Diagnosis and treatment of cancer involving cancer stem cells

Abstract

This application relates to the diagnosis and treatment of cancer involving cancer stem cells. The present invention provides methods for diagnosing or treating cancer diseases involving cancer stem cells, including targeting CLDN6. Specifically, the present invention provides methods for identifying cancer stem cells, including detecting cells expressing CLDN6. Furthermore, the present invention provides methods for treating or preventing cancer, including inhibiting and / or eliminating cancer stem cells by administering an antibody capable of binding to CLDN6 to a cancer patient.

Description

[0001] This application is a divisional application of Chinese patent application No. 201480042769.8 entitled "Diagnosis and Treatment of Cancer Involving Cancer Stem Cells". The original application was PCT international application PCT / EP2014 / 066330 filed on July 30, 2014, which entered the Chinese national phase on January 28, 2016. Technical Field

[0002] This application relates to the diagnosis and treatment of cancer stem cells. Background Technology

[0003] Conventional cancer treatments primarily attempt to selectively detect and eradicate most rapidly growing cancer cells (i.e., cells that form tumor masses) and exert their toxicity on cancer cells mainly by interfering with the cellular mechanisms involved in cell growth and DNA replication. Furthermore, standard oncology protocols are largely designed to administer the highest possible dose of irradiation or chemotherapy without excessive toxicity; this is commonly referred to as the “maximum tolerated dose” (MTD).

[0004] Chemotherapy regimens often involve the administration of combinations of chemotherapy agents in an attempt to enhance the efficacy of the treatment. Although many types of chemotherapy agents are available, these treatments have several drawbacks. For example, chemotherapy agents can cause significant and often dangerous side effects due to their nonspecific effects on rapidly growing cells, whether normal or malignant.

[0005] Other types of cancer treatments include surgery, hormone therapy, immunotherapy, epigenetic therapy, anti-angiogenic therapy, targeted therapy, and radiation therapy to eradicate neoplasmic cells in patients.

[0006] However, all conventional methods used to treat cancer have significant drawbacks for patients, including lack of efficacy (especially in terms of long-term outcomes) and toxicity. Therefore, new therapies are needed for treating cancer patients.

[0007] Mounting evidence suggests the existence of a subset of cancer cells within tumors that retain stem cell-like characteristics. This subset is termed cancer stem cells (CSCs). Cancer stem cells possess similar properties to normal stem cells, exhibiting self-renewal capabilities and forming all heterogeneous cell types characteristic of tumors. An effective assay for analyzing CSC-like characteristics in tumor cells is the colony formation assay. This assay allows for easy examination of the self-renewal capacity and tumorigenetic potential of individual tumor cells.

[0008] Cancer stem cells are believed to initiate tumor formation, maintain tumor growth, and potentially spread to distant organs within the body. Cancer stem cells comprise a distinct subpopulation of tumor cells that are more tumorigenic, grow relatively slower or remain quiescent, and tend to be relatively more chemoresistant than the rest of the tumor mass. Because conventional cancer treatments target rapidly proliferating cells (i.e., the cells that form the tumor mass), these treatments are considered relatively ineffective in targeting and damaging cancer stem cells. Cancer stem cells can exhibit other characteristics that make them relatively more chemoresistant, such as multidrug resistance and anti-apoptotic pathways. The inability to adequately target and eradicate cancer stem cells constitutes a key reason why standard oncology treatment regimens cannot be implemented to ensure long-term benefit for many cancer patients. Therefore, cancer stem cells may not only be a major cause of cancer recurrence and drug ineffectiveness after treatment but also a major cause of malignant cancer metastasis. Thus, one opportunity to cure cancer lies in eliminating cancer stem cells.

[0009] Claudins are integrated membrane proteins located within tight junctions between the epithelium and endothelium. Claudins are predicted to have four transmembrane segments, two extracellular loops, and N-termini and C-termini located in the cytoplasm. The claudin (CLDN) family of transmembrane proteins plays a crucial role in the maintenance of epithelial-endothelial tight junctions and may also play a role in the maintenance of the cytoskeleton and cell signal transduction. CLDN6 is expressed in a range of different human cancer cells, while expression in normal tissues is limited to the placenta.

[0010] The data we present here demonstrate that CLDN6 expression is upregulated during the generation of pluripotent cells. Furthermore, CLDN6 is closely associated with known markers of cancer stem cells, and CLDN6-positive tumor cells exhibit increased colony formation. This also demonstrates that treatment with CLDN6-specific antibodies can overcome chemotherapy resistance in tumors (e.g., ovarian cancer), and that the combination of chemotherapy and CLDN6 antibody therapy has a significant synergistic effect.

[0011] The results presented in this paper indicate that CLDN6 is a novel biomarker for cancer stem cells, and that cancer stem cells can be targeted by targeting CLDN6 for diagnostic and therapeutic purposes. Summary of the Invention

[0012] In one aspect, the present invention relates to a method for identifying cancer stem cells, comprising detecting cells expressing CLDN6.

[0013] In one embodiment, the presence of cells expressing CLDN6 indicates the presence of cancer stem cells and / or the amount of cells expressing CLDN6 is correlated with the amount of cancer stem cells. In one embodiment, cells expressing CLDN6 are detected in samples obtained from a cancer patient, for example, before, during, and / or after cancer treatment. In one embodiment, the method includes quantitative and / or qualitative determination of cells expressing CLDN6. In one embodiment, the method includes comparing the amount of cells expressing CLDN6 with the amount of cells expressing CLDN6 in a reference sample or with a predetermined reference range. The reference sample may be a sample from a patient diagnosed as cancer-free. The predetermined reference range may be based on a patient population diagnosed as cancer-free. In one embodiment, the method includes monitoring the amount of cancer stem cells in a cancer patient, wherein monitoring 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 with the amount of cancer stem cells in a sample obtained earlier from the cancer patient. In one embodiment, the sample obtained from the cancer patient is a sample taken from the cancer patient during or after the administration of cancer treatment.

[0014] In another aspect, the present invention relates to a method for monitoring the efficacy of cancer treatment in cancer patients, comprising: i) determining the amount of cancer stem cells in a sample obtained from the cancer patient during or after administration of cancer treatment; and (ii) comparing the amount of cancer stem cells in the sample obtained from the cancer patient with the amount of cancer stem cells in a previously obtained sample from the cancer patient, wherein determining the amount of cancer stem cells in the sample obtained from the cancer patient and / or determining the amount of cancer stem cells in a previously obtained sample from the cancer patient includes determining the amount of cells expressing CLDN6.

[0015] In one implementation, the sample obtained earlier from the cancer patient is a sample taken from the cancer patient before, during, or after the administration of cancer treatment.

[0016] In one embodiment of the method of all aspects of the present invention, a stable or decreasing amount of cancer stem cells indicates effective cancer treatment. In one embodiment of the method of all aspects of the present invention, an increase in the amount of cancer stem cells indicates ineffective cancer treatment. In one embodiment of the method of all aspects of the present invention, the cancer treatment is a cancer treatment targeting cancer stem cells. In one embodiment of the method of all aspects of the present invention, the sample obtained from the cancer patient is a biofluid or tumor tissue biopsy. In one embodiment of the method of all aspects of the present invention, the sample has undergone one or more pretreatment steps. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 or their amount are detected or determined by detecting or determining 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 or their quantity are detected or determined by using an immunoassay, wherein the immunoassay is preferably selected from: Western blot, immunohistochemistry, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, immunoradiometric assay, fluorescence immunoassay, immunofluorescence, protein A immunoassay, flow cytometry, and FACS analysis. In one embodiment of the method of all aspects of the present invention, cells expressing CLDN6 or their quantity are detected or determined by using an antibody capable of binding to CLDN6. In one embodiment of the method of all aspects of the present invention, the cells expressing CLDN6 are CLDN6-expressing cancer cells and / or cells present at tumor sites.

[0017] In another aspect, the present invention relates to a method for treating or preventing cancer, comprising inhibiting and / or eliminating cancer stem cells by administering an antibody capable of binding CLDN6 to a cancer patient.

[0018] In one embodiment, the cancer stem cells express CLDN6. In one embodiment, the method further includes administering chemotherapy and / or radiation therapy. In one embodiment, inhibiting and / or eliminating cancer stem cells enhances the anti-cancer effect of chemotherapy and / or radiation therapy, wherein the enhancement of the anti-cancer effect of chemotherapy and / or radiation therapy includes an extension of lifespan in cancer patients undergoing chemotherapy and / or radiation therapy.

[0019] In another aspect, the present invention relates to a method of treating or preventing cancer, comprising administering (i) an antibody capable of binding to CLDN6 and (ii) chemotherapy to a cancer patient.

[0020] In one embodiment, the cancer involves cancer stem cells expressing CLDN6. In one embodiment, administration of an antibody capable of binding CLDN6 results in the inhibition or elimination of cancer stem cells expressing CLDN6. In one embodiment, administration of an antibody capable of binding CLDN6 enhances the anticancer effect of chemotherapy, wherein the enhancement of the anticancer effect of chemotherapy preferably includes an extension of the lifespan of the cancer patient undergoing chemotherapy.

[0021] In one embodiment of the method of all aspects of the present invention, the elimination of cancer stem cells leads to cancer cure. In one embodiment of the method of all aspects of the present invention, an antibody capable of binding to CLDN6 and chemotherapy are administered in a synergistically effective amount. In one embodiment of the method of all aspects of the present invention, chemotherapy is administered at a dose below the maximum tolerated dose. In one embodiment of the method of all aspects of the present invention, chemotherapy comprises administering an agent selected from: taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, their prodrugs, their salts, and combinations thereof. In one embodiment of the method of all aspects of the present invention, chemotherapy comprises administering an agent selected from: paclitaxel, cisplatin, carboplatin, their prodrugs, their salts, and combinations thereof. In one embodiment of the method of all aspects of the present invention, cancer stem cells are located at the tumor site of a cancer patient. In one embodiment of the method of all aspects of the present invention, the cancer is resistant to chemotherapy, particularly if the chemotherapy is administered as a single treatment. In one embodiment of the method of all aspects of the present invention, an antibody capable of binding to CLDN6 has an inhibitory and / or cytotoxic effect on cancer stem cells, wherein the antibody capable of binding to CLDN6 exerts its inhibitory and / or cytotoxic effect on cancer stem cells by mediating one or more of the following: complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-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 coupled to a therapeutic portion, and may be an antibody-drug conjugate as described herein. In one embodiment, the therapeutic portion is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide. In one embodiment, the therapeutic portion acts on slow-growing 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 of all aspects of the present invention, the antibody capable of binding CLDN6 comprises: a heavy chain variable region (VH) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:5 and a light chain variable region (VL) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:4.

[0022] In another aspect, the present invention relates to a method of treating or preventing cancer, comprising administering an antibody-drug conjugate to a cancer patient, said antibody-drug conjugate comprising an antibody capable of binding CLDN6 covalently linked to at least one toxic drug moiety via a linker.

[0023] In one embodiment, the toxin drug portion is permeable to the cell membrane. In one embodiment, at least one of the toxin drug portions acts on slowly growing cells. In one embodiment, the toxin drug portion is maytansinoid or auristatin. In one embodiment, the maytansinoid is selected from DM1 and DM4. In one embodiment, the auristatin is selected from monomethylauristatin E (MMAE) and monomethylauristatin 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 a cysteine ​​sulfhydryl group of the antibody.

[0024] In one embodiment, the cancer involves cancer stem cells expressing CLDN6. In one embodiment, administration of the antibody-drug conjugate results in the inhibition or elimination of cancer stem cells expressing CLDN6. In one embodiment, the elimination of the cancer stem cells leads to cancer cure. In one embodiment, the cancer stem cells are located at the tumor site of the cancer patient. In one embodiment, the antibody-drug conjugate has inhibitory and / or cytotoxic effects on cancer stem cells, wherein the antibody-drug conjugate preferably exerts its inhibitory and / or cytotoxic effects on cancer stem cells by inducing apoptosis and / or inhibiting proliferation.

[0025] In one embodiment, the method further includes administering chemotherapy and / or radiation therapy.

[0026] In one embodiment, the administration of the antibody-drug conjugate enhances the anticancer effect of chemotherapy and / or radiotherapy, wherein the enhancement of the anticancer effect of chemotherapy and / or radiotherapy preferably includes an extension of the lifespan of cancer patients undergoing chemotherapy and / or radiotherapy.

[0027] In one embodiment, the antibody-drug conjugate and the chemotherapy are administered at a synergistically effective amount. In one embodiment, the chemotherapy is administered at a dose below the maximum tolerated dose. In one embodiment, the chemotherapy comprises administering an agent selected from: taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, their prodrugs, their salts, and combinations thereof. In one embodiment, the chemotherapy comprises administering an agent selected from: paclitaxel, cisplatin, carboplatin, their prodrugs, their salts, and combinations thereof. In one embodiment, the cancer is resistant to chemotherapy, particularly if the chemotherapy is administered as a single treatment.

[0028] In one embodiment, the antibody capable of binding CLDN6 (particularly when present in the antibody-drug conjugate) has affinity and / or specificity for CLDN6 to suitably enable endocytosis of the antibody and / or the antibody-drug conjugate. In one embodiment, the CLDN6-binding antibody in the antibody-drug conjugate binds to a first extracellular loop of the CLDN6. In one embodiment, the CLDN6-binding antibody in the antibody-drug conjugate comprises a heavy chain variable region (VH) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:5 and a light chain variable region (VL) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:4.

[0029] 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.

[0030] In another aspect, the present invention relates to a method for treating or preventing cancer, comprising: (i) identifying cancer stem cells in a cancer patient by the method of the present invention and (ii) administering a cancer treatment targeting the cancer stem cells to the cancer patient. In one embodiment, the cancer treatment targeting the cancer stem cells comprises implementing the method of treating or preventing cancer according to the present invention.

[0031] In another aspect, the present invention relates to methods for preventing cancer chemoresistance, cancer recurrence, or cancer metastasis (particularly during or after cancer treatment), said methods including treating cancer by the methods of the present invention.

[0032] In another aspect, the present invention provides a medical preparation for treating or preventing cancer, comprising (i) an antibody capable of binding CLDN6 and (ii) a chemotherapeutic agent. The antibody capable of binding CLDN6 and the chemotherapeutic agent may be present in the medical preparation in a mixture or separately from each other. The medical preparation may be in the form of a cassette comprising a first container containing the antibody capable of binding CLDN6 and a second container containing the chemotherapeutic agent. The medical preparation may also include printed instructions for using the preparation (particularly the preparation using the method of the present invention) to treat or prevent cancer. Different embodiments of the medical preparation (particularly the antibody and chemotherapeutic agent capable of binding CLDN6) are described herein.

[0033] In one specific aspect, the present invention provides a pharmaceutical product comprising (i) an antibody capable of binding CLDN6 and (ii) paclitaxel. The antibody capable of binding CLDN6 and paclitaxel may be present in the pharmaceutical product in a mixture or separately from each other. The pharmaceutical product may be used to treat or prevent cancer, such as ovarian cancer. The pharmaceutical product may be in the form of a cassette comprising a first container containing the antibody capable of binding CLDN6 and a second container containing paclitaxel. The pharmaceutical product may also include printed instructions for using the product (particularly the product used in the methods of the present invention) to treat or prevent cancer (e.g., ovarian cancer). Different embodiments of the pharmaceutical product (particularly the antibody capable of binding CLDN6) are described herein.

[0034] In another aspect, the present invention provides an antibody-drug conjugate comprising an antibody capable of binding CLDN6 covalently linked to at least one toxin drug moiety via a linker.

[0035] In one embodiment, the toxin drug portion is permeable to the cell membrane. In one embodiment, at least one of the toxin drug portions acts on slowly growing cells. In one embodiment, the toxin drug portion is maytansine alkaloid or olipattin. In one embodiment, the maytansine alkaloid is selected from DM1 and DM4. In one embodiment, the olipattin is selected from monomethylolipattin E (MMAE) and monomethylolipattin 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 a cysteine ​​sulfhydryl group of the antibody.

[0036] In one embodiment, the antibody capable of binding CLDN6 (particularly when present in the antibody-drug conjugate) has affinity and / or specificity for CLDN6 to suitably cause endocytosis of the antibody and / or the antibody-drug conjugate. In one embodiment, the CLDN6-binding antibody in the antibody-drug conjugate binds to a first extracellular loop of the CLDN6. In one embodiment, the CLDN6-binding antibody in the antibody-drug conjugate comprises a heavy chain variable region (VH) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:5 and a light chain variable region (VL) containing the amino acid sequence or a fragment thereof shown in SEQ ID NO:4.

[0037] In another aspect, the present invention provides a pharmaceutical formulation comprising the antibody-drug conjugate of the present invention, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0038] In another aspect, the present invention provides a pharmaceutical product comprising the antibody-drug conjugate of the present invention and a chemotherapeutic agent. Preferably, the pharmaceutical product is used to treat or prevent cancer. The antibody-drug conjugate and the chemotherapeutic agent may be present in the pharmaceutical product as a mixture or separately from each other. The pharmaceutical product may be in the form of a cassette comprising a first container containing the antibody-drug conjugate and a second container containing the chemotherapeutic agent. The pharmaceutical product may also include printed instructions for using the product (particularly the product used in the method of the present invention) to treat or prevent cancer. Different embodiments of the pharmaceutical product (particularly the antibody-drug conjugate and the chemotherapeutic agent) are described herein.

[0039] In one specific aspect, the present invention provides a pharmaceutical product comprising the antibody-drug conjugate of the present invention and paclitaxel. The antibody-drug conjugate and paclitaxel may be present in the pharmaceutical product as a mixture or separately from each other. The pharmaceutical product may be used to treat or prevent cancer, such as ovarian cancer. The pharmaceutical product may be in the form of a medicine box comprising a first container containing the antibody-drug conjugate and a second container containing paclitaxel. The pharmaceutical product may also include printed instructions for using the product (particularly the product used in the method of the present invention) to treat or prevent cancer (e.g., ovarian cancer). Different embodiments of the pharmaceutical product (particularly the antibody-drug conjugate) are described herein.

[0040] The present invention also provides pharmaceutical agents and compositions described herein, such as antibody-drug conjugates, antibodies capable of binding CLDN6, and / or chemotherapeutic agents for use in the methods described herein. For example, the present invention also provides antibody-drug conjugates or antibodies capable of binding CLDN6 for administration in conjunction with chemotherapeutic agents (e.g., paclitaxel).

[0041] In one embodiment, the antibody capable of binding CLDN6 is a monoclonal antibody, a chimeric antibody, or a humanized antibody or a fragment of an antibody. In one embodiment, when binding to CLDN6 of a cell (particularly CLDN6 expressed on the cell surface), the antibody mediates cell killing, wherein the cell is preferably a cancer stem cell, such as a cancer stem cell of the cancer described herein.

[0042] According to the present invention, the cancers are preferably selected from: ovarian cancer (especially ovarian adenocarcinoma and ovarian teratoma), lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (especially squamous cell lung cancer and adenocarcinoma), large cell carcinoma (LCC), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer (especially basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (especially malignant pleomorphic adenoma), sarcoma (especially synovial sarcoma and carcinosarcoma), bile duct cancer, bladder cancer (especially transitional cell carcinoma and papillary carcinoma), kidney cancer (especially renal cell carcinoma) including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small bowel cancer including ileal cancer (especially small bowel adenocarcinoma and ileal adenocarcinoma), placental choriocarcinoma, cervical cancer, testicular cancer (especially testicular seminoma, testicular teratoma and testicular embryonal carcinoma), uterine cancer, germ cell tumors such as teratoma or embryonal carcinoma (especially germ cell tumors of the testes and ovaries), and their metastatic forms.

[0043] According to the present invention, cancer cells and / or cancer stem cells expressing CLDN6 are preferably cancer cells as described herein.

[0044] 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.

[0045] In one embodiment, the cancer described herein includes primary cancer, advanced cancer, metastatic cancer, recurrent cancer, or combinations thereof, such as a combination of primary cancer and metastatic cancer. In one embodiment, the cancer is partially or completely refractory to chemotherapy (e.g., paclitaxel monotherapy). In one embodiment, the cancer is ovarian cancer, particularly ovarian cancer that is partially or completely refractory to chemotherapy (e.g., paclitaxel monotherapy).

[0046] Other features and advantages of the invention will become apparent from the following detailed description and claims. Attached Figure Description

[0047] Figure 1 CLDN6 mRNA is expressed in human iPS cells.

[0048] Human foreskin fibroblasts (HFF) were transfected with Lipofectamine RNAiMAX (Life Technologies) using either RNA-free (RNA-free control) or a reprogramming cocktail (unmodified OSKMNL+EBK+miR-mix), and cells were collected on days 5, 12, and 19 post-treatment. RNA was extracted, transcribed into cDNA, and then analyzed by quantitative real-time RT-PCR using the ABIPRISM 7300 sequence detection system and software (AppliedBiosystems (Qiagen) with QuantiTect SYBR green kit). The fold induction of CLDN6 expression is shown between cells treated with the reprogramming cocktail (black column) and HFF cells from day 1 treatment (gray column). CLDN6 mRNA expression was normalized to the mRNA expression of the housekeeping gene HPRT1. OSKMNL = transcription factors OCT4, SOX2, KLF4, cMYC, NANOG and (und)LIN28, EBK = IFN-escape protein E3, K3 and B18R, miR-mix = miRNA-302a / b / c / d and 367.

