0362] The tumor growth inhibition observed with IMAB027-vcMMAE may be caused by the activation and death of target cells by bystander cells.

[0363] Treatment of advanced human PA-1 ovarian intraperitoneal xenograft tumor models: In addition to treating sc xenograft tumors, the antitumor activity of toxin-conjugated IMAB027 antibodies was also tested in an ibuprofen xenograft tumor model using PA-1 cells that ectopically express luciferase for in vivo observation (Figure 22). Mice were administered either 16 mg / kg IMAB027-DM1 or 16 mg / kg IMAB027-vcMMAE by a single bolus IV injection 14 days after tumor cell transplantation.

[0364] In vivo bioluminescence intensity measurements revealed inhibition of tumor growth in peritoneal PA-1 metastases after treatment with IMAB027-DM1. Furthermore, IMAB027-vcMMAE showed significantly higher antitumor activity than IMAB027-DM1 or the vehicle, resulting in complete regression of peritoneal tumors in 100% of the animals (Figure 22).

[0365] In summary, IMAB027-vcMMAE and IMAB027-DM1 are highly effective within the concentration range tested in vivo, without toxic side effects. IMAB027-DM1 significantly inhibited tumor growth in subcutaneous xenograft tumors and reduced tumor growth in peritoneal xenograft tumors. IMAB027-vcMMAE significantly inhibited tumor growth and extended survival in animals carrying subcutaneous or peritoneal human xenograft tumors with homogeneous or even heterogeneous CLDN6 expression. Most impressively, the majority of tumor-carrying animals were cured after treatment with MMAE-conjugated antibodies. The outstanding antitumor activity of IMAB027-vcMMAE, particularly observed in animals with heterogeneous CLDN6 expression, suggests that the IMAB027-vcMMAE conjugate is suitable for treating tumors with low percentage CLDN6 positivity.

[0366] Example 9.3: Endocytosis of CLDN6-specific antibodies depends on their affinity and CLDN6-binding epitope. The cytotoxic effect of toxin-binding antibodies is strictly dependent on their target-mediated potential for internalization. Therefore, the creation of antibodies with high endocytosis rates is an essential factor in the development of toxin-binding antibodies.

[0367] The efficiency of endocytosis was tested in vitro by incubating human cancer cells endogenously expressing CLDN6 with a CLDN6-reactive monoclonal chimeric antibody and an anti-human Fab fragment conjugated to a toxin saporin. Internalization of the CLDN6-conjugated antibody / Fab-saporin complex induced specific cell death, which can be measured using a cell viability assay. Screening of various CLDN6-reactive antibodies revealed that endocytosis depends not only on the antibody binding affinity but also on the antigen epitope. We observed that binding of a CLDN6-specific antibody to an epitope in the first extracellular loop of CLDN6 supported endocytosis in OV-90 and PA-1 human cancer cells. Notably, the CLDN6-reactive antibody 5F2D2, which binds to a different epitope with similar or higher affinity, showed lower cytotoxic potential in this assay (Figure 23).

[0368] (Example 10) Test materials and methods used in Examples 7-9 above Cell culture: COV362(Luc) and PA-1(Luc) cells that stably express the luminescence reporter gene were generated by stable transfection of the cell lines COV362 (ECACC, 07071910) and PA-1 (ATCC, CRL-1572) with firefly luciferase, respectively.

[0369] NEC14 (JCRB, 0162) and MKN74 (JCRB, 0255) cells were cultured in RPMI1640 medium (Gibco, 61870-010) supplemented with 10% thermally inactivated FCS (Gibco, 10270-106). COV318 (ECACC, 07071903) and COV362 (Luc) cells were cultured in DMEM (Gibco, 41965-039) containing 2 mM GlutaMAX (Gibco, 35050-038) and 10% thermally inactivated FCS. PA-1 and PA-1(Luc) cells were cultured in MEM (Gibco, 31095-029) supplemented with 1.5 g / l sodium bicarbonate (Invitrogen, 25080), 1 mM sodium pyruvate (Invitrogen, 11360), 1% non-essential amino acids (Gibco, 11140-035), and 10% thermo-inactivated FCS. OV90 (ATCC, CRL-11732) cells were cultured in a 1:1 mixture of MCB105 (Sigma, M6395) and 199 medium (Sigma, M4530) supplemented with 1.5 g / l sodium bicarbonate and 15% thermo-inactivated FCS. Cells were grown at 37°C and 5% CO2.

[0370] Measurement of CLDN6 expression by flow cytometry: Cells were collected using 0.05% trypsin / EDTA (Gibco, 25300-054), washed with FACS buffer (PBS containing 2% FCS (Gibco, 10270-106) and 0.1% sodium azide (Applichem, A1430)), and then 2 × 10⁻⁶ cells were collected. 6Cells were resuspended in FACS buffer at a concentration of cells / ml. 100 μl of the cell suspension was incubated at 4°C for 30 minutes with 2.5 μg / ml of anti-CLDN6 antibody IMAB027 or isotype control human IgG1 antibody (Sigma, I5154). Cells were washed three times with FACS buffer and incubated at 4°C for 30 minutes with APC-conjugated F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch, 109-136-170) diluted 1:200 in FACS buffer. Cells were washed twice and resuspended in FACS buffer. Binding was analyzed by flow cytometry using BD FACSArray (BD Biosciences) and FlowJo software (Tree Star Inc.). Dead cells were excluded from the analysis using the live / dead dye propidium iodide (Sigma, P4864).

[0371] Treatment of COV318 cells with platinum derivatives: COV318 cells (1.2 × 10⁶ cells per 100mm cell culture dish) 6 Cells were grown under standard conditions. After 24 hours, the cells were treated with 0.5 μg / ml cisplatin or 2 μg / ml carboplatin and incubated for 96 hours. The medium was changed, and the treated cells were grown under standard growth conditions. At 3 and 6 days, the cells were analyzed for CLDN6 expression by flow cytometry.

[0372] Antibody-dependent cell-mediated cytotoxicity (ADCC) after treatment with cytotoxic agents: The effects of carboplatin and paclitaxel on IMAB027-mediated ADCC were measured using the human ovarian cancer cell line COV362, which was stably transfected with luciferase as a reporter. COV362 (Luc) cells (3 × 10⁶ cells per 150 mm cell culture dish) 6Cells were grown under standard conditions. After 24 hours, the cells were treated with 5 ng / ml paclitaxel, 20 μg / ml carboplatin, 25 ng / ml gemcitabine, 20 ng / ml doxorubicin, or 7.5 ng / ml topotecan and incubated for 4 days. The medium was changed, and the treated cells were grown under standard growth conditions for an additional 3 days for carboplatin and gemcitabine, or 10 days for paclitaxel, doxorubicin, or topotecan.

[0373] Cells were collected using 0.05% trypsin / EDTA (Gibco, 25300-054) and 2 × 10⁶ cells were collected in DMEM containing 2 mM glutamine (Gibco, 25030-081) and 20 mM HEPES (Gibco, 15630-056). 5 The concentration was adjusted to 1 × 10⁻¹⁰ cells / ml. 4 Cells / well were inoculated into a white 96-well PP plate and incubated at 37°C and 5% CO2 for approximately 5 hours.

[0374] Peripheral blood mononuclear cells (PBMCs) were isolated from human donor blood samples by density gradient centrifugation using a Ficoll Hypaque (GE Healthcare, 17144003). PBMCs including intermittent cells were isolated and washed three times with PBS containing 2 mM EDTA. PBMCs were then divided into 1.6 × 10⁶ cells. 7 The cells were resuspended in X-Vivo 15 medium (Lonza, BE04-418Q) at a concentration of cells / mL and stored at 37°C and 5% CO2 until the time of analysis.

[0375] IMAB027 and 25 μl of isotype control antibody were added to the cells at the indicated concentrations. Subsequently, 25 μl of PBMC suspension was added, and the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0376] 10 μl of 8% Triton X-100 (Sigma, T8787) in PBS was added to the total lysis control, and 10 μl of PBS was added to the maximum viable cell control and sample. Then, 50 μl of luciferin mixture (3.84 mg / ml D-luciferin (Sigma Aldrich, 50227) and 160 mM HEPES in ddH2O) was added, and the cells were incubated in the dark at room temperature for 90 minutes. Bioluminescence was measured using a luminometer (Infinite M200, TECAN). The results are shown as integrated digital relative luminescence units.

[0377] Specific dissolution is calculated as follows:

number

[0378] Intraperitoneal transplantation of COV318 cells in athymic nude mice: The in vivo tumorigenicity and accumulation of CLDN6-positive cells of the human ovarian cancer cell line COV318 were tested in mice. Therefore, 2 × 10⁶ cells were resuspended in PBS. 7 COV318 cells were collected from 6-8 week old female HSD: athymic nude Foxn1 nuMice were injected intraperitoneally. Mice were observed daily. Animals were euthanized once life-threatening symptoms became pronounced. Tumor and ascites cells were isolated and cultured for further analysis. Tumors were mechanically dissociated, passed through a mesh, and washed with DMEM medium (Gibco, 41965-039). To obtain a single-cell suspension, tumor cells were treated with acetase (Life Technologies, A11105-01) at 37°C for 30 minutes, passed through a 40 μm cell strainer, and washed with DMEM medium. Ascites fluid was collected and contaminating erythrocytes were removed using ACK (potassium ammonium chloride) lysis buffer (Invitrogen, A10492-01). Tumor and ascites cells were grown under standard conditions using standard COV318 medium (Gibco, 15140) supplemented with penicillin / streptomycin. Cells were screened for CLDN6 expression by flow cytometry.