[0049] Figure 2 CLDN6 is expressed on the surface of human iPS cells.

[0050] HFF cells were transfected with RNA-free (RNA-free control) or with a reprogramming mixture (unmodified OSKMNL+EBK+miR-mix) and cells were collected on days 5 (A), 12 (B), and 19 (C) post-treatment. Cells were stained for 30 minutes at 4°C with 1 μg / ml of CLDN6-specific IMAB027-AF647 and SSEA-4-V450 antibodies (2.5 μl per test, purchased from BD) and surface expression was analyzed by flow cytometry. Experiments were performed twice 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.

[0051] Figure 3 : Surface expression of CLDN6 in ovarian cancer cell lines.

[0052] To analyze CLDN6 expression, 1E6 cells were stained with 1 μg / ml IMAB027-AF647 at 4°C for 30 min, and surface expression was analyzed by flow cytometry. COV318 cells are shown in (A). The experiment was performed in triplicate, and a representative dot plot is shown. In (B), PA-1 cells stably transfected with the control vector (PA-1 76) or PA-1 cells transfected with a vector expressing shRNA targeting CLDN6 (clones PA-1 50 and PA-1 54) are shown. The experiment was performed in triplicate, and a representative dot plot is given. shRNA = hairpin RNA.

[0053] Figure 4 CLDN6 is important for the colony formation of ovarian cancer cells.

[0054] To analyze clonogenic behavior, COV318, PA-1 50, and PA-1 54 cells were stained with 1 μg / ml IMAB027-AF647 at 4°C for 30 minutes. Then, 700 (COV318) or 500 (PA-150 / 54) CLDN6-positive or CLDN6-negative cells were sorted into 6-well plates. Cell colonies were allowed to form for 14 days, followed by staining 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. Means and standard deviations from three independent experiments are shown.

[0055] Figure 5 In the ovarian cancer cell line COV318, CLDN6 is co-expressed with CSC markers CD24, CD90, and CD44.

[0056] At 4°C, 1E6COV318 cells were stained for 30 minutes with antibodies targeting different surface markers from the FACS panels shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triplicate. (A) shows a representative dot plot of colocalization of CLDN6 with different established CSC markers. (B) shows the percentage of CD44, CD24, and CD90 colocalized with CLDN6-positive cells, calculated using different gating strategies shown on the x-axis of this plot. The mean and standard deviation of the three replicates are shown.

[0057] Figure 6 The enrichment of cells expressing CLDN6 led to the accumulation of established CSC markers.

[0058] COV318 cells were stained with 0.5 μg / ml IMAB027 and APC-conjugated goat anti-human IgG secondary antibody (1:300), and then separated into CLDN6-positive and CLDN6-negative fractions by FACS sorting. Cells from both fractions were allowed to expand for 10 days. At 4°C, 1E6 cells from each fraction were stained for 30 minutes with antibodies targeting different surface markers according to the FACS groups shown in Table 1. The experiment was performed in triplicate. (A) shows a representative dot plot of the expression levels of different CSC markers in the CLDN6-positive and CLDN6-negative fractions, as well as their co-localization with CLDN6. (B) shows the percentage of CSC marker expression levels, and the enrichment factors (fold increases) of the relevant markers CD44, CD90, and CD24 were calculated by comparing the percentage of positive cells in the CLDN6-positive and CLDN6-negative fractions.

[0059] Figure 7 Compared with cells with low CLDN6 expression, cell lines with high CLDN6 expression showed enrichment of CSC markers.

[0060] At 4°C, 1E6 cells of ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) with high CLDN6 expression were stained for 30 minutes with antibodies targeting different surface markers in the FACS groups shown in Table 1, and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triplicate and representative dot plots are shown.

[0061] Figure 8 Antitumor effects of the combination of IMAB027 and paclitaxel in early xenograft tumor models.

[0062] Subcutaneous human ES-2 xenograft tumors expressing human CLDN6 were treated with paclitaxel via intraperitoneal (ip) injection at days 3, 10, and 17 post-transplantation. Antibody maintenance therapy was initiated on day 4, with IMAB027 administered three times weekly via intravenous (iv) / ip / ip alternation. (A) Mean tumor growth kinetics (±SEM) after treatment with IMAB027 (white squares), paclitaxel (gray circles), a combination of IMAB027 and paclitaxel (black squares), or a load-controlled (white circle). Arrows indicate the time point at which treatment began. (B) Survival curves of treated mice. Group size: n = 12.

[0063] Figure 9 Antitumor effects of IMAB027 combined with cisplatin in advanced xenograft tumor models.

[0064] Before treatment begins, the subcutaneous human NEC14 xenograft tumor is allowed to grow to approximately 100 mm. 3 The median size. Mice were treated daily with intraperitoneal injection of 1 mg / kg cisplatin from day 6 to day 10 post-transplantation, and maintenance therapy was initiated from day 6 with 35 mg / kg IMAB027 three times weekly (alternating IV / IP / IP). (A) Mean tumor growth kinetics (±SEM) after treatment with IMAB027 (solid circles), cisplatin (hollow squares), a combination of IMAB027 and cisplatin (solid squares), or a load-controlled (hollow circle) regimen. Arrows indicate the time points at which treatment began. (B) Size of a single tumor in mice on day 24 post-transplantation (mean ± standard deviation). (C) Survival curves of treated mice. Group size: n = 19. P values: *, P < 0.05; **, P < 0.01 and ***, P < 0.001.

[0065] Figure 10 Antitumor effects of the combination of IMAB027 and carboplatin in an advanced xenograft tumor model.

[0066] like Figure 9 The study described the treatment of advanced human NEC14 xenograft tumors with IMAB027 alone or in combination with a cell growth inhibitor. Mice were treated with 30 mg / kg carboplatin (instead of cisplatin) via rapid intraperitoneal bolus injection on days 6, 13, and 20. (A) Mean tumor growth kinetics (±SEM) after treatment with IMAB027 (solid circles), carboplatin (hollow squares), IMAB027 in combination with carboplatin (solid squares), or load control (hollow circles). Arrows indicate the time point at which treatment began. (B) Size of a single tumor in mice on day 24 post-transplantation (mean ± standard deviation). (C) Survival curves of treated mice. Group size: n = 19. P values: *, P < 0.05; **, P < 0.01 and ***, P < 0.001.

[0067] Figure 11 CLDN6 is important for the spheroidization behavior of ovarian cancer cells.

[0068] To analyze the effect of CLDN6 on spheroid 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 attachment plates under spheroid 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 spheroids from CLDN6-positive (CLDN6+) and CLDN6-negative (CLDN6-) COV318 cells at days 3, 8, and 19 post-sorting. (B) Representative images of second-generation spheroids obtained from single cells of the first-generation CLDN6+ spheroids in (A) at day 22 post-sorting.

[0069] Figure 12 Enrichment of CLDN6 positive cells after treatment with platinum derivatives.

[0070] COV318 cells were treated with 500 ng / ml cisplatin or 2000 ng / ml carboplatin for 4 days. Following treatment, cells were regrowth for 3 days (white column) and 6 days (black column) respectively, without the use of cell growth inhibitors. CLDN6 expression was analyzed by flow cytometry using the CLDN6-specific antibody IMAB027 and an isotype control antibody. The expression of treated COV318 cells relative to untreated cells is shown. For evaluation, the isotype control value was subtracted from the CLDN6 staining.

[0071] Figure 13 Enrichment of CLDN6-positive cells after intraperitoneal transplantation.

[0072] 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. CLDN6 expression in isolated cells was analyzed immediately after preparation and after several passages in culture. (A) Flow cytometry analysis of CLDN6 expression in parental COV318 cells using the CLDN6-specific antibody IMAB027 and an isotype control. (B) CLDN6 expression in cells derived from ascites and solid tumors from the ovary, liver, stomach, pancreas, and diaphragm at different time points after isolation (*: ascites at days 5 and 35; **: solid tumors at days 12 and 29). Fluorescence intensity is shown on the X-axis. The number of events shown on the Y-axis is scaled as a percentage of the maximum number of events.

[0073] Figure 14CLDN6 is associated with ovarian cancer stem cell markers in primary tumor samples.

[0074] The mRNA expression level of CLDN6 and several described ovarian cancer stem cell biomarkers in 42 ovarian cancer samples were analyzed by qRT-PCR using the Fluidigm detection system and software. Spearman correlation analysis was performed to analyze the correlation between CLDN6 and cancer stem cell-specific biomarkers. A scatter plot of significant correlations (P ≤ 0.05) is shown in (A). A summary of all correlations is shown in (B).

[0075] Figure 15 : IMAB027-mediated ADCC following treatment with carboplatin and paclitaxel.

[0076] ADCC activity of the combination of IMAB027 and chemotherapy was analyzed using COV362 (Luc) target cells. Cells were treated for 4 days with specified concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan. Following treatment, cells were grown for 3 days (AD) and 10 days (EJ) without cell growth inhibitors. Control cells were cultured without cell growth inhibitors. ADCC experiments (A, C, E, G, I) were performed using PBMCs from healthy donors with IMAB027 (black line) or an allotype control antibody (gray line) at a ~40:1 ratio of effector (PBMC) to target cells. Data points (n = 4 replicates) were plotted as mean ± SD. CLDN6 expression was analyzed by flow cytometry using IMAB027 (B, D, F, H, J). Black dashed lines indicate CLDN6 expression in untreated cells, and gray solid histograms represent CLDN6 expression after treatment.

[0077] Figure 16 Antitumor effects of the combination of IMAB027 and PEB treatment in a very late-stage xenograft tumor model.

[0078] Subcutaneous human NEC14 xenograft tumors were induced to grow to a very late 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 cisplatin at 1 mg / kg and etoposide at 5 mg / kg on days 13, 14, 15, 16, and 17, and with bleomycin at 10 mg / kg on days 13, 17, and 21 via intraperitoneal injection. IMAB027 was administered via 35 mg / kg via alternating IV / IP / IP three times weekly from day 13 to day 101 post-transplantation. The load-controlled group received 0.9% NaCl solution and drug substance buffer instead. Mice were monitored for a total of 220 days. (A), (B) Mean tumor growth kinetics (±SEM) in untreated mice and mice treated with IMAB027, PEB, or a combination of PEB and IMAB027. Arrows indicate the time points at which treatment began (Dunn multiple comparison test: ***, P<0.001). (C) Survival curves of untreated mice and mice treated with IMAB027, PEB, or a combination of PEB and IMAB027 (Mantel-Cox test: *, P<0.05; **, P<0.01). Group size: n=14.

[0079] Figure 17 The relative binding affinity and cytotoxicity of IMAB027, IMAB027-DM1 and IMAB027-vcMMAE.

[0080] (A) Flow cytometry analysis was used to measure the binding of IMAB027, IMAB027-DM1, and IMAB027-vcMMAE to 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. The reduction in cell viability was measured using an XTT-based viability assay. Data points (n = 3 replicates) are plotted as mean ± SD. MFI: mean fluorescence intensity.

[0081] Figure 18 Antitumor effect of IMAB027-DM1 conjugate on advanced xenograft tumors.

[0082] Ten days post-transplantation, nude mice with established subcutaneous human OV90 xenograft tumors were treated with a single intravenous injection of 1.78 mg / kg, 5.33 mg / kg, or 16 mg / kg IMAB027-DM1, or a loading control. Subcutaneous tumor size (mean + SEM) was measured twice weekly. Group size: n = 5, *: P < 0.05, **: P < 0.01.

[0083] Figure 19 Determination of the dose range of IMAB027-DM1 and IMAB027-vcMMAE conjugates for advanced OV90 xenograft tumors.

[0084] Ten days post-transplantation, nude mice with established subcutaneous human OV90 xenograft tumors were treated with single-dose intravenous injections of IMAB027-DM1, IMAB027-vcMMAE, the loading agent, or multiple-dose injections of IMAB027. (A) Tumor growth in mice treated with 1.33, 2.67, and 5.33 mg / kg IMAB027-DM1 i.v. (top) or 4, 8, or 16 mg / kg IMAB027-vcMMAE iv (bottom) compared to the loading control and IMAB027 (35 mg / kg, iv / ip / ip weekly). Subcutaneous tumor size (mean + SEM) was measured twice weekly. (B) Kaplan-Meier survival curves in mice treated with the loading agent or 4, 8, or 16 mg / kg IMAB027-vcMMAE. When the tumor volume reached 1400 mm²... 3 Mice were euthanized if the tumor became ulcerative. Group size: n = 10, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0085] Figure 20 Determination of the dose range of the IMAB027-vcMMAE conjugate for advanced PA-1 xenograft tumors.

[0086] Fifteen days post-transplantation, nude mice with established subcutaneous human PA-1 xenograft tumors were treated with single-dose intravenous injection of IMAB027-vcMMAE, a load-controlled agent, or multiple-dose injections of IMAB027. (A) Mean tumor growth (±SEM) and (B) Kaplan-Meier survival curves of mice treated with load-controlled agent, IMAB027 (35 mg / kg, weekly iv / ip / ip), or 4, 8, or 16 mg / kg of IMAB027-vcMMAE. When the tumor volume reached 1400 mm², the tumor growth was significantly reduced. 3Mice were euthanized if the tumor became ulcerative. Group size: n = 8, *: P < 0.05, **: P < 0.01. (C) Representative immunohistochemical staining of CLDN6 in PA-1 xenograft tumor sections at different time points post-transplantation.

[0087] Figure 21 The antitumor effect of IMAB027-vcMMAE on advanced MKN74 xenograft tumors.

[0088] Seven days post-transplantation, nude mice with established subcutaneous human MKN74 xenograft tumors were treated with intravenous injection of 16 mg / kg IMAB027-vcMMAE or a load-controlled agent. (A) Mean tumor growth (±SEM) and (B) Kaplan-Meier survival curves of mice treated with the load-controlled agent or IMAB027-vcMMAE. When the tumor volume reached 1400 mm²... 3 Mice were euthanized if the tumor became ulcerative. Group size: n = 10. (C) Flow cytometry analysis of CLDN6 expression on MKN74 tumor cells before transplantation and representative immunohistochemical staining of untreated MKN74 xenograft tumors on day 31 post-transplantation. **: P < 0.01, ***: P < 0.001.

[0089] Figure 22 The antitumor effects of IMAB027-DM1 and IMAB027-vcMMAE on advanced intraperitoneal metastatic human ovarian tumors.

[0090] Nude mice were implanted intraperitoneally with the human ovarian cancer cell line PA-1 (Luc) expressing luciferase. After intraperitoneal metastatic xenograft tumors formed, animals were treated on day 14 post-transplantation with intraperitoneal injections of 16 mg / kg IMAB027-DM1, IMAB027-vcMMAE, or a loading control. Metastatic tumor growth was determined by luminescent administration of luciferase using an IVIS Lumina imaging system. (A) Quantification of metastasis load in mice treated with IMAB027-DM1, IMAB027-vcMMAE, or a loading control. (B) Whole-body luminescent images of nude mice on day 28 post-transplantation. Group size: n = 8 (loading) or n = 9 (IMAB027-DM1, IMAB027-vcMMAE), **: P < 0.01, **: P < 0.0001.

[0091] Figure 23 : Endocytosis of human cancer cells against antibodies binding to CLDN6.

[0092] Cytotoxicity-based assays were used to determine endocytosis of CLDN6-binding IMAB027, chimAB5F2D2, or allotype control antibodies, relying on co-internalization of the target antibody with a saporin-conjugated anti-human IgG Fab fragment (FabZap). PA-1, OV90, or NEC14 human cancer cells were incubated with IMAB027, chimAB5F2D2, or allotype control antibodies and anti-human FabZap for 72 hours. (A) Dose-response curves of reduced viability of PA-1, OV90, and NEC14 cells mediated by IMAB027 / FabZap and chimAB5F2D2 / FabZap, respectively. Data points (n = 3 replicates) are plotted as mean ± SD. (B) Comparison of IMAB027-normalized EC50 (relative EC50) and maximum (relative maximum) for binding and endocytosis by flow cytometry. Detailed Implementation

[0093] Although the invention has been described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these methods, schemes, and reagents can be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0094] The elements of the invention will be described below. These elements are listed alongside specific embodiments; however, it should be understood that they can be combined in any manner and in any number to form other embodiments. The various embodiments and preferred embodiments described herein should not be construed as limiting the invention to the explicitly described embodiments. This description should be understood to support and cover embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any arrangement and combination of all elements described herein should be considered as disclosed in this specification unless the context otherwise indicates.

[0095] Preferably, the terminology used herein is such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. Leuenberger, B. Nagel, and H. The definition is as described in Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

[0096] Unless otherwise specified, the present invention will be carried out using conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology as explained in the literature in this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0097] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprising / including" and its variations shall be understood to mean including the stated members, integers or steps, or groups of members, integers or steps, but not excluding any other members, integers or steps, or groups of members, integers or steps, although in some embodiments such other members, integers or steps, or groups of members, integers or steps may be excluded; that is, the subject matter is to include the stated members, integers or steps, or groups of members, integers or steps. Unless otherwise specified herein or clearly contradicted by the context, nouns without quantifiers and similar references used in the context describing the invention (especially in the context of the claims) shall be interpreted to cover both singular and plural forms. The description of numerical ranges herein is intended only as a shorthand method for individually referring to each independent value falling within that range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually referenced herein. Unless otherwise specified herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise required, the use of any and all embodiments or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the invention and not to limit the scope of the invention. No language in this specification should be construed as representing any non-claimed element necessary for carrying out the invention.

[0098] This specification contains numerous references. Each reference cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, guidelines, etc.), whether mentioned above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that this invention is not a prior invention preceding these disclosures.

[0099] Claudins are a family of proteins that are among the most important components of tight junctions, forming a paracellular barrier that controls molecular flow in the intercellular space between epithelial cells. Claudins are four-terminal transmembrane proteins, with both their N-terminus and C-terminus located in the cytoplasm. The first extracellular loop, called EC1 or ECL1, consists of an average of 53 amino acids, while the second extracellular loop, called EC2 or ECL2, consists of approximately 24 amino acids. Cell surface proteins of the claudin family (such as CLDN6) are expressed in tumors from a variety of sources and are particularly well-suited as target structures for antibody-mediated cancer immunotherapy due to their selective expression (absence in toxicity-associated normal tissues) and localization to the plasma membrane.

[0100] CLDN6 has been identified as differentially expressed in tumor tissues. The only normal tissue expressing CLDN6 is the placenta, where small amounts of CLDN6 were detected at the RNA level. CLDN6 has been found 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.

[0101] In various embodiments of the present invention, cancers associated with CLDN6 expression include ovarian cancer (particularly ovarian adenocarcinoma and ovarian teratoma), lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (particularly squamous cell lung cancer and adenocarcinoma), 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 transitional cell carcinoma and papillary carcinoma), kidney cancer (particularly renal cell carcinoma) including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small bowel cancer including ileal cancer (particularly small bowel adenocarcinoma and ileal adenocarcinoma), testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer (particularly testicular seminoma, testicular teratoma and embryonal testicular carcinoma), uterine cancer, germ cell tumors such as teratomas or embryonal carcinomas (particularly germ cell tumors of the testis), and their metastatic forms. In one embodiment, the cancer associated with CLDN6 expression is selected from ovarian cancer, lung cancer, metastatic ovarian cancer, and metastatic lung cancer. Preferably, the ovarian cancer is carcinoma or adenocarcinoma. Preferably, the lung cancer is carcinoma or adenocarcinoma, and more preferably, bronchiolar carcinoma, such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0102] As used herein, the term "CLDN" refers to clathrin and includes CLDN6. Preferably, the clathrin is human clathrin.

[0103] The term "CLDN6" preferably refers to human CLDN6, and more particularly to a protein comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a variant thereof, preferably a protein consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a variant thereof. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80, more preferably amino acids 28 to 76, of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, more preferably amino acids 141 to 159, and more preferably amino acids 145 to 157, of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. The first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0104] According to the present invention, the term "variant" specifically refers to mutants, splicing variants, conformations, isotypes, allele variants, species variants, and species homologs, particularly those that are naturally occurring. An allele variant refers to an alteration in the normal sequence of a gene, the significance of which is usually not obvious. For a given gene, whole-genome sequencing typically identifies multiple allele variants. A species homolog is a nucleic acid or amino acid sequence that has a different species origin than a given nucleic acid or amino acid sequence. The term "variant" should encompass any post-translational modification variants and conformational variants.

[0105] According to the present invention, the term "dimin-positive cancer" or similar terms refer to cancer involving cancer cells expressing dimin, preferably dimin expressed on the surface of said cancer cells. If CLDN6 is located on the surface of a cell, CLDN6 is expressed on the cell surface and readily binds to CLDN6-specific antibodies added to the cell.

[0106] "Cell surface" is used according to its usual meaning in the art and therefore includes the outer part of the cell that is readily bound to proteins and other molecules. For example, transmembrane proteins having one or more extracellular portions are considered to be expressed on the cell surface.