[0379] Treatment of very advanced human NEC14 xenograft tumors: For transplantation, we need a 6-8 week old female HSD: athymic nude Foxn1. nu 2 × 10⁶ mice in 200 μl of PBS 7 NEC14 cells were subcutaneously inoculated into the flank. In a treatment study for very advanced tumors, the tumor was reduced to a maximum size of 170 mm. 3 The mice were allowed to grow to a certain volume for 13 days, and before treatment, they were divided into control, IMAB027, PEB (cisplatin, etoposide, bleomycin), and IMAB027 / PEB groups (n=14 for each group).

[0380] On day 13 post-transplant, drugs were administered as follows: bolus ip injection of 1 mg / kg cisplatin on days 13, 14, 15, 16, and 17; bolus ip injection of 5 mg / kg etoposide on days 13, 14, 15, 16, and 17; and bolus ip injection of 10 mg / kg bleomycin on days 13, 17, and 21. IMAB027 was administered by alternating bolus IV / IP / IP injections of 35 mg / kg IMAB027 three times a week from day 13 to day 101. As vehicle controls, mice were given drug substance buffer instead of antibody or 0.9% NaCl solution instead of PEB, respectively.

[0381] Tumor volume and the health status of the animals were observed twice a week. The tumor was 1400 mm. 3 Mice were euthanized when the tumor reached a certain volume or when it became ulcerative. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the Posthoch-Dunn multiple comparison test.

[0382] sphere formation assay: For the sphere formation assay, COV318 cells were stained for CLDN6 with 0.5 μg / ml IMAB027 at 4°C for 30 minutes, then incubated with goat anti-human IgG secondary antibody (1:300) at 4°C for 10 minutes, and subsequently sorted for their CLDN6 expression using a BD FACSAria cell sorter. Next, 1 × 10⁻⁶ cells were sampled. 6 CLDN6-positive or CLDN6-negative selected cells were inoculated into wells of a 6-well Ultra Low Attachment Plate (Corning) in serum-free DMEM / F12 medium containing 0.4% bovine serum albumin, 20 ng / ml basic fibroblast growth factor, 10 ng / ml epidermal growth factor, and 5 μg / ml insulin. The cells were allowed to form spheres under these stem cell-specific conditions for 21 days. The medium was changed every other day without destroying the spheres, and representative photographs were taken periodically.

[0383] To generate second-generation spheres, first-generation spheres (22 days post-inoculation) of CLDN6-positive COV318 cells were dissociated into single cells and then plated again in wells of a 6-well Ultra Low Attachment Plate. Again, the culture medium was changed every other day without disturbing the formed spheres, and representative photographs were taken periodically.

[0384] Quantitative real-time RT-PCR using BioMark® HD System (Fluidigm): Forty-two human ovarian cancer samples were analyzed using the Bio Mark® HD System (Fluidigm) for selected ovarian cancer stem cell-specific factors (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1A1, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch 1, Pou5F1, CD133, Snail, Sox 2) by TaqMan® gene expression assay (Life technologies) and qRT-PCR. RNA isolation from ovarian cancer samples was performed using the RNeasy Mini Kit (Qiagen) according to the respective manufacturers' instructions, and cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara Bio Inc.). Samples were prepared and analyzed according to the Fluidigm® Advanced Development Protocol 28-Fast Gene Expression Analysis using TaqMan® GE Assays rev A2. The 96.96 Gene Expression Dynamic Array IFC was filled using the IFC Controller HX. The chip array was analyzed using the Fluidigm BioMark® HD system. TaqMan PreAmp MasterMix was purchased from Applied Biosystems. The dataset was evaluated according to the ΔΔCt method. Correlation analysis between selected ovarian cancer stem cell markers and CLDN6 was performed using Spearman r. The significance of the correlation values ​​was evaluated by a test of correlation coefficients. The p-value was adjusted for multiple testing using the Benjamini-Hoshberg method, and an adjusted p-value ≤ 0.05 was considered significant.

[0385] Toxin binding of CLDN6 antibody: Monoclonal antibody toxin conjugation was performed at Piramal Healthcare (Grangemouth, UK).

[0386] For DM1 binding, the naked antibody was modified with SMCC (6 × molar concentration), which reacts with the free NH2 residue of the lysine group by incubation in PBS (pH 7.2) at room temperature for 1 hour. The modified antibody was then dialyzed to 35 mM citrate buffer (pH 5.0), and the linker-to-antibody ratio was measured using a reverse Elman assay. DM1 (6 × molar concentration) was conjugated to the maleimide portion of the SMCC linker via its sulfhydryl group by incubation at room temperature for 17 hours. The conjugated antibody was dialyzed to pharmaceutical buffer (20 mM His, 85 mg / ml sucrose, pH 5.8) and stored at -80°C. The drug-to-antibody ratio was analyzed by ultraviolet spectroscopy, monomer content was analyzed by SEC-HPLC, and free drug content was analyzed by RP-HPLC.

[0387] For MMAE binding, the naked antibody was dialyzed to PBS (pH 7.2) and modified by thiolation of the free NH2 group of the lysine residue using Trout's reagent (2-iminothiolane) (20 × molar concentration) at room temperature for 2 hours. The thiolated antibody was then dialyzed to 35 mM citrate buffer (pH 5.5), and the linker-to-antibody ratio was measured using a reverse Elman assay. vcMMAE (6 × molar concentration) was conjugated to the sulfhydryl group of the thiolated antibody via valine in a cathepsin-cleavable linker by incubation at room temperature for 15 hours. The conjugated antibody was dialyzed to pharmaceutical buffer (20 mM His, 85 mg / ml sucrose, pH 5.8) and stored at -80°C. The drug-to-antibody ratio was analyzed by ultraviolet spectroscopy, monomer content by SEC-HPLC, and free drug content by RP-HPLC.

[0388] Measurement of relative binding affinity by flow cytometry: Cells were collected using 0.05% trypsin / EDTA (Gibco, 25300-054), washed with FACS buffer (PBS containing 2% FCS (Gibco, 10270-106) and 0.1% sodium azide (Applichem, A1430)), and then 2 × 10⁻⁶ cells were collected. 6Cells were resuspended in FACS buffer at a concentration of cells / ml. 100 μl of the cell suspension was incubated with IMAB027, IMAB027-DM1, or IMAB027-vcMMAE (titer series ranging from 0.1 ng / ml to 20 μg / ml) at 4°C for 30 minutes. The cells were then washed three times with FACS buffer and incubated with anti-human IgG (Jackson ImmunoResearch, 109-136-170) diluted 1:200 in FACS buffer for 30 minutes at 4°C. The cells were then washed twice and resuspended in 100 μl of FACS buffer. Binding was analyzed by flow cytometry using BD FACSArray (BD Biosciences) and FlowJo software (Tree Star Inc.).

[0389] Survival efficacy assay for toxin-binding IMAB027: The effects of IMAB027-DM1 and IMAB027-vcMMAE on the viability of human tumor cell lines were measured in vitro using a colorimetric assay (Cell Proliferation Kit XTT from AppliChem) to detect cellular metabolic activity.

[0390] OV90 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054), and 2500 cells were seeded into 50 μl of growth medium in a 96-well culture. After 24 hours, a series of concentrations of DM1-conjugated IMAB027 and MMAE-conjugated IMAB027, or isotype control antibodies diluted in 50 μl of medium, were added. Cells were cultured for 3–7 days until untreated cells reached a concentration of approximately 80%. Cell viability was analyzed using the AppliChem Cell Proliferation Kit II (AppliChem, A8088-1000) as directed by the manufacturer. After incubation with XTT reagent for 3–5 hours, 100 μl of the cell supernatant was transferred to a new 96-well assay plate, and absorbance was measured at 480 nm (reference 630 nm) using a spectrophotometer (Tecan).

[0391] Survival rate was calculated using the following equation.

number

[0392] The EC50 value was determined by nonlinear regression using GraphPad Prism 6 software.

[0393] Treatment of advanced subcutaneous OV90 xenograft tumors: Human ovarian cancer cell line OV90 was cultured under standard conditions. For transplantation, 6-8 week old female HSD: athymoid nude Foxn1 cells were used. nu 1 × 10⁶ mice in 200 μl of PBS 7 OV90 cells were subcutaneously inoculated into the flank. In the post-progression treatment study, the tumors were allowed to grow for 10 days until they reached 50-150 mm. 3 Mice with tumors of established volume were randomly assigned to either a vehicle or antibody group (n=10) before treatment. Tumor volume (TV = (length × width) 2 ) / 2) was observed twice a week. TV was set to mm 3 This was used to create a tumor growth curve over time.

[0394] In the first dose-ranging study, animals received a single intravenous injection of vehicle and IMAB027-DM1 (1.78 mg / kg, 5.33 mg / kg, or 16 mg / kg) and were dissected 35 days post-transplant. In the second dose-ranging study, animals received a single intravenous injection of vehicle and IMAB027-vcMMAE (4 mg / kg, 8 mg / kg, or 16 mg / kg) or IMAB027-DM1 (1.33 mg / kg, 2.67 mg / kg, or 5.33 mg / kg). IMAB027 was administered three times a week by alternating bolus intravenous / intravenous / intravenous injections of IMAB027 at 35 mg / kg. The tumor size was 1400 mm. 3 Mice were euthanized when the tumor reached a larger volume or became ulcerated. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the Posthoch-Dunn multiple comparison test. Survival rates were analyzed using the Mantel-Cox test.

[0395] Treatment of subcutaneous PA-1 xenograft tumors and immunohistochemistry of tumor sections: The human ovarian cancer cell line PA-1 was cultured under standard conditions. For transplantation, 6- to 8-week-old female Hsd: nude Foxn1 nu mice were subcutaneously inoculated with 1×10 7 PA-1 cells in 200 μl of PBS on the flanks. In the post-treatment study, tumors were allowed to grow for 15 days, and mice with tumors of established volume of 40-120 mm 3 were randomly assigned to vehicle and antibody groups (n = 10) before treatment. Tumor volume (TV = (length × width 2 ) / 2) was observed twice a week. TV was expressed in mm 3 and a tumor growth curve over time was created.