[0107] In the context of this invention, the term "extracellular portion" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of a cell, and preferably, for example, is accessible to the outside of the cell by an antigen-binding molecule (e.g., an antibody) located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0108] The terms “part” or “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure (such as an amino acid sequence or a protein) refers to a continuous element of said structure. A part, fraction, or fragment of a structure preferably contains one or more functional properties of said structure. For example, a part, fraction, or fragment of an epitope or peptide is preferably equivalent to the epitope or peptide immunology from which it is derived. A part or fragment of a protein sequence preferably contains at least six, particularly at least eight, at least ten, at least twelve, at least fifteen, at least twenty, at least thirty, at least fifty, or at least one hundred consecutive amino acids of said protein sequence.

[0109] According to the present invention, CLDN6 is substantially not expressed in cells if the expression level is lower than that in placental cells or placental tissue. Preferably, the expression level is less than 10% of that expressed 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 the expression level does not exceed twice, preferably 1.5 times, and preferably does not exceed the expression level in said non-cancerous tissues (excluding the placenta). Preferably, CLDN6 is substantially not expressed in cells if the expression level is below the detection limit and / or if the expression level is too low to be bound by a CLDN6-specific antibody added to the cells.

[0110] According to the present invention, CLDN6 is expressed in cells if its expression level is preferably more than twice, preferably more than 10 times, 100 times, 1000 times, or 10000 times higher than that in non-cancerous tissues (excluding the placenta). Preferably, CLDN6 is expressed in the cells if its expression level is above the detection limit and / or if its expression level is high enough to allow binding by a CLDN6-specific antibody added to the cells. Preferably, the CLDN6 expressed in the cells is expressed or exposed on the cell surface.

[0111] It has been found that CLDN6 expression is detectable only as mRNA in the placenta, and not as protein at all. Therefore, statements made herein regarding CLDN6 expression in the placenta preferably refer to mRNA expression.

[0112] According to the present invention, the term "disease" refers to any pathological state, including cancer, particularly those forms of cancer described herein. Any cancer or particular form of cancer mentioned herein also includes its metastasis. In a preferred embodiment, according to this application, the disease to be treated involves cells expressing CLDN6, particularly cancer stem cells expressing CLDN6.

[0113] According to the present invention, "disease associated with cells expressing CLDN6" or similar expressions mean that CLDN6 is expressed in cells of a diseased tissue or organ. In one embodiment, CLDN6 expression in cells of a diseased tissue or organ is increased compared to its state in a healthy tissue or organ. An increase is defined as an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is found only in the diseased tissue, while expression in the corresponding healthy tissue is suppressed. According to the present invention, diseases associated with cells expressing CLDN6 include cancerous diseases. Furthermore, according to the present invention, cancerous diseases are preferably those in which cancer cells express CLDN6.

[0114] As used herein, “cancer disease” or “cancer” includes diseases characterized by abnormal regulatory cell growth, proliferation, differentiation, adhesion, and / or migration. “Cancer cell” means an abnormal cell that grows through rapid, uncontrolled cell proliferation and continues to grow after the stimulus that initiated new growth has ceased. Preferably, “cancer disease” is characterized by cells expressing CLDN6, particularly cancer stem cells expressing CLDN6.

[0115] According to the present invention, the term "cancer" 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 and their metastases. Examples include lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the above-mentioned cancer types or tumors. According to the present invention, the term cancer also includes cancer metastasis.

[0116] According to the present invention, "cancer" is a malignant tumor derived from epithelial cells. This group represents the most common forms of cancer, including common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0117] Adenocarcinoma is a cancer originating from glandular tissue, which is part of a large class of tissues called epithelial tissue. Epithelial tissue includes the skin, glands, and various other tissues lining the body's cavities and organs. Embryologically, epithelium originates from the ectoderm, endoderm, and mesoderm. Cells classified as adenocarcinoma do not necessarily have to be part of a gland, as long as they have secretory properties. This form of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated adenocarcinomas may not. By staining cells from tissue biopsies, pathologists determine whether a tumor is adenocarcinoma or some other type of cancer. Due to the ubiquitous nature of glands in the body, adenocarcinoma can arise in many tissues. Although each gland may not secrete the same substances, as long as the cells have exocrine functions, it can be considered glandular, and thus its malignant form is named adenocarcinoma. Given sufficient time, malignant adenocarcinomas invade other tissues and often metastasize. Ovarian adenocarcinoma is the most common type of ovarian cancer. These include serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0118] "Metastasis" refers to the spread of cancer cells from their initial site to another part of the body. The formation of metastasis is a highly complex process that depends on malignant cells detaching from the primary tumor, invading the extracellular matrix, penetrating the endothelial basement membrane into body cavities and blood vessels, and subsequently infiltrating target organs via blood transport. Finally, the growth of the new tumor at the target site depends on angiogenesis. Because tumor cells or components can remain and develop metastatic potential, tumor metastasis often occurs even after the removal of the primary tumor. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis," which refers to metastasis far from the primary tumor and the local lymph node system. In one embodiment, the term "metastasis" according to the invention refers to lymph node metastasis.

[0119] Refractory cancer is a malignant tumor that does not respond to specific treatments; it is unresponsive to treatment from the outset or becomes unresponsive over time. The terms “refractory,” “unresponsive,” or “resistant” are used interchangeably in this article.

[0120] As used herein, the term "cancer stem cell" refers to cells that can be the progenitors of highly proliferating cancer cells. Cancer stem cells have the ability to regrow tumors, as demonstrated by their ability to form tumors in immunodeficient mice. Cancer stem cells are typically slow-growing relative to a tumor mass; that is, they are usually quiescent. In some (but not all) implementations, cancer stem cells may represent only a portion of the tumor, for example, about 0.1% to 10%. Cancer stem cells may have one or more of the following characteristics or properties: (i) they may have the ability to initiate and / or maintain tumor growth; (ii) they may generally have fewer mutations than tumor masses (e.g., due to slow growth and therefore fewer DNA replication-related errors, improved DNA repair and / or epigenetic / non-mutagenic changes that promote malignancy); (iii) they may have many of the characteristics of (a) normal stem cells (e.g., similar cell surface antigens and / or intracellular expression profiles, self-renewal programs, multidrug resistance, immature phenotypes, etc., characteristics of normal stem cells) and may be derived from (a) normal stem cells; (iv) they may be a source of metastases; (v) they may grow slowly or be quiescent; (vi) they may be tumorigenic (e.g., as determined by NOD / SCID implantation experiments); (vii) they may be relatively resistant to conventional treatment (i.e., chemoresistant); and (viii) they may include a subpopulation of tumors (e.g., relative to tumor masses).

[0121] "Treatment" means applying treatment (e.g., a compound or composition, or a combination of compounds or compositions) to a subject to prevent or eliminate disease, including reducing the size or number of tumors in the subject, stopping or slowing the progression of disease in the subject, inhibiting or slowing the development of new disease in the subject, reducing the frequency or severity of symptoms and / or recurrences in a subject currently suffering from or previously suffering from disease, and / or prolonging, i.e., increasing or extending the subject's lifespan. In particular, the term "treatment of disease" includes curing, shortening the duration, improving, preventing, slowing or inhibiting the progression or worsening of disease, or preventing or delaying the onset of disease or its symptoms.

[0122] In the context of this invention, terms such as “protection” or “prevention” refer to the prevention or treatment of both the occurrence and / or spread of disease in a subject, and in particular, to minimizing the chance of the subject developing a disease or delaying the development of a disease. For example, a subject at risk of cancer would be a candidate for treatment to prevent cancer.

[0123] "At risk" refers to individuals identified as having a higher likelihood of developing a disease (especially cancer) than the general population. Furthermore, individuals who have had or currently have a disease (especially cancer) have an increased risk of developing the disease because they are more likely to continue developing it. Individuals who currently have or have had cancer also have an increased risk of cancer metastasis.

[0124] According to the present invention, the term "patient" means a person to be treated, particularly a person suffering from an illness, including humans, non-human primates or other animals, especially mammals, such as cattle, horses, pigs, sheep, goats, dogs, cats or rodents (e.g., mice and rats). In a particularly preferred embodiment, the patient is a human.

[0125] In the context of treatment administration, the term "combination," as used herein, refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which treatments or therapeutic agents are administered to the subject. Treatments or therapeutic agents may be administered before, simultaneously with, or after administering a second treatment or therapeutic agent to the patient. Preferably, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that the treatments or therapeutic agents can work together. In one specific embodiment, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that they provide an increased benefit compared to if administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0126] "Target cells" should refer to any unwanted cells, such as cancer cells, especially cancer stem cells. In some preferred embodiments, the target cells express CLDN6.

[0127] According to the present invention, the term "chemotherapy" refers to treatment using one or more chemotherapeutic agents or a combination of chemotherapeutic agents (e.g., cell growth inhibitors or cytotoxic agents). Chemotherapy agents according to the present invention include cell growth inhibitory compounds and cytotoxic compounds.

[0128] According to the present invention, the term "chemotherapy agent" includes taxanes (e.g., paclitaxel and docetaxel) and platinum compounds (e.g., cisplatin and carboplatin) and combinations thereof. Preferred combinations (particularly for the treatment of ovarian cancer) may include combinations of taxanes and platinum compounds, such as paclitaxel and carboplatin. More preferred combinations (particularly for the treatment of ovarian cancer, particularly ovarian germ cell tumors and / or for the treatment of germ cell tumors, particularly ovarian cancer and testicular germ cell tumors) may include combinations of platinum compounds (e.g., cisplatin) with etoposide and / or bleomycin. According to the present invention, references to chemotherapy agents include any prodrug such as esters, salts, or derivatives (e.g., conjugates of the agent). An example is a conjugate of the agent with a carrier substance, such as protein-bound paclitaxel (e.g., albumin-bound paclitaxel). Preferably, the salt of the agent is a pharmaceutically usable salt.

[0129] Taxanes are a class of diterpenoid compounds originally derived from natural sources (such as plants in the genus *Taxus*), but some have been synthesized artificially. The primary mechanism of action for taxane drugs is to disrupt microtubule function, thereby inhibiting cell division. Taxanes include docetaxel and taxol.

[0130] According to the present invention, the term "docetaxel" refers to a compound having the following formula:

[0131]

[0132] Specifically, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl-4-ethyl ester-2-benzoate-13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropyl ester}.

[0133] According to the present invention, the term "paclitaxel" refers to a compound having the following formula:

[0134]

[0135] Specifically, the term "paclitaxel" refers to the compound (2α,4α,5β,7β,10β,13α)-4,10-bis-(acetoxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropionyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate.

[0136] According to the present invention, the term "platinum compound" refers to a compound whose structure contains platinum, such as platinum complexes, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

[0137] The term "cisplatin" refers to the compound cis-diamminedichloroplatinum(II) (CDDP):

[0138]

[0139] The term "carboplatin" refers to the compound cis-diamine(1,1-cyclobutanedicarboxylic acid)platinum(II) of the following formula:

[0140]

[0141] The term "oxaliplatin" refers to a platinum compound that is complexed with a diaminocyclohexane support ligand of the following formula:

[0142]

[0143] Specifically, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato)-O,O')platinum(II). Oxaliplatin for injection is also marketed under the trade name Eloxatine.

[0144] Other chemotherapeutic agents contemplated for use in this invention (whether used 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.

[0145] The term "nucleoside analogue" refers to structural analogues of nucleosides, including both purine and pyrimidine analogues.

[0146] The term "gemcitabine" refers to a nucleoside analogue compound of the following formula:

[0147]

[0148] Specifically, the term refers to the compound 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythrofuranosyl)pyrimidin-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidin-2-one.

[0149] The term "nucleoside analogue" includes fluoropyrimidine derivatives, such as fluorouracil and its prodrugs. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the trade names Adrucil, Carac, Efudix, Efludex, and Fluoroplex) refers to compounds that are pyrimidine analogues of the following formula:

[0150]

[0151] Specifically, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0152] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent, which is a prodrug converted to 5-FU in tissues. Orally administered capecitabine has the following formula:

[0153]

[0154] Specifically, the term refers to the compound [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

[0155] The term "leucovorin" or "formyltetrahydrofolate" refers to a compound used in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Therefore, if the administration of 5-fluorouracil or its prodrug is mentioned herein, such administration in one embodiment may include administration with leucovorin. Leucovorin has the following formula:

[0156]

[0157] Specifically, the term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}glutaric acid.

[0158] According to the present invention, the term "camptothecin analogue" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7] indenzo[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analogue" refers to a compound comprising the following structure:

[0159]

[0160] According to the present invention, preferred camptothecin analogues are inhibitors of DNase topoisomerase I (topo I). According to the present invention, preferred camptothecin analogues are irinotecan and topotecan.

[0161] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. Chemically, it is a semi-synthetic analog of the natural alkaloid camptothecin, having the following formula:

[0162]

[0163] Specifically, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H-pyrano[3',4':6,7]-medium-nitrogen indo[1,2-b]quinoline-9-yl-[1,4'-dipiperidine]-1'-carboxylic acid ester.

[0164] Topotecan is a topoisomerase inhibitor of the following formula:

[0165]

[0166] Specifically, the term "topotecan" refers to the nitrogen-indo[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride of the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7].

[0167] 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 and typically require glycosylation at specific sites.

[0168] Anthracyclines preferably act through one or more of the following mechanisms of action: 1. inhibiting DNA and RNA synthesis by inserting between base pairs in DNA / RNA chains, thereby preventing the replication of rapidly growing cancer cells; 2. inhibiting topoisomerase II, preventing the relaxation of supercoiled DNA and thus blocking DNA transcription and replication; 3. generating iron-mediated free oxygen radicals that damage DNA and cell membranes.

[0169] According to the present invention, the term "anthracycline" preferably refers to an agent that induces apoptosis by inhibiting the rebinding of DNA with topoisomerase II, and is preferably an anticancer agent.

[0170] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (Daunorubicin), doxorubicin (Adriamycin), epirubicin, idarubicin, erythromycin, pyrarubicin, pentorubicin, N-trifluoroacetyl-daunorubicin-14-valerate, aclarubicin, morpholino-daunorubicin (morpholino-DOX), cyanomorpholino-daunorubicin (cyanomorpholino-DOX), 2-pyrrolo-daunorubicin (2-PDOX), 5-iminodaunorubicin, mitoxantrone, and aclarubicin A (aclarubicin). Mitoxantrone is a member of the anthracendione class of compounds, which is an anthracycline analog lacking the sugar moiety of anthracyclines but retaining a planar polycyclic aromatic ring structure that allows insertion into DNA.

[0171] In the context of this invention, epirubicin is particularly contemplated. Epirubicin is an anthracycline drug having the following formula:

[0172]

[0173] It is marketed under the trade names Ellence (USA), Pharmorubicin, or Epirubicin Ebewe (elsewhere). Specifically, the term "epirorubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl- Epirubicin is a 2-alkyl-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetraphenyl-5,12-dione. In some chemotherapy regimens, epirubicin is more advantageous than the most common anthracycline, doxorubicin, because epirubicin appears to cause fewer side effects.

[0174] 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 double-strand DNA breaks and prevents repair by topoisomerase II binding. Accumulated DNA breaks prevent entry into the mitotic phase of cell division and lead to cell death. Etoposide has the following formula:

[0175]

[0176] Specifically, the term refers to the compound 4'-demethyl-epiotatoxin 9-[4,6-O-(R)-ethylidene-β-D-glucopyranoside],4'–(dihydrophosphate).

[0177] The term "bleomycin" refers to a glycopeptide antibiotic produced by the bacteria *Streptomyces verticillus*. When used as an anticancer agent, its mechanism of action is to induce breaks in DNA. Bleomycin preferably comprises compounds having the following formula:

[0178]

[0179] If the chemotherapy according to the invention is administered in combination with an antibody capable of binding CLDN6 (which may be present in a conjugate with at least one toxic drug component, i.e., as an antibody-drug conjugate), the chemotherapy is preferably administered before and / or simultaneously with the antibody (as a mixture or as a separate component). Preferably, chemotherapy is initiated before the antibody is administered. Preferably, the chemotherapy enhances the expression of CLDN6 in cancer cells (e.g., cancer stem cells) and is initiated or administered before the antibody to enhance the antitumor activity of the antibody. Preferably, chemotherapy is initiated 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. Chemotherapy may continue during antibody administration or be stopped before or during antibody administration, for example, 1 to 3 days, 1 to 7 days, 1 to 10 days, or 1 to 14 days before antibody administration. Preferably, the chemotherapy agent comprises taxanes (e.g., paclitaxel or docetaxel) and / or platinum compounds (e.g., cisplatin or carboplatin).

[0180] The term "antigen" refers to a substance, such as a protein or peptide, that contains an epitope against which it elicits and / or will elicit an immune response. In a preferred embodiment, the antigen is a tumor-associated antigen (e.g., CLDN6), which is derived from cancer cells derived from the cytoplasm, cell surface, and cell nucleus, particularly those antigens that are produced in large quantities intracellularly or as surface antigens on cancer cells.

[0181] In the context of this invention, the term "tumor-associated antigen" or "tumor antigen" preferably refers to a protein that is specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage, as well as a protein that is expressed or abnormally expressed in one or more tumor or cancerous tissues. In the context of this invention, tumor-associated antigens preferably associate with the cell surface of cancer cells and are preferably not expressed or only rarely expressed in normal tissues.

[0182] The term "epitope" refers to an antigenic determinant in a molecule, that is, a portion of the molecule that is recognized by the immune system (e.g., by antibodies). For example, an epitope is a discrete three-dimensional site on an antigen recognized by the immune system. Epitopes typically consist of chemically active surface groups of a molecule (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural features and specific charge features. The difference between conformational epitopes and non-conformational epitopes is that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not lost. Epitopes of proteins (e.g., CLDN6) preferably comprise continuous or discontinuous portions of the protein, and their length is preferably 5 to 100, more preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids. For example, the length of an epitope may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0183] The term "antibody" includes a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and any molecule comprising the antigen-binding portion of said glycoprotein. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, fragments or derivatives of antibodies, including but not limited to single-chain antibodies (e.g., scFv's) and antigen-binding antibody fragments (e.g., Fab and Fab' fragments), and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain consists of a heavy chain variable region (abbreviated 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 hypervariable regions called complementarity-determining regions (CDRs), and more conserved regions called frame regions (FRs) are interspersed between the complementarity-determining regions. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0184] As used herein, the term "monoclonal antibody" refers to a preparation of a single-component antibody molecule. Monoclonal antibodies exhibit single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma comprising B cells obtained from a non-human animal (e.g., a mouse) fused with indefinitely proliferating cells.

[0185] As used herein, the term “recombinant antibody” includes all antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from hybridomas of animals (e.g., mice) where the immunoglobulin gene is transgenic or transchromosomally derived, (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfected tumors), (c) antibodies isolated from recombinant, combined antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing immunoglobulin gene sequences with other DNA sequences.

[0186] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through in vitro random or site-specific mutagenesis or through in vivo somatic mutations).

[0187] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from a non-human immunoglobulin, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity-determining region (CDR) grafted onto an appropriate scaffold 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 make it more similar to a human immunoglobulin. Some forms of humanized antibodies retain the entire CDR sequence (e.g., humanized mouse antibodies containing all six CDRs derived from mouse antibodies). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0188] The term "chimeric antibody" refers to an antibody in which a portion of the amino acid sequence of each heavy and light chain is homologous to a corresponding sequence from an antibody of a particular species or class, while the remaining segments of the chain are homologous to a corresponding sequence from another species or class. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammalian species, while the constant regions are homologous to antibody sequences from another species. A significant advantage of this chimeric form is that the variable regions can be conveniently generated from known sources using readily available B cells or hybridomas from non-human host organisms, with the combined constant regions derived, for example, from human cell preparations. The variable regions are easy to prepare and their specificity is unaffected by their source, while because the constant regions are human, the antibody is less likely to elicit an immune response in a human subject upon injection than when the constant regions are from non-human sources. However, the definition is not limited to this specific example.

[0189] Antibodies can be derived from different species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0190] The antibodies described herein include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In several embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, κ, or IgG1,λ isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ).

[0191] As used herein, “heterologous antibody” is defined in the context of a transgenic organism that produces such an antibody. The term refers to an antibody having an amino acid sequence or a nucleic acid-coding sequence that corresponds to an amino acid sequence or a nucleic acid-coding sequence visible in an organism not composed of said transgenic organism, and typically originating from a species different from said transgenic organism.

[0192] As used in this article, the term "heterohybrid antibody" refers to an antibody containing light and heavy chains of different biological origins. For example, an antibody containing a human heavy chain associated with a mouse light chain is a heteroohybrid antibody.

[0193] The antibodies described herein are preferably isolated. As used herein, "isolated antibody" is intended to mean an antibody substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN6 substantially free of antibodies that specifically bind to antigens other than CLDN6). Isolated antibodies specifically bind to epitopes, isoforms, or variants of human CLDN6; however, they may have cross-reactivity with other relevant antigens (e.g., those from other species (e.g., CLDN6 species homologs)). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies refers to antibodies with different specificities combined with a well-defined composition or mixture.

[0194] The term “antigen-binding portion” (or simply “binding portion”) or “antigen-binding fragment” (or simply “binding fragment”) of an antibody, or similar terms, refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments covered in the term “antigen-binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, HL, CL, and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds at the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341:544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked together using a recombinant approach via a synthetic linker to form a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a 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). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" in the term antibody. Another example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to said hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to said CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Domain-binding immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for intended use in the same manner as intact antibodies.