[0396] Animals were injected i.v. once with vehicle, IMAB027-vcMMAE (4 mg / kg, 8 mg / kg or 16 mg / kg) or IMAB027-DM1 (4 mg / kg, 8 mg / kg or 16 mg / kg). IMAB027 was applied three times a week by alternating bolus i.v. / i.p. / i.p. injection of 35 mg / kg IMAB027. Mice were sacrificed when tumors reached a volume larger than 1400 mm 3 or became ulcerated. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and post hoc Dunn's multiple comparison test. Survival rates were analyzed using the Mantel-Cox test.

[0397] To analyze CLDN6 expression during tumor establishment and progression, PA-1 tumors from untreated mice were excised at days 7, 14, and 56, respectively, fixed in formalin, and embedded in paraffin. 4 μm tissue sections were prepared from the FFPE (formalin-fixed paraffin-embedded) blocks of each sample, mounted on adhesive slides (SuperFrost Ultra Plus, Thermo Fisher Scientific), and calcined at 60°C for 60 minutes. Before staining, the FFPE tissue sections were deparaffinized. The sections were boiled at 120°C for 10 minutes in 10 mM citrate with 0.05% Tween-20 (pH 6.0). Endogenous peroxidase was quenched by incubation in PBS with 0.3% H2O2. After washing with PBS, nonspecific antibody binding sites were blocked with blocking buffer (10% goat serum in PBS) for 30 minutes at room temperature, and then incubated overnight with 0.2 μg / ml primary rabbit anti-claudin 6 antibody (IBL-America, 18865) diluted in blocking buffer. Next, the samples were washed three times with PBS and incubated with each secondary diluted antibody (Power Vision HRP goat anti-rabbit; Immunologic) for 30 minutes at room temperature. Visualization was performed for 4 minutes and 30 seconds using substrate chromogenic solution (VectorRed; Vector Laboratories). After counterstaining with hematoxylin, dehydration, and mounting, sections were analyzed using a Leica DM2000 microscope.

[0398] Treatment of advanced subcutaneous MKN74 xenograft tumors and immunohistochemistry of tumor sections: Human gastric cancer cell line MKN74 was cultured under standard conditions. For transplantation, 6-8 week old female HSD: athymic nude Foxn1 cells were used. nu 1 × 10⁶ mice in 200 μl of PBS 7 MKN74 cells were subcutaneously inoculated into the flank. In the post-progression treatment study, the tumor was allowed to grow for 7 days until it reached 200 ± 30 mm. 3 Mice with tumors of established volume were randomly assigned to either a vehicle or antibody group (n=10) before treatment. Tumor volume (TV = (length × width) 2 ) / 2) was observed twice a week. TV was set to mm 3This was used to create a tumor growth curve over time.

[0399] Animals were administered either vehicle control buffer or 16 mg / kg IMAB027-vcMMAE by a single bolus IV injection on day 8. Tumors measuring 1400 mm² were administered. 3 Mice were euthanized when the tumor reached a larger volume or became ulcerated. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the Post-Hock-Dun multiple comparison test. Survival rates were analyzed using the Mantell-Cox test.

[0400] CLDN6 target expression in MKN74 cells was analyzed by flow cytometry before transplantation and by histochemistry of untreated MKN74 xenograft tumors excised at day 31.

[0401] For immunohistochemistry, 3 μm thick tissue sections were prepared, placed on glass slides, and air-dried at room temperature for 90 minutes. All tissue sections were fixed in acetone at -20°C for 10 minutes and washed in PBS for 5 minutes. Endogenous peroxidase was quenched by incubation with 0.03% hydrogen peroxide (Dakocytomation EnVision System, K4011) for 15 minutes. After washing with PBS, nonspecific antibody binding sites were blocked with blocking buffer (10% goat serum in PBS) at room temperature for 30 minutes, followed by incubation with 5 μg / ml IMAB027-FITC at room temperature for 60 minutes. The samples were then washed three times with PBS and incubated with their respective secondary diluted antibodies (Bright Vision polyHRP anti-rabbit IgG, Immunologic, DPVR-110HRP) at room temperature for 30 minutes. Visualization was performed for 2 minutes and 30 seconds using substrate chromogenic solution (VectorRed; Vector Laboratories). The sections were counterstained with hematoxylin, dehydrated, and mounted, then analyzed using a Leica DM2000 microscope.

[0402] Treatment of intraperitoneal PA-1 (Luc) xenograft tumors performed: A human ovarian teratocarcinoma cell line PA-1 (Luc) that stably expresses firefly luciferase as a luminescent reporter gene was used as an intraperitoneal xenograft tumor model to examine the antitumor activity of the toxin-conjugated IMAB027 antibody in vivo. Previous transplantation experiments have revealed that intraperitoneal inoculation of PA-1 (Luc) cells gives rise to intraperitoneal tumor nodules.

[0403] 1×10 resuspended in PBS 7 PA-1 (Luc) cells were intraperitoneally injected into female Hsd: nude Foxn1 athymic nu mice. Bioluminescence imaging was initiated on day 14 after tumor cell inoculation and then performed once a week until the end of the study. D-luciferin (PerkinElmer, 122796) was dissolved in sterile water and intraperitoneally injected (150 mg / kg, injection volume 200 μl) 5 minutes before imaging with an IVIS Lumina Imaging System (Advanced Molecular Vision). The mice were anesthetized with isoflurane and placed in the dark chamber of the IVIS Lumina, and the emitted photons were quantified for an integration time of 1 minute. The intensity of the transmitted light generated from luciferase expressing PA-1 cells in the animal's body was displayed as a pseudo-color image, where blue is the weakest intensity and red is the strongest bioluminescence signal. A grayscale photographic image of the mouse was also obtained under LED low-light illumination. The images were overlaid using Living Image software (Xenogen). The same illumination settings were used for all images. To quantify bioluminescence, regions of interest (ROIs) were determined and the total flux of each ROI was measured in photons / second (p / s). The background bioluminescence value obtained from the non-signal emitting region of the animal was subtracted from the bioluminescence value of each animal.

[0404] On day 14, mice were randomly assigned and treated with either 16 mg / kg IMAB027-DM1 or IMAB027-vcMMAE intraperitoneally. Control animals were given vehicle buffer. Tumor growth was observed weekly by bioluminescence imaging from ventral views, and then the total flow rate (photons / second) within the region of interest covering the abdomen of the mice was analyzed.

[0405] Endocytosis: Endocytosis of CLDN6-conjugated antibodies was measured using a cytotoxicity-based endocytosis assay based on co-internalization of a target-binding antibody with a saporin-conjugated anti-human IgG Fab fragment (Fab-ZAP Human, Advanced Targeting Systems, IT-51). Saporins are ribosome-inactivating proteins that impair protein biosynthesis after internalization, thus leading to cell death.

[0406] PA-1 cells were isolated using 0.05% trypsin / EDTA (Gibco, 25300-054) and measured in 2.5 × 10⁶ units. 3 Cells / well were seeded in 50 μl of growth medium in a 96-well culture plate. After 24 hours, Fab-ZAP and then IMAB027 or isotype control antibody were added in 25 μl volumes each. CLDN6 antibody was administered in 6 or 8 serial dilutions, while Fab-ZAP was applied at a constant concentration (Fab-ZAP:antibody ratio 3:1 to 6561:1). Cells were further cultured for 72 hours in a humidified CO2 incubator at 37°C. Subsequently, cell viability was analyzed using the Cell Proliferation Kit II from AppliChem (AppliChem, A8088-1000) according to the manufacturer's instructions. Absorbance was measured at 480 nm (reference 630 nm) using a spectrophotometer (Tecan).

Claims

1. A method for measuring cancer stem cells, including the detection of cells expressing CLDN6.

2. The method according to claim 1, wherein the presence of cells expressing CLDN6 indicates the presence of cancer stem cells and / or the amount of cells expressing CLDN6 correlates with the amount of cancer stem cells.

3. The method according to claim 1 or 2, wherein cells expressing CLDN6 are detected in a sample obtained from a cancer patient.

4. The method according to any one of claims 1 to 3, wherein the method comprises quantitative and / or qualitative measurement of cells expressing CLDN6.

5. The method according to any one of claims 1 to 4, comprising comparing the amount of cells expressing CLDN6 with the amount of cells expressing CLDN6 in a reference sample or with a predetermined reference range.

6. The method according to claim 5, wherein the reference sample is a sample from a patient who has never been diagnosed with cancer.

7. The method according to claim 5, wherein the predetermined reference range is based on a population of patients who have never been diagnosed with cancer.

8. The method according to any one of claims 1 to 7, comprising observing the amount of cancer stem cells in a cancer patient.

9. The method according to claim 8, wherein observing the amount of cancer stem cells in a cancer patient includes comparing the amount of cancer stem cells in a sample obtained from the cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient.

10. The method according to claim 9, wherein the sample obtained from the cancer patient is a sample taken from the cancer patient during or after the administration of cancer therapy.

11. A method for observing the effects of cancer therapy in cancer patients, (i) Measuring the amount of cancer stem cells in a sample obtained from the cancer patient during or after the administration of cancer therapy; and (ii) Comparing the amount of cancer stem cells in a sample obtained from the cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient. A method comprising, wherein measuring the amount of cancer stem cells in a sample obtained from the cancer patient and / or measuring the amount of cancer stem cells in a sample obtained earlier from the cancer patient, is a method for measuring the amount of cells expressing CLDN6.