[0195] The term "binding domain" characterizes a structure relevant to the present invention (e.g., an antibody) that binds to / interacts with a given target structure / antigen / epitope. Therefore, according to the present invention, a binding domain signifies an "antigen interaction site".

[0196] For the purposes of this invention, the term "antibody" encompasses all antibodies and antibody derivatives (e.g., antibody fragments) as described herein. The term "antibody derivative" refers to any modified form of an antibody (e.g., a conjugate of an antibody with another pharmaceutical agent or antibody) or antibody fragment.

[0197] Naturally occurring antibodies are typically monospecific, meaning they bind to a single antigen. This invention includes antibodies that bind to target cells (by binding to CLDN6) and to a second entity, such as cytotoxic cells (e.g., by binding to the CD3 receptor). The antibodies of this invention can be bispecific or multispecific, such as trispecific, tetraspecific, etc.

[0198] The term "bispecific molecule" is intended to include substances having two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen and (b) a receptor (e.g., an Fc receptor on the surface of effector cells). The term "multispecific molecule" is intended to include substances having more than two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen, (b) a receptor (e.g., an Fc receptor on the surface of effector cells), and (c) at least one other component. Therefore, the term "antibody capable of binding CLDN6" includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules targeting CLDN6 and other targets (e.g., Fc receptors on effector cells). The term "bispecific antibody" also includes biantibodies. Biantibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow the two domains on the same chain to pair, thus prompting the domain to pair with the complementary domain of the other chain and generate 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).

[0199] In the context of this invention, an "antibody capable of binding CLDN6" is preferably capable of eliciting an immune effector function as described herein. Preferably, the immune effector function targets cells (e.g., cancer stem cells) carrying the tumor-associated antigen CLDN6 on their surface.

[0200] In the context of this invention, the term "immune effector function" includes any function mediated by components of the immune system that results in, for example, inhibition of tumor growth and / or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably, the immune effector function results in the killing of cancer cells, particularly cancer stem cells. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying tumor-associated antigens, cell lysis of cells carrying tumor-associated antigens, and / or inhibition of proliferation of cells carrying tumor-associated antigens. The bound substance also exerts its effect solely by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies block the function of tumor-associated antigens or induce apoptosis solely by binding to tumor-associated antigens on the surface of cancer cells.

[0201] According to the present invention, antibodies may be conjugated to therapeutic portions or pharmaceutical agents (e.g., toxic pharmaceutical portions, particularly cytotoxins, drugs (e.g., immunosuppressants), or radioisotopes). Cytotoxins or cytotoxic agents include any agent that is harmful to cells and particularly kills cells. Examples include: taxol, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthracin dione, mitoxantrone, scintillans, actinomycin D, muscarinic acid, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogues or homologues. Therapeutic agents suitable for forming antibody conjugates include, but are not limited to: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thioepachlorambucil, melphalan, carmustine (BSNU) and romustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II)(DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly actinomycin), bleomycin, scintillan and ampicillin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In one preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In one preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A. According to the invention, a particularly preferred toxin drug portion is a compound that inhibits microtubule assembly and has antiproliferative and / or cytotoxic effects.

[0202] According to the invention, antibodies conjugated with a therapeutic component or agent (e.g., cytotoxin) and acting on slowly growing or quiescent cells (e.g., cancer stem cells) are particularly preferred. Such therapeutic components include those acting 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, inserts into DNA to inhibit the initiation phase of transcription. Flavopiridol targets the elongation phase of transcription. α-Amanita phalloidin binds directly to RNA polymerase II, resulting in inhibition of both initiation and elongation phases.

[0203] Antibodies can also be conjugated with radioactive isotopes (e.g., iodine-131, yttrium-90, or indium-111) to produce cytotoxic radiopharmaceuticals.

[0204] The antibody conjugates of the present invention can be used to modify a given biological response, and the pharmaceutical portion should not be construed as limited to classical chemotherapeutic agents. For example, the pharmaceutical portion may be a peptide, protein, or polypeptide having the desired biological activity. Such proteins may include, for example, enzymatically active toxins or their active fragments, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins, such as tumor necrosis factor or interferon-γ; or biological response regulators, 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 pharmaceutical portions according to the invention are curcumin, salinomycin, and sulforaphane.

[0205] Techniques for conjugating these therapeutic components to antibodies are well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), pp. 623–53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies '84: Biological And Clinical Applications, Pincheraet et al. (eds.), pp. 475–506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Editors), 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).

[0206] In a preferred embodiment, the antibody according to the invention is conjugated to one or more maytansine alkaloid molecules.

[0207] Maytansine alkaloids are potent microtubule-targeting compounds that inhibit cell proliferation during mitosis. Maytansine alkaloids are derivatives of maytansine, which is a 19-membered stalk-type macrolide (ansa macrolide) structure linked to a chlorinated benzene ring. Maytansine has the following formula:

[0208]

[0209] Certain microorganisms have been found to also produce maytansin alkaloids, such as maytansinol and C-3 maytansinol ester (US Patent No. 4,151,042). For example, in US Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4 Synthesized maytanyl alcohol and maytanyl alcohol analogues have been reported in ,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 (which is incorporated herein by reference).

[0210] Maytansine alkaloids are well known in the art and can be synthesized or isolated from natural sources using known techniques. According to the invention, particularly preferred maytansine alkaloids are thiol-containing derivatives of maytansine, such as DM1 and DM4. These thiol-containing derivatives of maytansine comprise compounds in which a methyl group bound to a carbonyl group is replaced by a group containing a free thiol group (e.g., the group -R-SH, where R represents an alkylene or other carbon-containing atom).

[0211] DM1, also known as mertansine, is a maytansine alkaloid with the following formula:

[0212]

[0213] Specifically, the terms "mertansine" or "DM1" refer to compound N. 2' -Deacetylated N 2' -(3-Mercapto-1-oxopropyl)-Maytansine.

[0214] "DM4" refers to compound N 2' -Deacetylated-N 2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansin.

[0215] Anti-CLDN6 antibody-matenidine alkaloid conjugates are prepared by chemically linking an anti-CLDN6 antibody to a maytansine alkaloid molecule without significantly attenuating the biological activity of either the antibody or the maytansine alkaloid molecule. Each antibody molecule is conjugated to an average of 3 to 4 maytansine alkaloid molecules, although it is expected that even a single toxin / antibody molecule could enhance cytotoxicity compared to the use of naked antibodies.

[0216] In this regard, the term "antibody covalently linked to at least one toxin drug" includes cases where one or more molecules of the same drug are covalently linked to an antibody molecule, and cases where different drugs are covalently linked to antibody molecules. In the latter case, one or more molecules of each different drug may be linked to an antibody molecule or a combination thereof (e.g., one molecule of one drug is linked while several molecules of another drug are linked).

[0217] In some embodiments of the invention, the antibody is conjugated to dolastatin or dolastatin peptide analogs and derivatives—orrestatins (US Patent Nos. 5,635,483; 5,780,588, which are incorporated herein by reference). Orrestatins are synthetic analogs of dolastatin 10 (a natural product from the marine mollusc Dolabela auricularia). Like maytansine alkaloids, orrestatins are microtubule disruptors. The dolastatin or orrestatin drug moiety can be linked to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety.

[0218] Exemplary oprestatin implementations include a monomethyl oprestatin pharmaceutical portion, such as MMAE and MMAF, which is preferably N-terminally linked.

[0219] MMAE, also known as monomethyl guanystatin E, has the following formula:

[0220]

[0221] Specifically, the term "MMAE" refers to the compound (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohepane-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butamido)butyramide. MMAE is actually demethylated olistatin E, meaning the N-terminal amino group has only one methyl substituent, instead of the two substituents found in olistatin E itself.

[0222] According to the invention, antibody-vc olistatin conjugates, such as antibody-vc MMAE conjugates, are particularly preferred. According to the invention, the terms "antibody-vc olistatin" or "vcMMAE" refer to such antibody-drug conjugates (ADCs) comprising olistatin (e.g., MMAE) linked to an antibody via a lysosomal cleavable dipeptide valine-citrulline (vc).

[0223] MMAF, also known as monomethyl auratestatin M, 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)butamido)butamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropionic acid.

[0224] Many linker groups known in the art are used to prepare antibody-drug conjugates.

[0225] In one embodiment of the invention, an antibody is linked to a drug via a bifunctional crosslinking agent. As used herein, a “bifunctional crosslinking agent” refers to a reagent having two reactive groups, one of which reacts with the antibody and the other with the drug to link the antibody to the drug, thereby forming a conjugate. Any suitable bifunctional crosslinking agent can be used with the present invention, provided that the linking agent provides for maintaining, for example, the cytotoxicity of the drug and the targeting properties of the antibody. Preferably, the linker molecule links the drug and antibody via chemical bonds, such that the drug and antibody are chemically coupled (e.g., covalently bound) to each other.

[0226] In one embodiment, the bifunctional crosslinking agent comprises an inclevable linker. An inclevable linker is any chemical moiety capable of stably, covalently linking a drug (e.g., maytandemin alkaloids) to an antibody. Preferably, the inclevable linker is inclevable under physiological conditions (particularly intracellular). Therefore, the inclevable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under which the drug or antibody remains active. Suitable crosslinking agents for forming inclevable linkers between drugs and antibodies are well known in the art. In one embodiment, the drug is linked to the antibody via a thioether bond. Examples of inclevable linkers include linkers having a maleimide-based or haloacetyl moiety for reacting with the drug (e.g., with the thiol group of maytandemin alkaloids). Such bifunctional crosslinking agents are well known in the art and include, but are not limited to, N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC) and N-succinimide-4-(N-maleimidemethyl)-cyclohexane-1-carboxyl-(6-amidohexanoate), which are “long-chain” analogs of SMCC (LC-SMCC). Preferably, the bifunctional crosslinking agent is SMCC. Using such a linker, a drug (e.g., mertansine) can be linked to the amino group (e.g., the free NH2 group of a lysine residue) of an antibody via 4-(3-mercapto-2,5-dioxo-1-pyrrolidinylmethyl)-cyclohexanecarboxylate. Each antibody-drug conjugate molecule may comprise a single antibody molecule bound to several molecules of mertansine.

[0227] In a particularly preferred embodiment, the ligation agent is a cleavable linker. Preferably, the cleavable linker is cleavable under physiological conditions, particularly intracellularly. Examples of suitable cleavable linkers include disulfide linkers, acid-labile linkers, photostable linkers, peptidase-labile linkers, and esterase-labile linkers. Disulfide-containing linkers are cleavable linkers generated through disulfide exchange, which can occur under physiological conditions. Acid-labile linkers are cleavable linkers at acidic pH. For example, certain intracellular compartments (e.g., endosomes and lysosomes) have acidic pH values ​​(pH 4-5) and provide conditions suitable for cleaving acid-labile linkers. Photostable linkers are useful on body surfaces and in many light-accessible body cavities. Furthermore, infrared light can penetrate tissues. Peptidase-labile linkers can be used to cleave certain peptides, either intracellularly or extracellularly. In one embodiment, the cleavable linker is cleaved under mild conditions, i.e., intracellular conditions in which the activity of the cytotoxic agent is unaffected.

[0228] In a particularly preferred embodiment, the linker is a linker comprising or composed of dipeptide valine (Val)-citrulline (Cit)(vc), which is cleaved by cathepsins within tumor cells.

[0229] According to the present invention, the term "cancer treatment targeting cancer stem cells" refers to any treatment that can be used to target and preferably kill and / or impair the proliferation or viability of cancer stem cells. Such treatments include: i) targeting naked antibodies, antibody fragments, and proteins (e.g., antibodies or antibody conjugates capable of binding to CLDN6 as described above) on certain cell surfaces of cancer stem cells (e.g., CLDN6) or ii) small molecules that impair the proliferation or viability of cancer stem cells. In one specific embodiment, the expression level of the antigen-binding substance on cancer stem cells is higher than that on normal stem cells. In one specific embodiment, the substance specifically binds to a cancer stem cell antigen.

[0230] According to the present invention, the term "binding" preferably refers to specific binding.

[0231] According to the present invention, in a standard assay, if an antibody has a significant affinity for a predetermined target and binds to the predetermined target, then the antibody is capable of binding to the predetermined target. "Affinity" or "binding affinity" is generally determined by the equilibrium dissociation constant (K0). D The term "significant affinity" is preferably measured using a ratio of 10. -5 M or lower, 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower or 10 -12 M or a lower dissociation constant (K) D It combines with the predetermined target.

[0232] In standard assays, if an antibody does not have significant affinity for the target and does not bind significantly to the target, particularly not detectably, then the antibody is (substantially) unable to bind to the target. Preferably, if present at a concentration up to 2 μg / ml, preferably 10 μg / ml, more preferably 20 μg / ml, particularly 50 or 100 μg / ml or higher, the antibody is unable to bind detectably to the target. Preferably, if the antibody binds to the target at a Kx of a predetermined target (to which the antibody is capable of binding), the antibody is unable to bind to the target. D Compared to at least 10 times, 100 times, 10 3 times, 10 4 times, 10 5 times or 106 K times D If the antibody binds to the target, then the antibody does not have a significant affinity for the target. For example, if the antibody binds to a target that the antibody can bind to, then the antibody does not have a significant affinity for the target. D 10 -7 M, then the antibody binds to K, which has no significant affinity for it. D For at least 10 -6 M, 10 -5 M, 10 -4 M, 10 - 3 M, 10 -2 M or 10 -1 M.

[0233] In standard assays, an antibody is specific to the predetermined target if it can bind to the target but not to other targets, i.e., it has no significant affinity for other targets and does not bind significantly to other targets. According to the invention, an antibody is specific to CLDN6 if it can bind to CLDN6 and (substantially) not to other targets. Preferably, the antibody is specific to CLDN6 if its affinity and binding to other targets do not significantly exceed the affinity or binding to proteins unrelated to CLDN6 (e.g., bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-dense transmembrane proteins such as MHC molecules or transferrin receptors or any other specified polypeptides). Preferably, if the antibody binds to the target at a Kk ratio to non-specific targets... D Compared to at least 10 times, 100 times, or 10 times lower 3 times, 10 4 times, 10 5 times or 10 6 K times D If the antibody binds to its specific target, then the antibody becomes specific for the predetermined target. For example, if the antibody binds to K of its specific target... D 10 -7 M, then the K of the antibody binding to its non-specific target. D For at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 M.

[0234] The binding of the antibody to the target can be experimentally determined using any suitable method; see, for example, Berzofsky et al., “Antibody-Antigen Interactions”, Fundamental Immunology, eds. Paul, WE, Raven Press, New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company, New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, for example, by equilibrium dialysis; by using a BIAcore 2000 instrument, using a generic method described by the manufacturer; by using radioimmunoassay of a radiolabeled target antigen; or by other methods known to the technician. For example, affinity data can be analyzed by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). The affinity of the specific antibody-antigen interaction being measured can vary if measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other antigen-binding parameters (e.g., K) will affect the specific antibody-antigen interaction. D IC 50 The measurement of ) is preferably performed using standardized solutions of antibody and antigen, as well as standardized buffer solutions.

[0235] As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by genes in the heavy chain constant region.

[0236] The term "isotype switching" as used in this article refers to the phenomenon where the antibody class or isotype changes from one Ig class to another.

[0237] As used in this article, the term "naturally occurring" refers to the fact that an object is present in nature. For example, a polypeptide or polynucleotide sequence that exists in organisms (including viruses) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0238] As used in this article, "rearrangement" refers to the conformation of a heavy-chain or light-chain immunoglobulin locus in which the V region is located immediately adjacent to the DJ or J region in a conformation encoding a substantially intact VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison with germline DNA; rearranged loci will possess at least one recombinant heptamer / nonamer homology element.

[0239] When referring to the V segment, the term "unrearranged" or "phylogenetic configuration" used in this paper refers to a configuration in which the V segment has not been rearranged and is not adjacent to the D or J segment.

[0240] Preferably, binding an antibody capable of binding to CLDN6 to cells expressing CLDN6 induces or mediates the killing of CLDN6-expressing cells. Cells expressing CLDN6 are preferably cancer stem cells, and particularly cells of the cancer diseases described herein, such as ovarian cancer stem cells. Preferably, the antibody induces or mediates cell killing by inducing one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of the proliferation of CLDN6-expressing cells. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, which in a particular embodiment are selected from monocytes, mononuclear cells, NK cells, and PMNs. Inhibition of cell proliferation can be measured in vitro using bromodeoxyuridine (5-bromo-2-deoxyguanosine, BrdU) by determining cell proliferation in the assay. BrdU is a synthetic nucleoside, an analogue of thymidine, and can be incorporated into newly synthesized DNA of replicating cells (in the S phase of the cell cycle) during DNA replication, thereby replacing thymidine. The presence of incorporated chemicals, for example, is detected using antibodies specific to BrdU, indicating that the cell is actively replicating its DNA.

[0241] In some preferred embodiments, the antibody described herein may be characterized by one or more of the following properties:

[0242] a) Specific to CLDN6;

[0243] b) The binding affinity to CLDN6 is about 100 nM or less, preferably about 5 to 10 nM or less, more preferably about 1 to 10 nM or less;

[0244] c) The ability to induce or mediate CDC on CLDN6-positive cells;

[0245] d) The ability to induce or mediate ADCC on CLDN6-positive cells;

[0246] e) The ability to inhibit the growth of CLDN6-positive cells;

[0247] f) The ability to induce apoptosis in CLDN6-positive cells.

[0248] In one embodiment, the antibody capable of binding CLDN6 has the ability to bind to epitopes present in CLDN6, preferably epitopes located within the extracellular domain of CLDN6, particularly within the first extracellular loop, preferably amino acids 28 to 76 of CLDN6 or the second extracellular loop, preferably amino acids 141 to 159 of CLDN6. In some specific embodiments, the antibody capable of binding CLDN6 binds to epitopes present on CLDN6 but not on CLDN9. Preferably, the antibody capable of binding CLDN6 binds to epitopes present on CLDN6 but not on CLDN4 and / or CLDN3. Most preferably, the antibody capable of binding CLDN6 binds to epitopes present on CLDN6 but not on other CLDN proteins besides CLDN6.

[0249] Antibodies capable of binding CLDN6 preferably bind to CLDN6 but not to CLDN9, and preferably not to CLDN4 and / or CLDN3. Preferably, antibodies capable of binding CLDN6 are specific for CLDN6. Preferably, antibodies capable of binding CLDN6 bind to CLDN6 expressed on the cell surface. In some particularly preferred embodiments, antibodies capable of binding CLDN6 bind to the natural CLDN6 epitope present on the surface of living cells.

[0250] In a preferred embodiment, the antibody capable of binding CLDN6 comprises a heavy chain variable region (VH) containing an amino acid sequence selected from SEQ ID NO:3, 5, 7, 9 and fragments thereof.

[0251] In a preferred embodiment, the antibody capable of binding CLDN6 comprises a light chain variable region (VL) containing an amino acid sequence selected from SEQ ID NO:4, 6, 8, 10, 11, 12 and fragments thereof.

[0252] In some preferred embodiments, the antibody capable of binding CLDN6 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possible cases (i) to (vii):

[0253] (i) VH contains the amino acid sequence shown in SEQ ID NO:3 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:4 or a fragment thereof.

[0254] (ii) VH contains the amino acid sequence shown in SEQ ID NO:5 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:6 or a fragment thereof.

[0255] (iii) VH contains the amino acid sequence shown in SEQ ID NO:7 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:8 or a fragment thereof.

[0256] (iv) VH contains the amino acid sequence shown in SEQ ID NO:9 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:10 or a fragment thereof.

[0257] (v) VH contains the amino acid sequence shown in SEQ ID NO:5 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:4 or a fragment thereof.

[0258] (vi) VH contains the amino acid sequence shown in SEQ ID NO:5 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:11 or a fragment thereof.

[0259] (vii) VH contains the amino acid sequence shown in SEQ ID NO:5 or a fragment thereof and VL contains the amino acid sequence shown in SEQ ID NO:12 or a fragment thereof.

[0260] In a particularly preferred embodiment, the antibody capable of binding CLDN6 comprises a combination of the following heavy chain variable region (VH) and light chain variable region (VL):

[0261] VH contains the amino acid sequence shown in SEQ ID NO:5 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO:4 or a fragment thereof.

[0262] The term "segment" specifically refers to one or more complementary determinant regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and / or the light chain variable region (VL). In one embodiment, the one or more complementary determinant regions (CDRs) are selected from a set of complementary determinant regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "segment" refers to the complementary determinant regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and / or the light chain variable region (VL).

[0263] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of CDR groups, as described herein, comprises the CDRs and their intervening framework regions. Preferably, the portion will also comprise at least about 50% of one or both of the first and fourth framework regions, said 50% being 50% of the C-terminus of the first framework region and 50% of the N-terminus of the fourth framework region. Antibody construction using recombinant DNA technology may result in the introduction of adapter-encoded residues at the N-terminus or C-terminus of the variable region, the introduction of the adapter being to facilitate cloning or other manipulative steps, including the introduction of the adapter to link the variable region of the present invention to other protein sequences, said other protein sequences including immunoglobulin heavy chains, other variable domains (e.g., in the production of biantibodies), or protein tags.

[0264] In one implementation, an antibody comprising one or more CDRs, a set of CDRs, or a combination of groups of CDRs, as described herein, is included in the CDRs within the human antibody framework.

[0265] The term "antibody containing a specific chain, region, or sequence in its heavy chain" as used herein preferably refers to an antibody in which all heavy chains contain the specific chain, region, or sequence. This also applies to the light chains of the antibody.

[0266] It should be understood that the antibodies described herein can be delivered to a patient by administering a nucleic acid (e.g., RNA) encoding the antibody and / or by administering a host cell containing a nucleic acid (e.g., RNA) encoding the antibody. Therefore, when administered to a patient, the nucleic acid encoding the antibody can be present in a naked form or in the form of a suitable delivery carrier (e.g., in the form of liposomes or viral particles), or within a host cell. Over a longer period, the provided nucleic acid can sustainably generate antibodies, thereby mitigating at least partially the instability observed with therapeutic antibodies. The nucleic acid delivered to the patient can be generated recombinantly. If the nucleic acid, which is not present in the host cell, is administered to the patient, it is preferably taken up by the patient's cells expressing an antibody encoded by said nucleic acid. If the nucleic acid is administered to the patient by cells in the presence of the nucleic acid in the host cell, it is preferably expressed by the host cells in the patient to generate an antibody encoded by said nucleic acid.

[0267] As used herein, the term "nucleic acid" is intended to include both DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0268] Nucleic acids may be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, phage vectors such as λ phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and a suitable DNA sequence required for expression of a coding sequence that is effectively linked 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 design and amplify a desired DNA fragment and may lack the functional sequences required for expression of the desired DNA fragment.

[0269] In the context of this invention, the term "RNA" refers to a molecule comprising ribonucleotide residues, preferably entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-furanose ribonucleotide. This term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides. Such alterations may include, for example, adding non-nucleotide material to the ends or interior of the RNA (e.g., at one or more nucleotides). The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0270] According to the present invention, the term "RNA" includes and preferably refers to "mRNA," meaning "messenger RNA," and also refers to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA typically contains 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 invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0271] In one embodiment of the invention, the RNA is self-replicating RNA, such as single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, the self-replicating RNA is alphaviral genomic RNA or RNA derived from alphaviral genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is a Semliki forest virus. In one embodiment, the self-replicating RNA contains one or more transgenes, at least one of which encodes an antibody described herein. In one embodiment, if the RNA is viral RNA or derived from viral RNA, the transgene may partially or completely replace a viral sequence, such as a viral sequence encoding a structural protein. In one embodiment, the self-replicating RNA is in vitro transcribed RNA.

[0272] The genome of alphaviruses is a positive-sense single-stranded RNA (ssRNA(+)) encoding two open reading frames (ORFs) of a large multiprotein. The ORF at the 5' end of the genome encodes non-structural proteins nSP1 to nSP4 (nsP1-4), which are translated and processed into RNA-dependent RNA polymerases (replicaases); the ORF at the 3' end encodes structural proteins—the capsid and glycoprotein. Both ORFs are separated by so-called subgenomic promoters (SGPs), which control the transcription of the structural ORFs. When used as gene vectors, the structural proteins following the SGPs are typically replaced by transgenes. To package these vectors into viral particles, the structural proteins are usually transexpressed by helper constructs. In the cytoplasm of infected cells, alphaviruses replicate exclusively at the RNA level. Post-infection, the ssRNA(+) genome acts as the mRNA for the translation of the nsP1234 polyprotein precursor, which is self-proteolytically processed into fragments nsP123 and nsP4 early in the viral life cycle. Fragments nsP123 and nsP4 form a (-) strand replicase complex that transcribes (-) strand RNA from a genomic RNA template. In a later stage, the nsP1234 polymer is completely cleaved into single proteins that assemble into a (+) strand replicase complex that synthesizes a new (+) strand genome, as well as subgenomic transcripts encoding structural proteins or transgenes. Both the subgenomic RNA and the new genomic RNA are capped and polyadenylated, thus being recognized as mRNA after infection of target cells. Only the new genomic RNA contains packaging signals that ensure the specific packaging of the genomic RNA into and out of budding virions. The appeal of alphavirus replicons to vectors is based on the positive orientation of the capped and polyadenylated RNA genome. Transferable replicon RNA is readily synthesized in vitro, allowing capping to be achieved by adding a cap-analog to the in vitro transcription reaction, and the poly-A tail on the plasmid template can be encoded as a poly-T track. Transfection of in vitro transcribed (IVT) replicons using conventional transfection techniques is possible, and even small amounts of initiating IVT RNA are rapidly amplified. Within hours of transfer, the transgene downstream of the SGP was transcribed into approximately 40,000 to 200,000 very high copy numbers of subgenomic RNA per cell; therefore, the strong expression of the recombinant protein was not unexpected. Depending on the specific purpose, the IVT replicon can be directly transfected into target cells or packaged into alphavirus particles using a trans-providing helper vector. Transfer to the skin or muscle resulted in high and sustained local expression, while strongly inducing humoral and cellular immune responses.

[0273] To improve the expression and / or stability of the RNA used according to the present invention, it may be modified, preferably without altering the sequence of the expressed peptide or protein.

[0274] In the case of RNA, the term "modification" as used according to the present invention includes any modification of RNA that is not naturally present in said RNA.

[0275] In one embodiment of the invention, the RNA used according to the invention does not contain uncapped 5'-triphosphates. These uncapped 5'-triphosphates can be removed by treating the RNA with a phosphatase.

[0276] The RNA according to the invention may contain modified naturally occurring or synthetic ribonucleotides to improve its stability and / or reduce its cytotoxicity. For example, in one embodiment, 5-methylcytidine is partially or completely (preferably completely) substituted for cytidine in the RNA used according to the invention. Alternatively or additionally, in one embodiment, pseudouridine is partially or completely (preferably completely) substituted for uridine in the RNA used according to the invention.

[0277] In one embodiment, the term "modification" refers to providing RNA having 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 and generally consists of a guanosine nucleotide linked to the mRNA via an unusual 5'-to-5'-triphosphate bond. In one embodiment, the guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m7G). In the context of this invention, the term "5'-cap" includes 5'-cap analogues that resemble RNA cap structures and are modified to have the ability to stabilize RNA if attached there (preferably in vivo and / or in cells).

[0278] In the presence of the 5'-cap or 5'-cap analogue, RNA with a 5'-cap or 5'-cap analogue can be provided by in vitro transcription of a DNA template, wherein the 5'-cap is co-transcribed into the resulting RNA chain, or, for example, RNA can be generated by in vitro transcription and the 5'-cap can be transcribed and attached to the RNA using a capping enzyme (e.g., a capping enzyme of vaccinia virus).

[0279] RNA may include further modifications. For example, further modifications to the RNA used in this invention may be the elongation or truncation of a naturally occurring poly(A) tail or changes to the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of the RNA, for example, the insertion of one or more 3'-UTRs, preferably from two copies of a globin gene (e.g., α2-globin, α1-globin, β-globin, preferably β-globin, more preferably human β-globin).

[0280] Therefore, to improve the stability and / or expression of the RNA used according to the present invention, it can be modified to appear in conjunction with a polyA sequence, preferably 10 to 500 residues in length, more preferably 30 to 300 residues, even more preferably 65 to 200 residues, and particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence is about 120 adenosine residues in length. Furthermore, the incorporation of two or more 3'-untranslated regions (UTRs) into the 3'-untranslated region of the RNA molecule can lead to enhanced translation efficiency. In a specific embodiment, the 3'-UTR is derived from the human β-globin gene.

[0281] Preferably, if the RNA is delivered to (i.e. transfected into) a cell, particularly a cell present in the body, the RNA expresses the protein or peptide it encodes.

[0282] The term "transfection" refers to the introduction of nucleic acids (particularly RNA) into cells. For the purposes of this invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, which may be present in a subject (e.g., a patient). Thus, according to the invention, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, cells that may form part of an organ, tissue, and / or organism of a patient. According to the invention, transfection can be transient or stable. For some applications of transfection, even transient expression of the transfected genetic material is sufficient. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the foreign nucleic acids will be diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the rate of dilution. Stable transfection must occur if it is desired that the transfected nucleic acids are actually retained in the genome of the cell and its progeny cells. RNA can be transfected into cells to transiently express the proteins it encodes.

[0283] The term "stability" of RNA refers to its "half-life." "Half-life" refers to the time period required to eliminate half of the molecular activity, amount, or number. In the context of this invention, the half-life of RNA indicates the stability of said RNA. The half-life of RNA may affect the "expression duration" of RNA. RNA with a long half-life can be expected to be expressed for a longer period.

[0284] In the context of this invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into protein. According to the invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to RNA (particularly mRNA) synthesized in vitro in a cell-free system, preferably using suitable cell extracts. Preferably, cloning vectors are used to produce transcripts. These cloning vectors are generally designated as transcription vectors and are covered by the term "vector" according to the invention.

[0285] According to the present invention, the term "translation" refers to the process in the ribosomes of a cell, in which the chain of messenger RNA guides the assembly of amino acid sequences to form peptides or proteins.

[0286] According to the present invention, the term "expression" is used in its most general sense and includes, for example, the production of RNA and / or peptides or proteins through transcription and / or translation. With respect to RNA, the term "expression" or "translation" specifically refers to the production of peptides or proteins. It also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "anomalous expression".

[0287] According to the present invention, "abnormal expression" or "alternative expression" means that the expression is altered, preferably increased, compared to a reference (e.g., a state in a subject that does not have a disease associated with abnormal or anomalous expression of a certain protein (e.g., a tumor antigen). An increase in expression means an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100%, or more. In one embodiment, the expression is found only in diseased tissues, while expression is suppressed in healthy tissues.

[0288] The term "specific expression" means that a protein is expressed essentially only in a specific tissue or organ. For example, specific expression of a tumor antigen in the placenta means that the protein is expressed primarily in the placenta and not expressed in other tissues or at a negligible level in other tissue or organ types. Thus, a protein that is specifically expressed in placental cells and expressed to a lesser extent in any other tissue is specifically expressed in placental cells. In some embodiments, the tumor antigen may also be specifically expressed in more than one tissue type or organ (e.g., in two or three tissue types or organs, but preferably in no more than three different tissue or organ types) under normal conditions. In this case, the tumor antigen is specifically expressed in these organs.

[0289] According to the present invention, the term "RNA encoding" means that, if present in a suitable environment, preferably within a cell, RNA can be expressed to produce the protein or peptide it encodes.

[0290] Some aspects of this invention rely on adoptive transfer of host cells, which are transfected in vitro with nucleic acids (e.g., RNA encoding antibodies as described herein) and transferred to a recipient (e.g., a patient), preferably after in vitro expansion from a low precursor frequency to a clinically relevant cell number. According to the invention, the host cells used for treatment can be autologous, allogeneic, or syngeneic with the recipient to be treated.

[0291] The term "autologous" is used to describe anything that comes from the same object. For example, an "autograft" refers to a graft from tissue or organ from the same object. Such a process is advantageous because it overcomes an immunological barrier that would otherwise lead to rejection.

[0292] The term "alien" is used to describe anything that comes from different individuals of the same species. Two or more individuals are said to be alliens when their genes at one or more loci are different.

[0293] The term "homogeneous" is used to describe anything that comes from an individual or tissue with the same genotype, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0294] The term "heterogeneous" is used to describe something composed of multiple different elements. As an example, transferring bone marrow from one individual to another constitutes a heterologous graft. Heterogeneous genes are genes that originate from sources different from the original organism.

[0295] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a peptide containing two or more amino acids covalently linked by peptide bonds, 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 at most preferably eight, ten, twenty, thirty, forty or fifty, particularly one hundred. 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 synonyms and are used interchangeably herein.

[0296] The teachings given herein regarding specific amino acid sequences (e.g., those shown in the sequence listing) should also be interpreted as referring to variants of the specific sequence that produce a sequence functionally equivalent to the specific sequence, such as amino acid sequences exhibiting the same or similar properties as the specific amino acid sequence. An important property is the preservation of antibody binding to its target or the maintenance of the antibody's effector function. Preferably, a variant sequence relative to the specific sequence, when it replaces the specific sequence in the antibody, preserves the antibody's binding to CLDN6 and preferably retains the antibody's function as described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage.

[0297] For example, the sequence shown in the sequence listing may be modified to remove one or more, preferably all, free cysteine ​​residues, particularly by replacing cysteine ​​residues with amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine or methionine, with alanine or serine being the most preferred.

[0298] Those skilled in the art will understand that, in particular, the sequences of the CDR, hypervariable regions, and variable regions can be modified without losing the ability to bind CLDN6. For example, the CDR region may be identical to or highly homologous to the region of the antibody specified herein. “Highly homologous” can be expected to involve 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2 substitutions in the CDR. Furthermore, the hypervariable and variable regions can be modified so that they show substantially homology to the antibody regions specifically disclosed herein.

[0299] For the purposes of this invention, "variants" of amino acid sequences include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants containing missing amino acids at the N-terminus and / or C-terminus of a protein are also referred to as N-terminal and / or C-terminal truncated variants.

[0300] Amino acid insertion variants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence; however, random insertions and appropriate screening of the resulting products are also possible.

[0301] Amino acid addition variants include the fusion of one or more amino acids (e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids) at the amino terminus and / or carboxyl terminus.

[0302] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can occur at any position in the protein.

[0303] Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue at that position. Modifications are preferably made at non-conserved positions between homologous proteins or peptides in the amino acid sequence, and / or by replacing the amino acid with another amino acid having similar properties. Preferably, the amino acid changes in the protein variants are conserved amino acid changes, i.e., the replacement of an amino acid with a similar charge or no charge. Conserved amino acid changes involve the replacement of one of the amino acid families associated with its side chain. Naturally occurring amino acids are generally classified into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

[0304] Preferably, the degree of similarity or identity between the given amino acid sequence and its variants 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%. Preferably, the degree of similarity or identity is given for amino acid regions that are at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, a degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In some preferred embodiments, a degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining 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, and Gap Extend 0.5.

[0305] "Sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0306] The term "percentage identity" is intended to represent the percentage of identical amino acid residues between two sequences to be compared after optimal alignment, a percentage that is entirely statistically significant, and where the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are routinely performed by comparing their sequences after optimal alignment, the comparisons being made by segments or by a "comparison window" to identify and compare sequence similarity in local regions. Besides manual generation, the best alignments for comparison can also be generated by: local homology algorithms of Smith and Waterman, 1981, Ads App. Math. 2, 482; local homology algorithms of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; similarity search methods of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444; or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0307] Percentage identity is calculated as follows: determine the number of identical positions between the two sequences being compared, divide that number by the number of positions being compared, and multiply the result by 100 to obtain the percentage identity between the two sequences.

[0308] The term "cell" or "host cell" preferably refers to an intact cell, i.e., a cell with an intact membrane that has not yet released its normal intracellular components (e.g., enzymes, organelles, or genetic material). An intact cell is preferably a living cell, i.e., a living cell capable of performing its normal metabolic functions. Preferably, according to the invention, the term refers to any cell that can be transfected with exogenous nucleic acids. Preferably, when a cell is transfected with exogenous nucleic acids and transferred to a recipient, the nucleic acids can be expressed in the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from Gram-negative bacterial strains, such as *Escherichia coli*, *Proteus*, and *Pseudomonas*, and Gram-positive bacterial strains, such as strains of *Bacillus*, *Streptomyces*, *Staphylococcus*, and *Lactococcus*. Suitable fungal cells include those from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*. Suitable yeast cells include those from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizo saccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia amethanolicd*), and *Hansenula*. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293HEK cells, etc. However, amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins may also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a wide range of tissue types and include primary cells and cell lines, such as cells of the immune system, particularly antigen-presenting cells (e.g., dendritic cells and T cells), stem cells (e.g., hematopoietic stem cells and mesenchymal stem cells), and other cell types. Antigen-presenting cells are cells that display antigens on their surface in the presence of a major histocompatibility complex. T cells can recognize this complex using their T cell receptors (TCRs).

[0309] The term "transgenic animal" refers to an animal having a genome containing one or more transgenes (preferably heavy chain and / or light chain transgenes) or transchromosomal transgenes (integrated or not integrated into the animal's natural genomic DNA), and preferably capable of expressing said transgenes. For example, a transgenic mouse may have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosomal transgene, such that when immunized with CLDN6 antigen and / or cells expressing CLDN6, the mouse produces human anti-CLDN6 antibodies. The human heavy chain transgene may be integrated into the mouse's chromosomal DNA (as in the case of transgenic mice (e.g., HuMAb mice (e.g., HCo7 or HCl2 mice))) or the human heavy chain transgene may be maintained extrachromosomally, as in the case of transchromosomal (e.g., KM) mice as described in WO 02 / 43478. Such transgenic and transchromosomal mice can be made to produce multiple isotypes (e.g., IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN6 through VDJ recombination and isotype conversion.

[0310] As used herein, “reduce,” “lower,” or “inhibit” means an overall reduction in a level (e.g., expression level or cell proliferation level) or the ability to cause an overall reduction, preferably 5% or higher, 10% or higher, 20% or higher, more preferably 50% or higher, and most preferably 75% or higher.

[0311] Terms such as “increase,” “enhance,” or “enhance” preferably refer to an increase or enhancement of about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more.

[0312] While some considerations regarding the mechanisms behind the efficacy of antibody therapies are provided below, they should not be considered as limiting the invention in any way.

[0313] The antibodies described herein preferably interact with components of the immune system, preferably via ADCC or CDC. These antibodies can also be used to directly kill tumor cells by targeting a payload (e.g., a radioactive isotope, a drug, or a toxin) or in synergy with conventional chemotherapy agents to attack tumors through complementary mechanisms of action, which may include impaired anti-tumor immune responses due to the cytotoxic side effects of chemotherapy agents on T lymphocytes. However, the antibodies described herein can also function simply by binding to CLDN6 on the cell surface, thereby, for example, blocking cell proliferation.

[0314] Antibody-dependent cell-mediated cytotoxicity

[0315] ADCC describes the cytotoxic capacity of effector cells (particularly lymphocytes) as described herein, preferably target cells that require antibody labeling.

[0316] ADCC preferably occurs when antibodies bind to antigens on tumor cells and when the antibody Fc domain engages with Fc receptors (FcRs) on the surface of immune effector cells. Several Fc receptor families have been identified, and specific cell populations characteristically express identified Fc receptors. ADCC can be viewed as a mechanism for directly inducing varying degrees of direct tumor destruction, which leads to antigen presentation and induces a tumor-directed T cell response. Preferably, in vivo induction of ADCC will elicit both a tumor-directed T cell response and a host-derived antibody response.

[0317] complement-dependent cytotoxicity

[0318] CDC is another antibody-guided method of cell killing. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also effective in guiding CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of the antigen-antibody complex leads to the exposure of multiple C1q binding sites adjacent to the CH2 domain of the antibody molecule (e.g., IgG molecule) (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites transform the previous low-affinity C1q-IgG interaction into a high-affinity interaction, which triggers a cascade of events involving a series of other complement proteins and results in the proteolytic release of effector cell chemokines / activators C3a and C5a. Preferably, this complement cascade ultimately forms a membrane attack complex, which creates pores in the cell membrane, facilitating the free movement of water and solutes inside and outside the cell.

[0319] The antibodies described herein can be generated using a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization is preferred in principle, other techniques can also be used to generate monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display using antibody gene libraries.

[0320] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma generation in mice is a well-established and well-established protocol. Immunoassay protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion protocols are also known.

[0321] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0322] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying a portion of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include mice referred to as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.” Human antibodies can be generated in such transgenic mice as described in detail in WO2004 035607 regarding CD20.

[0323] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding gene from lymphocytes that produce antibodies of defined specificities; for example, see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Details of recombinant antibody engineering can also be found in Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0324] To generate antibodies, mice can be immunized as described using a vector conjugated peptide derived from an antigen sequence (i.e., the sequence the antibody is to target), an enriched preparation of a recombinantly expressed antigen or a fragment thereof, and / or cells expressing the antigen. Alternatively, mice can be immunized using DNA encoding an antigen or a fragment thereof. If immunization with a purified or enriched preparation of the antigen does not produce antibodies, mice can be immunized with cells expressing the antigen (e.g., cell lines) to promote an immune response.

[0325] Immune responses can be monitored throughout the immunization protocol using plasma and serum samples obtained via tail vein or retroorbital sampling. Fusion can be performed using mice with sufficient immunoglobulin titers. Uptake immunizations of mice with antigen-expressing cells can be administered intraperitoneally or intravenously up to three days before sacrifice and spleen removal to increase the rate of hybridomas secreting specific antibodies.

[0326] To generate hybridomas that produce monoclonal antibodies, spleen cells and lymph node cells can be isolated from immunized mice and fused with a suitable indefinitely proliferating cell line (e.g., a mouse myeloma cell line). The resulting hybridomas can then be screened for antigen-specific antibody production. Antibody-secreting hybridomas can then be screened on individual wells using ELISA. Antigen-specific antibodies can be identified using antigen-expressing cells via immunofluorescence and FACS analysis. Antibody-secreting hybridomas can be replated and screened again, and if monoclonal antibodies remain positive, subcloning can be performed using limiting dilutions. Stable subclones can then be cultured in vitro in tissue culture media to produce antibodies for characterization.