12. The method according to any one of claims 9 to 11, wherein the sample obtained earlier from the cancer patient is a sample taken from the cancer patient before, during, or after the administration of cancer therapy.

13. The method according to any one of claims 10 to 12, wherein stabilization or reduction of the amount of cancer stem cells indicates that the cancer therapy is effective.

14. The method according to any one of claims 10 to 12, wherein an increase in the amount of cancer stem cells indicates that the cancer therapy is ineffective.

15. The method according to any one of claims 10 to 14, wherein the cancer therapy is a cancer therapy for cancer stem cells.

16. The method according to any one of claims 3 to 15, wherein the sample obtained from the cancer patient is a biological fluid or a tumor biopsy.

17. The method according to any one of claims 3 to 11, wherein the sample is subjected to one or more pretreatment steps.

18. The method according to any one of claims 1 to 17, comprising detecting cells expressing CLDN6 or measuring their quantity by using an immunoassay.

19. The method according to claim 18, wherein the immunoassay method is selected from the group consisting of Western blotting, immunohistochemistry, radioimmunoassay, ELISA (solid-phase enzyme immunoassay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, immunofluorescence assay, protein A immunoassay, flow cytometry, and FACS analysis.

20. The method according to any one of claims 1 to 19, comprising detecting cells expressing CLDN6 or measuring the amount of such cells by using an antibody having the ability to bind to CLDN6.

21. The method according to any one of claims 1 to 20, wherein the cells expressing CLDN6 are cancer cells expressing CLDN6 and / or cells present in a tumor site.

22. A method for treating or preventing cancer, comprising inhibiting and / or eliminating cancer stem cells by administering an antibody having the ability to bind to CLDN6 to a cancer patient.

23. The method according to claim 22, wherein the cancer stem cells express CLDN6.

24. The method according to claim 22 or 23, further comprising administering chemotherapy and / or radiotherapy.

25. The method according to any one of claims 22 to 24, wherein inhibiting and / or eliminating cancer stem cells enhances the anticancer effect of chemotherapy and / or radiotherapy.

26. The method according to claim 25, wherein the enhancement of the anticancer effect of chemotherapy and / or radiotherapy includes extending the survival time of cancer patients receiving chemotherapy and / or radiotherapy.

27. A method for treating or preventing cancer, comprising administering to a cancer patient (i) an antibody having the ability to bind to CLDN6 and (ii) chemotherapy.

28. The method according to claim 27, wherein the cancer comprises cancer stem cells expressing CLDN6.

29. The method according to claim 27 or 28, wherein administering an antibody having the ability to bind to CLDN6 results in the inhibition or elimination of cancer stem cells expressing CLDN6.

30. The method according to any one of claims 27 to 29, wherein administering an antibody having the ability to bind to CLDN6 enhances the anticancer effect of chemotherapy.

31. The method according to claim 30, wherein the enhancement of the anticancer effect of chemotherapy includes extending the survival time of cancer patients receiving chemotherapy.

32. The method according to any one of claims 22 to 26 and 29 to 31, wherein the elimination of cancer stem cells leads to a cure for cancer.

33. The method according to any one of claims 24 to 32, wherein the antibody having the ability to bind to CLDN6 and the chemotherapy are administered in synergistically effective amounts.

34. The method according to any one of claims 24 to 33, wherein the chemotherapy is administered at a dose lower than the maximum tolerated dose.

35. The method according to any one of claims 24 to 34, wherein the chemotherapy comprises administering an active substance selected from the group consisting of taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, their prodrugs, their salts, and combinations thereof.

36. The method according to any one of claims 24 to 35, wherein the chemotherapy comprises administering an active agent selected from the group consisting of paclitaxel, cisplatin, carboplatin, its prodrugs, its salts, and combinations thereof.

37. The method according to any one of claims 22 to 26 and 28 to 36, wherein the cancer stem cells are present in the tumor site of the cancer patient.

38. The method according to any one of claims 22 to 37, wherein the cancer is resistant to chemotherapy, particularly when administered as monotherapy.

39. The method according to any one of claims 22 to 38, wherein the antibody having the ability to bind to CLDN6 exerts an inhibitory and / or cytotoxic effect on cancer stem cells.

40. The method according to claim 39, wherein the antibody having the ability to bind to CLDN6 imparts an inhibitory and / or cytotoxic effect to cancer stem cells by mediating one or more of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, induction of apoptosis, and inhibition of proliferation.

41. The method according to any one of claims 22 to 40, wherein the antibody having the ability to bind to CLDN6 is linked to a therapeutic component.

42. The method according to claim 41, wherein the therapeutic component is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide.

43. The method according to claim 41 or 42, wherein the therapeutic component acts on slowly proliferating cells.

44. The method according to any one of claims 22 to 43, wherein the antibody having the ability to bind to CLDN6 binds to the first extracellular loop of CLDN6.

45. The method according to any one of claims 22 to 44, wherein the antibody having the ability to bind to CLDN6 contains a heavy chain variable region (VH) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO:

4.