[0327] The antibodies of the present invention can also be generated in host cell transfected tumors using, for example, a combination of recombinant DNA technology and gene transfection methods known in the art (Morrison, S. (1985) Science 229:1202).

[0328] For example, in one embodiment, a target gene (e.g., an antibody gene) may be ligated into an expression vector (e.g., a eukaryotic expression plasmid), for instance, using the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338 841, or other expression systems known in the art. The purified plasmid containing the cloned antibody gene may be introduced into eukaryotic host cells, such as CHO cells, NS / O cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells (e.g., cells from plants, fungi, or yeast). Methods for introducing these genes may be those described in the art, such as electroporation, lipofectine, lipofectamine, or others. After introducing these antibody genes into host cells, cells expressing the antibody may be identified and selected. These cells represent transfected tumors from which their expression levels can subsequently be amplified and scaled up for antibody production. Recombinant antibodies may be isolated and purified from these culture supernatants and / or cells.

[0329] Alternatively, the cloned antibody gene can be expressed in other expression systems, including prokaryotic cells, such as microorganisms like Escherichia coli. Furthermore, antibodies can be produced in non-human transgenic animals, for example, in the milk of sheep and rabbits or in eggs, or in transgenic plants; see, for example, Verma, R., et al. (1998) J. Immunol. Meth. 216:165-181; Pollock, et al. (1999) J. Immunol. Meth. 231:147-157; and Fischer, R., et al. (1999) Biol. Chem. 380:825-839.

[0330] Inlay

[0331] Mouse antibodies exhibit high immunogenicity in humans, leading to decreased therapeutic efficacy with repeated application. The primary immunogenicity is mediated by the heavy chain constant region. If the individual antibodies are chimeric or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided. Chimeric antibodies are antibodies whose different portions originate from different animal species, for example, those having variable regions derived from mouse antibodies and constant regions of human immunoglobulins. Chimeric antibodies are obtained by linking the variable regions of the mouse heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the human κ light chain constant region to the mouse light chain variable region. In another preferred embodiment, chimeric antibodies are generated by linking the human λ light chain constant region to the mouse light chain variable region. Preferred heavy chain constant regions for generating chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0332] Humanization

[0333] Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDRs are more diverse than those outside the CDRs among antibodies. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies mimicking the properties of a specific naturally occurring antibody by constructing expression vectors containing the CDR sequence from that specific naturally occurring antibody, which are then grafted onto frame sequences from different antibodies with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such frame sequences are available from public DNA databases that include germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes, which are formed during B cell maturation via V(D)J linkage. Germline gene sequences will also differ from the sequences of the high-affinity secondary repertoire antibody at individual locations that uniformly traverse the variable region.

[0334] Standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry) can be used to determine the ability of an antibody to bind to an antigen.

[0335] To purify the antibody, selected hybridomas can be cultured in 2-liter roller flasks to purify the monoclonal antibody. Alternatively, the antibody can be generated in a dialysis-based bioreactor. The supernatant can be filtered and concentrated (if necessary) before affinity chromatography with protein G-agarose or protein A-agarose. The eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be replaced with PBS, and the concentration can be determined using an extinction coefficient of 1.43 via OD280. The monoclonal antibody can be aliquoted and stored at -80°C.

[0336] Site-directed mutagenesis or multi-site-directed mutagenesis can be used to determine whether a selected monoclonal antibody binds to a unique epitope.

[0337] Antibody isotypes can be determined using a variety of commercially available kits (e.g., Zymed, Roche Diagnostics) via isotype ELISA. The wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is incubated with a monoclonal antibody or a purified isotype control at ambient temperature for two hours. The wells are then reacted with mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or mouse IgM specific peroxidase conjugated probes. After washing, the plate is developed with ABTS substrate (1 mg / ml) and analyzed at OD 405 to 650. Alternatively, the IsoStrip mouse monoclonal antibody isotype kit (Roche, Cat. No. 1493027) can be used as described by the manufacturer.

[0338] Flow cytometry can be used to demonstrate the presence of antibodies or the binding of monoclonal antibodies to antigens expressed on live cells in the serum of immunized mice. Cell lines expressing the antigen, either naturally or after transfection, and negative controls lacking antigen expression (cultured under standard growth conditions) can be mixed with different concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, APC- or Alexa647-labeled anti-IgG antibodies can be bound to the antigen-bound monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by gating individual live cells using FACS instruments based on light scattering and side scattering properties via flow cytometry. Co-transfection methods can be used to distinguish between antigen-specific monoclonal antibodies and non-specific binders in a single measurement. Cells transiently transfected with plasmids encoding antigens and fluorescent markers can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies preferentially bind to cells expressing the fluorescent marker, while non-specific antibodies bind to untransfected cells in a comparable proportion. Alternative assays using fluorescence microscopy can supplement or replace flow cytometry assays. Cells can be stained completely as described above and examined by fluorescence microscopy.

[0339] Immunofluorescence microscopy can be used to demonstrate the presence of antibodies or the binding of monoclonal antibodies to live cells expressing antigens in the serum of immunized mice. For example, cell lines spontaneously or post-transfection expressing the antigen and negative controls lacking antigen expression are cultured 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. Cells are then fixed with methanol or paraformaldehyde, or left untreated. The cells are then reacted with a monoclonal antibody against the antigen at 25°C for 30 minutes. After washing, the cells are reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells are then examined by fluorescence microscopy.

[0340] Cell extracts from appropriate negative controls and antigen-expressing cells can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked, and detected with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and visualized with ECL substrate.

[0341] The reactivity of antibodies to antigens can also be tested by immunohistochemistry in a manner known to those skilled in the art, for example, using paraformaldehyde- or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections obtained from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from xenograft tumors in mice carrying cell lines that spontaneously express or post-transfection express antigens. For immunostaining, antibodies that react with the antigen can be incubated, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the supplier's instructions.

[0342] The ability of antibodies to mediate phagocytosis and killing of cells expressing CLDN6 can be tested. In vitro assays of monoclonal antibody activity will provide preliminary screening before in vivo model testing.

[0343] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0344] In short, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells, or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation followed by lysis of contaminated red blood cells. The washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum, and at different effector cell:target cell ratios. 51Cr-labeled target cells expressing CLDN6 are mixed. Alternatively, target cells can be labeled with a fluorescently enhanced ligand (BATDA). The highly toxic fluorescent chelate formed by the enhanced ligand and europium released from dead cells can be measured using a fluorometer. Another alternative technique utilizes transfection of target cells with luciferase. The added fluorescent yellow can then be oxidized using only live cells. Purified anti-CLDN6 IgG is then added at different concentrations. Unrelated human IgG can be used as a negative control. Depending on the type of effector cells used, assays can be performed at 37°C for 4 to 20 hours. Measurements can be taken by measuring the concentration of CLDN6 in the culture supernatant. 51 The release of Cr or the presence of EuTDA chelates can be used to determine cell lysis in a sample. Alternatively, luminescence produced by the oxidation of fluorescent yellow can serve as a measure of viable cells.

[0345] Multiple combinations can also be used to test anti-CLDN6 monoclonal antibodies to determine whether using multiple monoclonal antibodies enhances cell lysis.

[0346] Complement-dependent cytotoxicity (CDC)

[0347] A variety of known techniques can be used to test the ability of monoclonal anti-CLDN6 antibodies to mediate CDC. For example, complement serum can be obtained from blood in a manner known to those skilled in the art. Different methods can be used to determine the CDC activity of the mAb. For example, it can be measured... 51 The release of Cr can be evaluated using a propidium iodide (PI) exclusion assay to assess increased membrane permeability. In short, it can wash target cells and release 5 × 10⁻⁶ Cr. 5 Incubate cells at different concentrations of mAb at room temperature or 37°C for 10 to 30 minutes. Then, add serum or plasma to a final concentration of 20% (v / v) and incubate at 37°C for 20 to 30 minutes. All cells from each sample can be added to the PI solution in the FACS tube. The mixture can then be analyzed immediately using FACSArray via flow cytometry.

[0348] In an alternative assay, CDC induction can be determined based on adherent cells. In one embodiment of this assay, 24 hours prior to the assay, 3 × 10⁻⁶ cells are injected with the culture medium. 4Cells were seeded at a density specified for each well in a flat-bottomed microtiter plate for tissue culture. The next day, growth medium was removed, and cells were incubated in triplicate with antibodies. Control cells were incubated separately with growth medium or growth medium containing 0.2% saponin to determine background and maximum lysis. After 20 minutes of incubation at room temperature, the supernatant was removed, and cells were added to 20% (v / v) human plasma or serum in DMEM (preheated to 37°C) and incubated for another 20 minutes at 37°C. All cells from each sample were added to propidium iodide solution (10 μg / ml). The supernatant was then replaced with PBS containing 2.5 μg / ml ethidium bromide, and fluorescence emission after excitation at 520 nm was measured using a Tecan Safire at 600 nm. The percentage of specific lysis was calculated as follows: % specific lysis = (sample fluorescence - background fluorescence) / (maximum lysis fluorescence - background fluorescence) × 100.

[0349] Inducing apoptosis and inhibiting cell proliferation using monoclonal antibodies.

[0350] The ability to induce apoptosis can be tested by incubating monoclonal anti-CLDN6 antibodies with, for example, CLDN6-positive tumor cells or tumor cells transfected with CLDN6 at 37°C for approximately 20 hours. Cells can be harvested, washed with annexin-V binding buffer (BD Biosciences), and incubated in the dark for 15 minutes with FITC- or APC-conjugated annexin-V (BD Biosciences). All cells from each sample can be added to PI solution (10 μg / ml in PBS) in FACS tubes and evaluated immediately by flow cytometry (as above). Alternatively, a commercially available kit can be used to detect the general inhibition of cell proliferation by monoclonal antibodies. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells in microplates. Europium-labeled monoclonal antibodies are used to detect the incorporation of BrdU. Cells were fixed using a fixation solution to denature the DNA, enabling antibody detection. Unbound antibodies were washed away, and a DELFIA inducer was added to dissociate europium ions from the labeled antibodies into solution, where they formed highly fluorescent chelates with the components of the DELFIA inducer. In the assay, fluorescence measured using time-resolved fluorometry was proportional to the amount of DNA synthesized in the cells of each well.

[0351] Preclinical research

[0352] The binding substances described herein can also be tested in in vivo models (e.g., in immunodeficient mice carrying xenograft tumors expressing CLDN6) to determine their efficacy in controlling the growth of CLDN-expressing tumor cells.

[0353] In vivo studies using the antibodies described herein can be conducted after xenografting of CLDN6-expressing tumor cells into immunocompromised mice or other animals. Antibodies can be administered to tumor-free mice followed by injection of tumor cells to measure the effectiveness of the antibodies in preventing tumor or tumor-related symptom formation. Antibodies can be administered to mice with tumors to determine the therapeutic effects of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibody administration can be combined with the administration of other substances (such as cell growth inhibitors, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies) to determine the synergistic potency and potential toxicity of the combination. Animals can be inoculated with antibodies or control reagents, and a thorough study can be conducted on symptoms that may be associated with CLDN6 antibody treatment to analyze antibody-mediated toxic side effects. Possible side effects of in vivo administration of CLDN6 antibodies include toxicity in CLDN6-expressing tissues (including the placenta). Antibodies that identify CLDN6 in humans and other species (e.g., mice) are particularly useful for predicting potential side effects mediated by the administration of monoclonal CLDN6 antibodies in humans.

[0354] Epitopes recognized by antibodies can be mapped according to the detailed descriptions in "Epitope Mapping Protocols (Methods in Molecular Biology) ISBN-089603-375-9" by Glenn E. Morris and "Epitope Mapping: A Practical Approach" by Olwyn MR Westwood and Frank C. Hay, Practical Approach Series, 248.

[0355] The compounds and agents described herein may be administered in any suitable pharmaceutical composition.

[0356] The pharmaceutical compositions of the present invention are preferably sterile and contain an effective amount of the antibody described herein and other optional pharmaceutical agents discussed herein to produce the desired response or desired effect.

[0357] Pharmaceutical compositions are typically provided in a homogeneous dosage form and can be prepared in a manner known per se. Pharmaceutical compositions may be, for example, in the form of a solution or suspension.

[0358] Pharmaceutical compositions may contain salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" means that the materials do not interact with the active ingredient of the pharmaceutical composition and are non-toxic.

[0359] Non-pharmaceutical salts can be used to prepare pharmaceutically acceptable salts, and are also included in this invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0360] Buffering substances suitable for use in pharmaceutical compositions include acetic acid, citric acid, boric acid, and phosphoric acid in salts.

[0361] Preservatives suitable for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thiomercurate.

[0362] Injectable formulations may contain pharmaceutically acceptable excipients, such as Ringer lactate.

[0363] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, wherein active components are combined to promote, enhance, or achieve application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a patient.

[0364] Carrier substances that can be used for parenteral administration include, for example, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and in particular, biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxy-propylene copolymers.

[0365] The term “excipient” as used herein is intended to refer to all substances that may be present in a pharmaceutical composition but are not the active ingredient, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents.

[0366] The pharmaceutical agents and compositions described herein can be administered via any conventional route, such as parenteral administration, including by injection or infusion. Parenteral administration is preferred, for example, intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular administration.

[0367] Compositions suitable for parenteral administration generally comprise sterile aqueous or non-aqueous preparations of the active compound, preferably isotonic with the recipient's blood. Examples of compatible carriers and solvents are Ringer solutions and isotonic sodium chloride solutions. Additionally, sterile immobilized oils are commonly used as solutions or suspension media.

[0368] The pharmaceutical agents and compositions described herein are administered in an effective amount. An "effective amount" refers to an amount, alone or in combination with other doses, that achieves the desired response or desired effect. In the treatment of a specific disease or condition, the desired response preferably involves inhibiting the progression of the disease. This includes slowing the progression of the disease and, in particular, interrupting or reversing the progression of the disease. The desired response in treating a disease or condition may also be delaying or preventing the onset of said disease or condition. In particular, the term "effective amount" refers to a therapeutic amount sufficient to result in the prevention of the development, recurrence, or onset of cancer and one or more of its symptoms, a reduction in the severity or duration of cancer, an improvement in one or more of the symptoms of cancer, prevention of cancer development, induction of cancer regression, and / or prevention of cancer metastasis. In one embodiment of the invention, the therapeutic amount effectively achieves stabilization, reduction, or elimination of cancer stem cell populations and / or eradication, removal, or control of primary cancer, metastatic cancer, and / or recurrent cancer.

[0369] The effective dose of the agents or compositions described herein will depend on the condition to be treated, the severity of the disease, the patient's individual parameters (including age, physical condition, size, and weight), the duration of treatment, the type of concomitant treatment (if present), the specific route of administration, and similar factors. Therefore, the dosage of the agents described herein may depend on several such parameters. Higher doses (or more effective doses obtained through different, more localized routes of administration) may be used if the patient's response to the initial dose is insufficient.

[0370] The agents and compositions described herein can be administered to patients to treat or prevent cancerous diseases, such as those characterized by the presence of cancer stem cells expressing CLDN6, as described herein.

[0371] The agents and compositions provided herein can be used alone or in combination with conventional treatment regimens such as surgery, irradiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0372] Cancer treatment represents a particularly desirable area where combination strategies are preferred because the combined action of two, three, four, or even more cancer drugs / therapies often produces a significantly stronger synergistic effect than a single treatment. Therefore, in another embodiment of the invention, cancer treatment can be effectively combined with a variety of other drugs, including, for example, conventional oncology treatments, multi-epitope strategies, additional immunotherapy, and treatments 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). The sequential administration of different agents can inhibit cancer cell growth at different checkpoints, while other agents can, for example, inhibit angiogenesis, malignant cell survival, or metastasis, potentially transforming cancer into a chronic disease. The following list provides some non-limiting examples of anticancer drugs and treatments that can be used in combination with the present invention:

[0373] 1. Chemotherapy

[0374] Chemotherapy is the standard of care for many types of cancer. The most common chemotherapeutic agents work by killing rapidly dividing cells (one of the main characteristics of cancer cells). Therefore, combinations with conventional chemotherapeutic agents (such as alkylating agents, antimetabolites, anthracyclines, phytoalkaloids, topoisomerase inhibitors, and other antitumor agents that affect cell division or DNA synthesis) can significantly improve the therapeutic effect of the present invention by clearing suppressor cells, restarting the immune system, making tumor cells more susceptible to immune-mediated killing, or by additionally activating cells of the immune system. The synergistic anticancer effects of chemotherapy and vaccine-based immunotherapy have been demonstrated in numerous 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; see also 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; see also Hirooka et al. 2009: A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38(3): e69-74). Hundreds of chemotherapy drugs are available, most of which are well-suited for combination therapy.Some (non-limiting) examples of chemotherapeutic agents that can be combined with the present invention are: carboplatin, cisplatin (Platinol, Platinol-AQ), crizotinib (Xalkori), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), and mesylate. Imatinib (Gleevec), irinotecan (Camptosar), liposome-encapsulated doxorubicin (DOXIL), methotrexate (Folex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), sorafenib (Nexavar), sunitinib (Sutent), topotecan (Hycamtin), trabectidin (Yondelis), vincristine (Oncovin, Vincasar PFS), and vinblastine (Velban).

[0375] 2. Surgery

[0376] Cancer surgery—the procedure of removing tumor cells—remains the foundation of cancer treatment. Surgery can be combined with other cancer treatments to remove any remaining tumor cells. Combining surgical approaches with subsequent immunotherapy has proven to be a promising method on several occasions.

[0377] 3. Radiation

[0378] Radiation therapy remains an important part 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 deprive cancer cells of their proliferative (cell division) potential. The types of radiation used to treat cancer are photon radiation (X-rays and gamma rays) and particle radiation (electron, proton, and neutron beams). There are two ways to deliver radiation to the site of cancer. External beam radiation is delivered from outside the body to the tumor site by targeting high-energy rays (photon, proton, or particle radiation). Internal radiation, or brachytherapy, is delivered directly to the tumor site from inside the body via a radiation source sealed in a catheter or seed. Suitable radiotherapy techniques for use in conjunction with this invention include, for example, fractionation (radiotherapy delivered in a fractionated manner, e.g., fractions of 1.5 to 3 Gy daily over several weeks), 3D conformal radiotherapy (3DCRT; delivering radiation to the total tumor volume), intensity-modulated radiotherapy (IMRT; intensity modulation of multiple radiation beams controlled by a computer), image-guided radiotherapy (IGRT; including techniques that allow for correction of pre-radiation imaging), and stereotactic body radiotherapy (SRBT; delivering extremely high individual doses of radiation in a small treatment fraction). 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.

[0379] 4. Antibodies

[0380] Antibodies (preferably monoclonal antibodies) achieve their therapeutic effects on cancer cells through a variety of mechanisms. They can play a direct role in inducing apoptosis or programmed cell death. They can block components of signal transduction pathways (e.g., growth factor receptors), effectively inhibiting the proliferation of tumor cells. In cells expressing monoclonal antibodies, they can induce the formation of anti-idiotypic antibodies. Indirect effects include recruiting cytotoxic cells, such as monocytes and macrophages. This type of antibody-mediated cell killing is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind to complement, leading to direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Combining surgical approaches with immunotherapeutic 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 combined with the present invention: abavoxumab (CA-125), axitumab (CD41), adalimumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), pentiazemab (CEA), amatuximab (MORAb-009), mamanomab (TAG-72), apoizumab (HLA-DR), axitumab (CEA), bavituzumab (phosphatidylserine), betuzumab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), butuximab (CD30). TNFRSF8), mocantuzumab (canAg mucin), rapamtuzumab (MUC1), caloumab pendipteptide (prostate cancer cells), carlumab (CNTO888), caputuzumab (EpCAM, CD3), cetuximab (EGFR), citatuzumab bogatox (EpCAM), citatuzumab (IGF-1 receptor), claaudiximab (mica), clivatuzumabtetraxetan (MUC1), sitatuzumab (TRAIL-R2), dacizumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), demuromumab (B-lymphoma cells), drozitumab (DR5), emeximab (GD3 ganglioside), epcam, erlotuzumab (EpCAM), erlotuzumab (SLAMF7), enostatin (PDL192), ensituximab (NPC-1C), epazolizumab (CD22), erlotuzumab (HER2 / neu, CD3), irexizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH) 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), ganitamumab (IGF-I), gemutumab oxazolidin (CD33), Gevokizumab (IL-1β), Girentuximab (carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), teimumab (CD20), Icrumumab (VEGFR-1), igovovomab (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Oxantuzumab (CD22), Ipilimumab (CD152), Iratumumab (CD30), Labezumab (CEA), Lesamumab (TRAIL-R2), Rivirex (Hepatitis B surface antigen), Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Lukamumab (CD40), Luximab (CD23), Mapamumumab (TRAIL-R1), Matozumab (EGFR), Mepolimab (IL-5), Milatuzumab (CD74), Mitomumab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumomab pasudotox (CD22), tanalocidab (C242 antigen), tanamumab (5T4), narnatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), oflamumab (CD20), olalatumab (PDGF-Rα), onartuzumab (human dispersive factor receptor kinase), oportuzumabMonatox (EpCAM), Ogvovomab (CA-125), Oxelumab (OX-40), Panitumab (EGFR), Patritumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pituitumab (adenocarcinoma antigen), Putomomumab (vimentin), Racotumomab (N-hydroxyacetylneuraminic acid), Radretumab (fibronectin extra domain-B), Raviolimab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sirolizumab (FAP), Cetuximab ( IL-6), Tabalumab (BAFF), Tazumab (alpha-fetoprotein), Patapromumab (CD19), Tenatumomab (tenosin C), Teprotumumab (CD221), Simimumab (CTLA-4), Garizumab (TRAIL-R2), TNX-650 (IL-13), Tosimomumab (CD20), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tesimomumab (CTLA-4), Simimumab (EpCAM), Ublituximab (MS4A1), Urelumab (4-1BB), Voloximab (integrin α5β1), Votomumab (tumor antigen CTAA16.88), Zatumumab (EGFR), Zatumumab (CD4).

[0381] 5. Cytokines, chemokines, co-stimulatory molecules, fusion proteins

[0382] Another embodiment of the invention involves combining the antigen-encoded pharmaceutical composition of the invention with cytokines, chemokines, co-stimulatory molecules, and / or their fusion proteins to induce beneficial immunomodulatory or tumor-suppressive effects. To increase the infiltration of immune cells into tumors and promote the movement of antigen-presenting cells to tumor-draining lymph nodes, a variety of chemokines having C, CC, CXC, and CX3C structures can be used. 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, co-stimulatory or regulatory molecules, such as B7 ligands (B7.1 and B7.2), are useful. Other cytokines are also useful, such as interleukins, especially (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 other stimulating molecules, including but not limited to: 4-1BB, 4-1BB-L, CD137, CD137L, CTLA-4GITR, GI TRL, Fas, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LT.β.R, 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-induced TNFR-related proteins, TNF receptor-associated apoptosis-mediating protein (TRAMP), and death receptor 6 (DR6). CD40 / CD40L and OX40 / OX40L are important targets for combination immunotherapy because they directly affect T cell survival and proliferation. For review, see Lechner et al. 2011: Chemokines, costimulatorymolecules and fusion proteins for the immunotherapy of solidtumors. Immunotherapy 3(11), 1317-1340.

[0383] 6. Bacterial treatment

[0384] Researchers have been using anaerobic bacteria, such as *Clostridium novyi*, to deplete the oxygen-poor interior of tumors. When anaerobic bacteria come into contact with the oxidative side of the tumor, they die, meaning they are harmless to the rest of the body. Another strategy utilizes anaerobic bacteria transformed with enzymes that can convert non-toxic prodrugs into toxic drugs. As the bacteria proliferate in the necrotic and hypoxic areas of the tumor, these enzymes are expressed only within the tumor. Therefore, systemically applied prodrugs are metabolized into toxic drugs only within the tumor. This has been proven effective using the non-pathogenic anaerobic bacterium *Clostridium sporogenes*.

[0385] 7. Kinase inhibitors

[0386] Another large group of potential targets for complementary cancer therapy includes kinase inhibitors, as cancer cell growth and survival are closely related to dysregulation of kinase activity. A wide range of inhibitors are being used to restore normal kinase activity, thereby reducing tumor growth. The group of targeted kinases includes receptor tyrosine kinases such as BCR-ABL, β-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, and ALK; cytoplasmic tyrosine kinases such as c-SRC, c-YES, Abl, and JAK-2; serine / threonine kinases such as ATM, Aurora A&B, CDK, mTOR, PKCi, PLK, β-Raf, S6K, STK11 / LKB1, and lipid kinases such as PI3K and SK1. Small molecule kinase inhibitors, such as PHA-739358, nilotinib, dasatinib, PD166326, NSC 743411, lapatinib (GW-572016), cannatinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), suten (SU11248), sorafenib (BAY 43-9006), and leflunomide (SU101). For more information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0387] 8. Toll-like receptors

[0388] Members of the Toll-like receptor (TLR) family are important links between innate and adaptive immunity, and the action of many adjuvants depends on TLR activation. Numerous established cancer vaccines incorporate TLR ligands to enhance vaccine responses. In addition to TLR2, TLR3, TLR4, and especially TLR7 and TLR8 have been tested in passive immunotherapy for cancer treatment. The closely related TLR7 and TLR8 contribute to antitumor responses by influencing immune cells, tumor cells, and the tumor microenvironment, and can be activated by nucleoside analogue structures. All TLRs have been used as adjuvants for standalone immunotherapies or cancer vaccines and can be synergistically combined with the formulations and methods of this invention. For more information, see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

[0389] 9. Angiogenesis inhibitors

[0390] In addition to therapies targeting tumor-mediated escape mechanisms and immunomodulatory receptors affected by immunosuppression, there are also therapies targeting the tumor environment. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) necessary for tumor survival. For example, angiogenesis, which is promoted to meet the increased nutrient and oxygen demands of tumor cells, can be blocked by targeting different 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 neurofeedin-1 and neurofeedin-2)1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, angiogenesis inhibitor, calreticulin, platelet factor 4, TIMP and CDAI, Meth-1 and Meth-2, IFN-α, IFN-β and IFN-γ, CXCL10, IL-4, IL-12 and IL-18, prothrombin (tricyclic domain-2), antithrombin III fragments, prolactin, VEGI, SPARC, osteopontin, mammary filamentin, canstatin, proliferation-related proteins, restin, and drugs such as bevacizumab, itraconazole, carboxytriazole, TNP-470, CM101, IFN-α, platelet factor-4, suramin, SU5416, platelet-reactive protein, VEGFR antagonists, angiogenesis inhibitory steroids + heparin, chondroitin-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, platelet-reactive protein, prolactin αVβ3 inhibitors, linolamine, tasquinimod, see Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9): 976-81.

[0391] 10. Small molecule targeted therapy drugs

[0392] Small molecule targeted therapies are typically inhibitors of the enzymatic domains of mutated, overexpressed, or other key proteins within cancer cells. Prominent and non-limiting examples are the tyrosine kinase inhibitors imatinib (Gleevec / Glivec) and gefitinib (Iressa). The use of small molecules targeting certain kinases (e.g., sunitinib malate and / or sorafenib tosylate) in combination with vaccines for cancer treatment is also described in previous patent application US2009004213.

[0393] 11. Virus-based vaccines

[0394] Numerous available or developing virus-based cancer vaccines can be used in combination with the formulations of this invention for therapeutic purposes. One advantage of using such viral vectors is their inherent ability to elicit an immune response, resulting in an inflammatory reaction as a consequence of viral infection, which serves as a danger signal required for immune activation. Ideally, the viral vector should be safe and should not introduce an anti-vector immune response to enhance the anti-tumor-specific response. Recombinant viruses such as vaccinia virus, herpes simplex virus, adenovirus, adeno-associated virus, retrovirus, and fowlpox virus have been used in animal tumor models and, based on encouraging results, have begun human clinical trials. Particularly important virus-based vaccines are virus-like particles (VLPs), which are small particles containing certain proteins from the outer shell of a virus. Virus-like particles do not contain any genetic material from the virus and do not cause infection, but they can be constructed to present tumor antigens on their outer shell. VLPs can be derived from a variety of viruses, such as hepatitis B virus or other viral families, including Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV), and Flaviviridae (e.g., hepatitis C virus). For a general review, see Sorensen and Thompson 2007: Virus-based immunotherapy of cancer: what do we know and where are we going? APMIS 115(11):1177-93; Virus-like particles targeting cancer are reviewed in Buonaguro et al. 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert RevVaccines 10(11):1569-83; and Guillén 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.

[0395] 12. Multi-epitope strategy

[0396] The use of multiple epitopes demonstrates promising results for vaccination. Rapid sequencing technology combined with intelligent algorithm systems enables the utilization of tumor mutanomes and can provide multiple epitopes for personalized vaccines that can be combined with this invention. For more 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 Castle et al. 2012: Exploiting the mutanome for tumor vaccination. Cancer Res 72(5):1081-91.

[0397] 13. Adoptive T cell transfer

[0398] For example, 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 describes the combination of tumor antigen vaccination and T-cell transfer.

[0399] 14. Peptide-based targeted therapy

[0400] Peptides can bind to cell surface receptors or to the affected extracellular matrix around the tumor. If the radionuclide decays near the cell, the radionuclide linked to these peptides (e.g., RGD) ultimately kills the cancer cells. Oligomers or multimers of these binding motifs are of particular interest because they can lead to enhanced tumor specificity and affinity. 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.

[0401] 15. Other treatments

[0402] Many other cancer treatments can be combined with this invention to produce synergistic effects. Non-limiting examples include apoptosis-targeting therapies, hyperthermia, hormone therapy, telomerase therapy, insulin-enhanced therapy, gene therapy, and photodynamic therapy.

[0403] Various methods known in the art can be used to detect and / or determine the amount of cells expressing CLDN6.

[0404] For example, immunoassays can be used to detect the expression of CLDN6 protein in cells or on the cell surface. According to the present invention, immunoassays include, but are not limited to: Western blot, immunohistochemistry, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioassay, fluorescence immunoassay, immunofluorescence, protein A immunoassay, flow cytometry, or FACS analysis.

[0405] In one implementation, the cells are bound to an antibody capable of binding one or more CLDN6 markers prior to detection and / or measurement.

[0406] Alternatively, the expression of CLDN6 mRNA can be detected or the amount of CLDN6 mRNA can be determined to detect and / or measure the number of cells expressing CLDN6.

[0407] In some embodiments of the invention, the sample obtained from the patient for detecting and / or determining the amount 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 tissue biopsy from a subject having or suspected of having cancerous tissue). Most preferably, the sample is a tissue biopsy of a tumor.

[0408] According to the method of the present invention, the sample may be a biological sample that has undergone one or more pretreatment steps before detecting and / or determining the amount of cells expressing CLDN6. In some embodiments, the biofluid is pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or by a combination of these pretreatment steps. In other embodiments, the tissue sample is pretreated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization, or homogenization, followed by centrifugation, filtration, precipitation, dialysis, or chromatography, or by a combination of these pretreatment steps.

[0409] The amount of cancer stem cells in a sample can be expressed as, for example, as a percentage of all cells or all cancer cells in the sample, or quantified relative to area (e.g., cells per field of view), volume (e.g., cells per ml), or weight (e.g., cells per ml).

[0410] The amount of cancer stem cells in the test sample can be compared to the amount of cancer stem cells in (a) a reference sample. In one embodiment, the reference sample is a sample obtained from a subject treated at an earlier time point (e.g., before receiving treatment as a baseline reference sample, or at an earlier time point during treatment). In this embodiment, the expected outcome of the treatment is a reduction 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 of the same type of cancer. In this embodiment, the expected outcome of the treatment is that the test sample has the same amount of cancer stem cells as or less than the amount of cancer stem cells detected in the reference sample. In a specific embodiment, a stable or decreasing amount of cancer stem cells relative to an earlier determined (previously detected) amount for the subject indicates an improvement in the subject's prognosis or a positive response to treatment, while an increase in the amount of cancer stem cells relative to an earlier determination indicates the same or worse prognosis and / or a failure to respond to treatment.

[0411] In some implementations, a combination of cell surface markers (e.g., CLDN6) with other markers typical of cancer stem cells is used to determine the amount of cancer stem cells in a sample.

[0412] The present invention also provides a kit comprising one or more containers filled with reagents for detecting, measuring, or monitoring cells expressing CLDN6. In one embodiment, the kit optionally includes instructions for using reagents (particularly those used in the methods of the present invention) to determine cancer stem cells or monitor the efficacy of cancer treatment by detecting and / or measuring the amount of cells expressing CLDN6. In one embodiment, the kit contains an agent that specifically binds to CLDN6 protein or CLDN6 mRNA. In some embodiments, the agent is an antibody or antibody fragment. In other embodiments, the agent is a nucleic acid. For nucleic acid detection, the kit generally contains (but is not limited to) a probe specific to CLDN6 mRNA. For quantitative PCR, the kit generally contains preselected primers specific to the CLDN6 nucleic acid sequence. The quantitative PCR kit may also contain an enzyme suitable for amplifying nucleic acids (e.g., a polymerase such as Taq) and deoxynucleotides and buffers required for amplifying the reaction mixture. The quantitative PCR kit may also contain a probe specific to the CLDN6 nucleic acid sequence. In some embodiments, the quantitative PCR kit also includes components suitable for reverse transcription of RNA, said components including enzymes (e.g., reverse transcriptase) and primers for the reverse transcription reaction, as well as deoxynucleotides and buffers required for the reverse transcription reaction.

[0413] In some embodiments, the reagent is detectably labeled. Furthermore, the kit may contain instructions for performing the assay and for interpreting and analyzing the data generated from the assay performance.

[0414] Based on the results obtained (i.e., whether cancer stem cells are present or whether their quantity is stable or decreasing), medical personnel can choose specific cancer treatments, such as cancer treatments targeting cancer stem cells, or they can choose to continue the treatment. Alternatively, based on the results, if cancer stem cells are not present or their quantity has not increased, medical personnel can choose to administer cancer treatments not targeting cancer stem cells or to continue, modify, or discontinue the treatment.

[0415] In some embodiments of the invention, if it is determined that the reduction in the cancer stem cell population is insufficient to compare the cancer stem cell population in a sample obtained from a patient undergoing cancer treatment with a sample previously obtained from said patient, then the medical personnel have several options to adjust the treatment. For example, the medical personnel may increase the dosage of the cancer treatment, the frequency of administration, the duration of administration, or any combination thereof. In one specific embodiment, after determination, additional cancer treatment may be administered to the patient in place of or in combination with the first treatment.

[0416] In other implementations, if it is determined that the reduction in the cancer stem cell population is useful for comparing the cancer stem cell population in a sample obtained from a patient undergoing cancer treatment with a sample previously obtained from said patient, then the medical personnel may choose not to adjust the cancer treatment. For example, the medical personnel may choose not to increase the dosage, frequency, duration of administration, or any combination thereof of the cancer treatment. Additionally, the medical personnel may choose to add other treatments or combinations thereof.

[0417] The present invention is further illustrated by the following embodiments, which are not to be construed as limiting the scope of the invention.

[0418] Example

[0419] Example 1: Expression of CLDN6 on the surface of induced human pluripotent stem cells

[0420] To analyze whether CLDN6 is expressed in human induced pluripotent stem cells (iPSCs), according to the protocol described in PCT / EP2012 / 04673, a reprogrammed mixture (unmodified OSKMNL+EBK+miR-mix; consisting of in vitro transcribed (IVT) RNA from OSKMNL = transcription factors OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28, IVT-RNA from EBK = IFN-escape proteins E3, K3, and B18R, and a miRNA mix consisting of miR-302a / b / c / d and 367) or simulated transfected HFFs (RNA-free control) was used at multiple time points during treatment. Quantitative real-time RT-PCR (qRT-PCR) was performed using an ABI PRISM 7300 sequence detection system and software (Applied Biosystems with QuantiTect SYBR green Kit (Qiagen)). Transcript expression of CLDN6 in *Bioscience*. Cells were cultured in Nutrisem serum-free medium (Stemgent, Cambridge (MA)) supplemented with 10 ng / ml bFGF and 0.5 μM Thiazovivin. The reprogramming mixture 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 only (RNA-free control). On day 19 of treatment, we detected a significant upregulation of CLDN6 of almost 6000-fold compared to untreated HFF, while on day 12 of treatment, we observed an upregulation of approximately 2000-fold. Figure 1Therefore, CLDN6 is expressed in human induced pluripotent stem cells (iPSCs).

[0421] Flow cytometry was used to examine whether CLDN6 was also expressed on the surface of iPSCs. Since iPSCs grow on HFF feeder cells, we combined this with staining analysis for SSEA-4 (a widely accepted stem cell marker) to ensure specific detection of iPSCs. For this purpose, HFF cells treated with a reprogramming mixture or a simulated control (RNA-free) were collected on days 5, 12, and 19 of treatment and stained with 1 μg / ml CLDN6-specific IMAB027-AF647 and 2 μl SSEA-4 antibody at 4°C for 30 min, and surface expression was analyzed by flow cytometry. Our staining protocol also included the viability dye 7-AAD to exclude dead cells from our analysis. The experiment was repeated twice, and 50,000 events were recorded from each sample using a BD CantoII flow cytometer. The recorded cells were analyzed using FlowJo software, and a representative dot plot is shown. Figure 2 ).

[0422] On day 5, CLDN6 was undetectable on the surface of HFFs treated with or without the reprogramming mixture. Unexpectedly, we found that 15% of the HFFs expressed SSEA-4 regardless of treatment with the reprogramming mixture. This can be explained by the fact that the HFFs used were newly generated fibroblasts, and these cells may retain some positivity for SSEA-4. On day 12 of treatment, approximately 63% of the treated HFFs were positive for SSEA-4, and we observed approximately 15% of the CLDN6-SSEA-4 double-positive fraction. On day 19 of treatment, 15% of the treated HFFs were positive for both CLDN6 and SSEA-4, representing distinct subsets. It is speculated that the CLDN6-SSEA-4 positive subset merely labels iPSCs, while the CLDN6-SSEA-4 negative subset is considered to be HFF feeder cells or non-reprogrammed cells, and the SSEA-4 single-positive cells represent cells present at the start of reprogramming.

[0423] Since we found that 15% of HFF cells were positive for SSEA-4 but not for CLDN6, we hypothesize that CLDN6 represents a more specific biomarker for human iPSCs than SSEA-4. SSEA-4 is also expressed in nascent HFFs, while CLDN6 appears to be specifically expressed only in all reprogrammed HFF cells representing the iPSC fraction.

[0424] Therefore, CLDN6 is specifically expressed on the surface of human iPSCs.

[0425] Example 2: CLDN6 plays an important role in the colony formation of ovarian cancer cells.

[0426] An effective 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 efficacy of individual tumor cells. To analyze whether CLDN6 plays a role in tumorigenesis, we selected COV318, an ovarian tumor cell line exhibiting only a CLDN6-positive cell subset, and PA-1, a uniformly CLDN6-expressing cell line carrying stable lentiviral small hairpin RNA (shRNA) that mediates CLDN6 knockdown (clones PA-1 50, PA-1 54); [Reference] Figure 3 .

[0427] Cells were stained with 1 μg / ml IMAB027-AF647 for CLDN6 at 4°C for 30 minutes, and then sorted for CLDN6 expression using a BDFACSAria cell sorter via FACS (Fluorescence Activated Cell Sorting). 500 (PA-1 50, PA-1 54) or 700 (COV318) cells from CLDN6-positive or negative subsets were directly sorted into the wells of 6-well plates and allowed to grow for 14 days until sufficient colonies formed. The culture medium was changed twice weekly. Colonies were stained with 10% ethanol containing 0.5% crystal violet and fixed for 20 minutes, 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 The image shows representative colony formation assays of COV318 and CLDN6-knockdown cell lines PA-150 and 54.

[0428] 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 colony-forming ability, an important characteristic of cancer stem cells.

[0429] Example 3: Co-expression of CLDN6 with CSC markers CD24, CD90 and CD44 in ovarian cancer cell lines

[0430] The use of specific surface marker expression profiles is a common strategy for identifying and isolating cell carcinoma cells (CSCs) from solid tumors and cell lines. Surface markers used in the literature for isolating CSCs from ovarian cancer include CD44, CD24, CD90, CD34, CD117, and CD133. To analyze whether we could identify CSC subsets in ovarian cancer cell lines containing small CLDN6-positive cell subsets, we established a FACS group containing antibodies against these surface markers (Table 1). Furthermore, we included detecting CLDN6 antibodies in the group to investigate the percentage of CLDN6 colocalization with established CSC markers, thereby assessing CLDN6's potential as a CSC marker. For this purpose, COV318 1E6 cells were stained with specified antibody amounts (see Table 1) at 4°C for 30 minutes, followed by flow cytometry analysis of their surface marker expression profiles. Our staining protocol also included viability dyes. This was to exclude dead cells from our analysis. The experiment was performed three times in duplicate, and 50,000 events were recorded from each sample using a BD Canto II flow cytometer. The recorded cells were analyzed using FlowJo software.

[0431]

[0432] Table 1: CSC FACS Groups. This table shows the FACS groups used to analyze CSC markers and CLDN6 expression in ovarian cancer cell lines. The amounts of antibodies used for the respective markers and the conjugated fluorescent dyes are listed.

[0433] FACS analysis showed that COV318 cells expressed the CSC markers CD44, CD90, and CD24 subsets, as well as CLDN6, which at least partially colocalized with all three markers. Figure 5A). We then used different gating strategies to calculate the percentage of colocalization for all four markers. First, we calculated the percentage of CD44, CD24, CD90, and CLDN6 positive cells in the entire viable cell population. We found that 0.18% of viable cells were positive for all four markers. Next, we calculated the percentage of CD44, CD24, and CD90 positive cells in the viable cell population, which represents the CSC fraction. We found that when set relative to the entire viable cell population, 0.23% of cells were positive for all three markers; however, when set relative to the CLDN6 positive subset, we found that 20.1% of the cell fraction was triple positive, indicating an 87-fold concentration of the three markers in the CLDN6 positive subset. In the final step, we calculated the percentage of CLDN6 positive cells in the entire viable cell population on one hand, and its percentage in the CD44 / CD24 / CD90 positive subset on the other. We found that the concentration of cells expressing CLDN6 increased from 0.91% in the total cell population to 66.87% in the CSC fraction, indicating a 74-fold increase. Figure 5 B).