46. A method for treating or preventing cancer, comprising administering to a cancer patient an antibody drug conjugate containing an antibody having the ability to bind to CLDN6, which is covalently bound to at least one toxic drug component by a linker.

47. The method according to claim 46, wherein the toxic drug component is permeable to cell membranes.

48. The method according to claim 46 or 47, wherein the toxic drug component is a meitansinoid or auristatin.

49. The method according to claim 48, wherein the maytansinoid is selected from the group consisting of DM1 and DM4.

50. The method according to claim 48, wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

51. The method according to any one of claims 46 to 50, wherein the linker is a severable linker.

52. The method according to any one of claims 46 to 51, wherein the linker is a linker that can be cut with cathepsin.

53. The method according to any one of claims 46 to 52, wherein the antibody is linked to the linker via the cysteine ​​thiol of the antibody.

54. The method according to any one of claims 46 to 53, wherein the cancer comprises cancer stem cells expressing CLDN6.

55. The method according to any one of claims 46 to 54, wherein administration of the antibody drug conjugate results in inhibition or elimination of cancer stem cells expressing CLDN6.

56. The method according to any one of claims 46 to 55, further comprising administering chemotherapy and / or radiotherapy.

57. The method according to any one of claims 46 to 56, wherein administration of the antibody drug conjugate enhances the anticancer effect of chemotherapy and / or radiotherapy.

58. The method according to any one of claims 46 to 57, wherein the antibody having the ability to bind to CLDN6 in the antibody-drug conjugate binds to the first extracellular loop of CLDN6.

59. The method according to any one of claims 46 to 58, wherein the antibody having the ability to bind to CLDN6 contains a heavy chain variable region (VH) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO:

4.

60. The method according to any one of claims 1 to 59, wherein CLDN6 has an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO:

2.

61. The method according to any one of claims 1 to 60, wherein the cancer includes primary cancer, advanced cancer, metastatic cancer, recurrent cancer, or a combination thereof.

62. A method for treating or preventing cancer, (i) Measuring cancer stem cells in a cancer patient by the method described in any one of claims 1 to 21 and (ii) Administering cancer therapy targeting cancer stem cells to the cancer patient. A method that includes this.

63. The method according to claim 62, wherein the cancer therapy for cancer stem cells comprises carrying out the method for treating or preventing cancer according to any one of claims 22 to 61.

64. A method for preventing cancer chemotherapy resistance, cancer recurrence or cancer metastasis, particularly during or after cancer treatment, comprising treating cancer by the method according to any one of claims 22 to 63.

65. A medical preparation for treating or preventing cancer, comprising (i) an antibody having the ability to bind to CLDN6 and (ii) a chemotherapeutic agent.

66. The medical preparation according to claim 65, which exists in the form of a kit comprising a first container containing the antibody having the ability to bind to CLDN6 and a second container containing the chemotherapeutic agent.

67. A medical preparation according to claim 65 or 66, further comprising printed instructions relating to the use of the preparation for the treatment or prevention of cancer.

68. An antibody drug conjugate containing an antibody capable of binding to CLDN6, covalently bound to at least one toxic drug component by a linker.

69. The antibody drug conjugate according to claim 68, wherein the toxic drug component is permeable to cell membranes.

70. The antibody drug conjugate according to claim 68 or 69, wherein the toxic drug component is a meitansinoid or auristatin.

71. The antibody drug conjugate according to claim 70, wherein the aforementioned maytansinoid is selected from the group consisting of DM1 and DM4.

72. The antibody drug conjugate according to claim 70, wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

73. The antibody drug conjugate according to any one of claims 68 to 72, wherein the linker is a cleavable linker.

74. The antibody drug conjugate according to any one of claims 68 to 73, wherein the linker is a linker that can be cleaved with cathepsin.

75. The antibody-drug conjugate according to any one of claims 68 to 74, wherein the antibody is linked to the linker via the cysteinethiol of the antibody.

76. The antibody-drug conjugate according to any one of claims 68 to 75, wherein the antibody having the ability to bind to CLDN6 in the antibody-drug conjugate binds to the first extracellular loop of CLDN6.

77. The antibody-drug conjugate according to any one of claims 68 to 76, wherein the antibody having the ability to bind to CLDN6 contains a heavy chain variable region (VH) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO: 5 and a light chain variable region (VL) comprising an amino acid sequence or fragment thereof represented by SEQ ID NO:

4.

78. A pharmaceutical formulation comprising an antibody drug conjugate according to any one of claims 68 to 77 and a pharmaceutically acceptable diluent, carrier, or excipient.

79. A medical preparation containing an antibody drug conjugate and a chemotherapeutic agent according to any one of claims 68 to 77.

80. The medical preparation according to claim 79, which exists in the form of a kit comprising a first container containing the antibody drug conjugate and a second container containing the chemotherapeutic agent.

81. A medical preparation according to claim 79 or 80, further comprising printed instructions relating to the use of the preparation for the treatment or prevention of cancer.

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