[0434] In summary, these data indicate that CLDN6 accumulates in the CSC gradation, while CSC biomarkers are enriched in the CLDN6-positive subgroup. These findings suggest that CLDN6 is a biomarker for CSC.

[0435] Example 4: Enrichment of cells expressing CLDN6 led to the accumulation of established CSC markers CD44, CD24, and CD90.

[0436] It has been shown that CSC fractions isolated from cell lines and tumors are often rich in CSC markers such as CD44 and CD24. To analyze the potential of CLDN6 as a novel CSC marker, we investigated whether CLDN6-positive fraction cell isolates from a large number of cells lead to the accumulation of established ovarian CSC markers.

[0437] For this purpose, COV318 cells were stained with 0.5 μg / ml IMAB027 at 4°C for 30 min, and then incubated with goat anti-human IgG secondary antibody (1:300) at 4°C for 10 min. CLDN6-positive and CLDN6-negative cell fractions were then separated from COV318 cells using a BD FACSAria cell sorter via FACS sorting. The selected cells were then expanded under standard growth conditions for 10 days. 1E6 cells from both subpopulations were stained at 4°C for 30 min against the CSC markers CD44, CD24, CD90, CD34, CD117, and CD133 (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 BDCanto II flow cytometer, and the recorded cells were analyzed using FlowJo software.

[0438] FACS analysis showed that after 10 days of culture under standard conditions, approximately 50% of cells in the CLDN6-positive fraction remained CLDN6-positive, while cells in the CLDN6-negative fraction were completely CLDN6-negative. Importantly, we found that the CLDN6-positive fraction showed accumulation of CSC markers CD44, CD24, and CD90 compared to the CLDN6-negative fraction of COV318 cells. Representative points from different samples are illustrated in the figure. Figure 6 Further quantification of the expression levels of these biomarkers revealed that when comparing CLDN6-positive and CLDN6-negative subsets, CD44 was enriched 99-fold, CD90 was enriched 8-fold, and CD24 was enriched 33-fold. Figure 6 B).

[0439] These results demonstrate that CLDN6 can be used as a selection marker for isolating CSC fractions from a large number of cell lines, indicating that CLDN6 is a novel CSC marker.

[0440] Example 5: Compared with cells expressing low CLDN6, cell lines expressing high CLDN6 showed enrichment of CSC markers.

[0441] CLDN6 has been shown to be highly expressed in germ cell tumors, ovarian adenocarcinoma, and some cancers with a primitive phenotype. In cases where CLDN6 is a CSC marker, we would expect the accumulation of CSC-like cells in such cell lines or tumors, thus resulting in the accumulation of CSC marker-positive cells.

[0442] We investigated the expression levels of established CSC markers in four CLDN6-high expression cell lines: ovarian cancer cell lines OV90 and PA-1, and testicular cancer cell lines NEC-8 and NEC-14. For this purpose, 1E6 cells from each cell line were stained at 4°C for 30 min against surface markers CD44, CD24, CD90, CD34, CD117, and CD133, as well as CLDN6 (see Table 1 for details), and the expression profiles of the cells were then analyzed by flow cytometry. Experiments were performed in triplicate, with 50,000 events recorded from each sample using a BD Canto II flow cytometer, and the recorded cells analyzed using FlowJo software. Viability staining was used to analyze the expression levels of CLDN6. Counterstaining was performed to exclude dead cells from the analysis. Representative spots from each sample are illustrated in the figure below. Figure 7 middle.

[0443] FACS analysis showed that approximately 95% of all studied cell lines were positive for CLDN6. As expected, in addition to CLDN6, these CLDN6-overexpressing cell lines also showed accumulation of established CSC markers, with OV90 cells showing high expression of CD44, CD133, CD24, and CD117, PA-1 cells showing high expression of CD44, CD133, CD90, and CD117, and NEC-8 and NEC-14 cells showing elevated expression levels of the markers CD133, CD90, CD24, and CD117.

[0444] These results indicate that CLDN6-expressing cell lines are rich in CSC-like cells and further support the view that CLDN6 is a CSC marker.

[0445] Example 6: Treatment of advanced human xenograft tumors using a combination of CLDN6 antibody and chemotherapy drugs to synergistically enhance the therapeutic effect. The same method inhibited tumor cell growth and prolonged survival.

[0446] Transplanting human cancer cell lines into Hsd: athymic naked-Foxn1 nu Mice. After tumor establishment, tumor-bearing mice were grouped and administered CLDN6-specific monoclonal antibody (IMAB027), chemotherapy, or a combination of both. The control group received antibody buffer (loador control).

[0447] Specifically, for the treatment of tumors xenografted with human ES-2 (CLDN6), the stable human ovarian cancer cell line ES-2 transfected with human CLDN6 was cultured in a humidified incubator at 37°C with 5% CO2 in a minimum 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, the tumor was transplanted into 6-week-old female Hsd: thymic naked-Foxn1 cells. nu Mice were subcutaneously inoculated in 200 μl PBS at a dose of 5 × 10⁻⁶ mg / L. 6 ES-2 (CLDN6) cells. Mice were treated with saline control, antibody or drug monotherapy, and antibody / cell growth inhibitor combination therapy (n=12 per group) on day 3 post-transplantation. Paclitaxel or saline control was administered at 15 mg / kg on days 3, 10, and 17 post-transplantation. Antibody maintenance therapy began on day 4 with rapid bolus injections of 35 mg / kg IMAB027 or a loading control (IMAB027 buffer) three times weekly (alternating IV / IP / IP). Tumor burden and animal health were monitored twice weekly. Tumor volume reached a maximum of 1400 mm². 3Mice were euthanized when the tumors became ulcerative. The Kruskal-Wallis test and post-hoc Dunn multiple comparison test were used to analyze tumor growth inhibition.

[0448] For the treatment of advanced human NEC14 xenograft tumors, according to the supplier's instructions, in a humidified incubator at 37°C with 5% CO2, in RPMI 1640 medium containing 10% FCS (Life Technologies) and GlutaMAX. TM Human testicular germ cell tumor cell line NEC14 was cultured at Life Technologies. For transplantation, the cell line was introduced into 6-8 week old female Hsd: thymic naked-Foxn1 cells. nu Mice were subcutaneously inoculated in 200 μl PBS at a dose of 2 × 10⁻⁶ mg / L. 7 NEC14 cells. In the advanced treatment study, the tumor grew to a volume of 50 mm. 3 Up to 150mm 3 Before treatment, mice were randomly assigned to a control group, an antibody group or a single-drug treatment group, and an antibody / drug combination treatment group (n=19 per group). Six days post-transplantation, mice were administered the following single-drug or combination or load control (saline): cisplatin 1 mg / kg intraperitoneally (IP) on days 6, 7, 8, 9, and 10; carboplatin 30 mg / kg intraperitoneally (IP) on days 6, 13, and 20; and antibody maintenance therapy, with rapid bolus injections of 35 mg / kg IMAB027 or load control (IMAB027 buffer) three times weekly (alternating IV / IP / IP). Tumor burden was monitored twice weekly. Tumors were classified as tumor burden when they reached a maximum size of 1400 mm². 3 Mice were euthanized when the tumors became ulcerative. The Kruskal-Wallis test and post-hoc Dunn multiple comparison test were used to analyze tumor growth inhibition.

[0449] Compared with the control group, paclitaxel treatment of human ES-2 (CLDN6) xenograft tumors expressing human CLDN6 in ectopic locations was ineffective and showed no antitumor activity. In contrast, IMAB027 inhibited tumor growth and prolonged survival in mice. Combination therapy with IMAB027 and paclitaxel synergistically inhibited tumor growth. Figure 8 ).

[0450] Furthermore, both cisplatin and IMAB027, as single agents, significantly reduced tumor growth in animals with NEC14 tumors. However, after initial tumor growth inhibition, we observed recurrent tumor growth in most animals. In the combination therapy approach, cisplatin and IMAB027 synergistically inhibited tumor growth and induced complete NEC14 tumor remission. Most impressively, survival data demonstrated the therapeutic efficacy of the IMAB027 combined with cisplatin. Compared to the single-agent approach, almost all mice treated with IMAB027 in combination with cisplatin survived for 90 days after tumor transplantation. Figure 9 ).

[0451] In late-stage treatment of mice with NEC14 xenograft tumors, other platinum derivatives (such as carboplatin) showed only very limited antitumor efficacy. However, the combination of carboplatin and IMAB027 resulted in a synergistic tumor-suppressive effect, as well as highly efficient tumor growth inhibition and prolonged survival. Figure 10 ).

[0452] Therefore, the combination of CLDN6-specific antibodies and chemotherapy drugs increased the inhibition of tumor growth and prolonged the survival of mice transplanted with human tumor cells. The combination of antibodies and chemotherapy drugs produced a synergistic effect in inhibiting tumor cell growth and prolonging survival.

[0453] Example 7: CLDN6 is a CSC marker

[0454] Example 7.1: CLDN6 is important for the sphere-forming behavior of ovarian cancer cells.

[0455] Another effective assay for analyzing CSC-like characteristics of tumor cells is the spheroidization assay. This assay readily examines the ability of cells to exhibit anchorage-independent growth (a hallmark of CSCs). To analyze whether CLDN6 plays a role in anchorage-independent growth of tumor cells, we selected COV318 cells (an ovarian tumor cell line containing only a CLDN6-positive cell subset). COV318 cells were sorted based on CLDN6 expression, and CLDN6-positive and negative cell populations were cultured under stem cell-specific conditions for 21 days to form spheroids. The spheroidization assay showed that CLDN6-positive COV318 cells exhibited the ability to form spheroids when cultured under stem cell-specific conditions, while CLDN6-negative cells almost completely died. Figure 11 A). These results indicate that the CLDN6 positive fraction represents a rich cluster of stem cells that exhibits the ability to anchor-independent growth.

[0456] To determine the ability of CLDN6-positive COV318 cells to undergo multiple cell division cycles while maintaining their undifferentiated state, we analyzed the ability of spheroids to generate second-generation spheroids. For this purpose, first-generation spheroids of CLDN6-positive COV318 cells (day 22 post-seeding) were dissociated into single cells and then replated. Twenty-three days after replateding, we could clearly observe the formation of second-generation spheroids, and these newly formed spheroids were morphologically more regular than the initial first-generation spheroids. Figure 11 B). These observations further confirm that the CLDN6 positive fraction of COV318 cells represents the stem cell fraction of this cell line.

[0457] In summary, these results clearly demonstrate that CLDN6 plays a significant role in anchorage-independent growth of ovarian cancer cells (an important characteristic of cancer stem cells).

[0458] Example 7.2: Enrichment of CLDN6-positive COV318 cells after in vitro treatment with chemotherapeutic agents.

[0459] The ovarian cancer cell line COV318 exhibits heterologous expression of CLDN6 and a very small cell subset (~0.3-0.5%) expressing CLDN6. In vitro treatment of COV318 cells with platinum derivatives under various conditions yielded a residual cell population with a higher percentage of CLDN6-positive cells (>2%). Figure 12 The specific accumulation of CLDN6-positive cells after treatment suggests that these cells may have a selective survival or growth advantage during chemotherapy. Resistance to conventional chemotherapy is a characteristic of CSCs.

[0460] Example 7.3: Enrichment of CLDN6-positive COV318 cells in vivo

[0461] In previous studies, we found that COV318 cells injected subcutaneously into the flanks of athymic nude mice were weakly tumorigenic. Conversely, mice receiving COV318 cells via intraperitoneal injection developed malignant ascites and peritoneal tumors over 100 days. Compared to parental COV318 cells (… Figure 13 Compared to A), most cells isolated from ascites and tumors were CLDN6 positive. Figure 13 B, top image). After culturing under standard conditions, COV318 cells again lost CLDN6 expression in vitro ( Figure 13 (B, below). This means that CLDN6-positive COV318 cells exhibit a higher tumorigenic capacity, indicating that the small CLDN6-positive subset contains CSCs.

[0462] Example 7.4: CLDN6 is associated with ovarian cancer stem cell markers in primary tumor samples.

[0463] Because our previous results showed that CLDN6 co-localizes with some CSC markers in ovarian cancer cell lines, we then posed the question: Is CLDN6 expression also associated with CSC markers in primary tumor samples from ovarian cancer patients?

[0464] For this purpose, the mRNA expression of CLDN6 and selected biomarkers (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1A1, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch1, Pou5F1, CD133, Snail, Sox2) specific to cancer stem cells in ovarian cancer were analyzed by qRT-PCR in 42 human ovarian cancer samples. Subsequent correlation analysis of CLDN6 with these biomarkers was performed using Spearman's r. Scatterplots. Scatterplots of significant correlations and summaries of all correlations are shown below. Figure 14 middle.

[0465] In the analyzed ovarian cancer samples, CLDN6 was found to be positively correlated with CTCFL, LIN28B, CD24, GNL3, and EPCAM, while CD44 was found to be negatively correlated with CLDN6. Figure 14 A). No significant correlation was observed for any of the other studied biomarkers. Figure 14 B).

[0466] These results further support the view that CLDN6 is a CSC marker.

[0467] Example 8: Enhanced antitumor activity of anti-CLDN6 antibody by combination with chemotherapy drugs

[0468] Example 8.1: Effects of chemotherapy agents on IMAB027-mediated ADCC

[0469] The effects of chemotherapy agents on IMAB027-mediated ADCC were analyzed using COV362 (Luc) cells pretreated with carboplatin, gemcitabine, paclitaxel, doxorubicin, and topotecan, respectively. As shown by flow cytometry, pretreatment of target cells led to an increase in CLDN6 protein levels on the cell surface. Figure 15 B, D, F, H, and J). Compared to untreated target cells, cells treated with chemotherapeutic agents showed a 3-fold increase in maximum cell lysis (B, D, F, H, and J). Figure 15 (A, C, F, G, and I). In summary, combination with chemotherapy drugs enhances the antitumor activity of IMAB027.

[0470] Example 8.2: The combination of multiple chemotherapy PEB (cisplatin, etoposide, and bleomycin) and IMAB027 effectively increased the inhibition of tumor growth and prolonged the survival of mice transplanted with the human tumor cell line NEC14.

[0471] Hsd: Athymic naked-Foxn1 nu Mice were subcutaneously transplanted with the human testicular germ cell tumor cell line NEC14. Mice with very advanced tumors were randomly assigned to groups and treated with antibody IMAB027, multiple chemotherapy for PEB (cisplatin, etoposide, and bleomycin), or a combination of IMAB027 and PEB.

[0472] Compared with untreated and IMAB027-treated mice, multichemotherapy significantly reduced tumor growth. Figure 16 A). However, in most animals, tumors began to grow on day 30 after the initial response to PEB treatment. Sustained inhibition of tumor growth was only achieved in the combination therapy where PEB and IMAB027 acted synergistically. Figure 16 B). Untreated mice showed a median survival of 30 days, while median survival of 34 and 97 days was observed in mice treated with IMAB027 and PEB, respectively. In the PEB-treated group, at the end of the study, 3 / 14 (21%) mice showed complete tumor regression, and 1 / 14 (7%) mice showed ~30 mm 3 Residual tumor masses were observed. Surprisingly, complete tumor regression was observed in 12 / 14 (86%) mice treated with the combination of PEB and IMAB027. Eleven mice were cured with no recurrence within 6 months. One tumor-free mouse was euthanized on day 93 due to poor overall health. Figure 16 C). This study showed that animals with very advanced human NEC14 testicular tumors who received the combination of PEB and IMAB027 had significantly longer survival and significantly higher response rates compared to animals treated with multiple chemotherapy alone.

[0473] Example 9: Anti-CLDN6 antibody-drug conjugates are highly effective in treating tumors expressing CLDN6.

[0474] Example 9.1: In vitro binding and antitumor activity of toxin-conjugated IMAB027

[0475] The relative binding affinity of the IMAB027-drug conjugates IMAB027-DM1 and IMAB027-vcMMAE was tested on the ovarian cancer cell line OV90 using flow cytometry. In saturation binding assays, antibody concentration was plotted against median fluorescence intensity (MFI) and EC50 (the concentration of antibody that binds to half of the binding sites at equilibrium), and maximum binding was calculated by nonlinear regression. Compared to unconjugated IMAB027, the IMAB027-drug conjugates showed similarly low EC50 values ​​and achieved saturation binding at low concentrations. Figure 17 A). The cytotoxic activity of the IMAB027-drug conjugate was determined using an XTT proliferation assay on OV90 cells. Dose-response curves showed similar inhibition of tumor cell growth by IMAB027-DM1 and IMAB027-vcMMAE in vitro. Figure 17 B). In summary, IMAB027 conjugated with DM1 or vcMMAE binds to CLDN6-positive target cells with similar relative affinity and efficiently induces tumor cell killing.

[0476] Example 9.2: Treatment of advanced human xenograft tumors with toxin-conjugated IMAB027 anti-CLDN6 antibody inhibited tumor cell growth, prolonged survival, and mediated complete tumor regression.

[0477] The antitumor activity of the CLDN6-specific antibody IMAB027, conjugated to a cytotoxic drug, was tested in mice transplanted with CLDN6-positive human cancer cell lines. In this targeting approach, IMAB027 was linked to either maytansine alkaloid DM1 or olistatin E (MMAE) and then conjugated to thymic naked-Foxn1 in mice with advanced xenograft tumors. nu Monitoring tumor growth in mice.

[0478] Treatment of advanced human OV90 subcutaneous xenograft ovarian tumor models :

[0479] The antitumor effect of toxin-conjugated IMAB027 was tested in mice with advanced human xenograft tumors expressing homologous CLDN6. Treatment began on day 10 post-tumor cell transplantation. Following a single rapid intravenous bolus, IMAB027-DM1 and IMAB027-vcMMAE significantly inhibited tumor growth in xenografts of OV90 ovarian cancer cells expressing homologous CLDN6. Figure 18 and 19 A). More importantly, a single application of 16 mg / kg IMAB027-vcMMAE resulted in complete tumor regression in 60% of treated mice. Figure 19 B).

[0480] Treatment of advanced human PA-1 subcutaneous xenograft ovarian tumor model :

[0481] The antitumor effect of toxin-conjugated IMAB027 was also tested in a late-stage xenograft tumor model with xenogeneic CLDN6 expression. Following subcutaneous transplantation, PA-1 xenograft tumors lost CLDN6 expression after a certain period of time. Figure 20 C). Treatment began on day 15. At that time, the PA-1 xenograft tumors began to lose CLDN6 expression. Animals received IMAB027-vcMMAE via a single rapid intravenous bolus of 4, 8, or 16 mg / kg. Control animals received unconjugated IMAB027 or load control buffer instead.

[0482] Treatment with IMAB027-vcMMAE significantly inhibited tumor growth in PA-1 xenografts and prolonged survival in tumor-bearing mice, while IMAB027 or IMAB027-DM1 (data not shown) did not affect PA-1 tumor growth. Figure 20 (A and B).

[0483] The reduced level of CLDN6-positive tumor cells in the tumor mass may explain the weak antitumor activity of IMAB027 and IMAB027-DM1 in this in vivo tumor model. IMAB027-DM1 conjugates to the membrane-impermeable toxin DM1 via an incleavable linker. Conversely, IMAB027-vcMMAE conjugates to the cell membrane-permeable toxin MMAE via a cathepsin-cleavable linker. Following cell treatment, the release of the membrane-permeable form of MMAE favors the killing 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.

[0484] In summary, IMAB027-vcMMAE effectively kills human xenograft tumors with heterologous CLDN6 expression by targeting cell activation and killing by bystander cells.

[0485] Treatment of advanced human MKN74 subcutaneous gastric xenograft tumor model :

[0486] 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 the gastric cancer cell line MKN74 was CLDN6 positive in vitro. Interestingly, in athymic nude mice, a large number of tumor cells showed CLDN6 expression after subcutaneous transplantation. Figure 21C). Treatment of established MKN74 xenograft tumors with 16 mg / kg IMAB027-vcMMAE resulted in highly significant inhibition of tumor growth and prolonged survival. Figure 21 (A and B).

[0487] The tumor growth inhibition observed with IMAB027-vcMMAE may be caused by the target cell activation and killing by bystander cells.

[0488] Treatment of advanced human PA-1 intraperitoneal xenograft tumor models of ovarian tumors :

[0489] In addition to subcutaneous (sc) injection therapy for xenograft tumors, the antitumor activity of toxin-conjugated IMAB027 antibodies was also tested in vivo using PA-1 cells ectopically expressing luciferase in xenograft tumor models via intraperitoneal (ip) injection. Figure 22 On day 14 after tumor cell transplantation, mice were given a single rapid intravenous injection of 16 mg / kg IMAB027-DM1 or 16 mg / kg IMAB027-vcMMAE.

[0490] In vivo bioluminescence intensity measurements indicated that peritoneal PA-1 metastases were inhibited after treatment with IMAB027-DM1. Furthermore, IMAB027-vcMMAE showed significantly higher antitumor efficacy than IMAB027-DM1 or its carrier, with complete regression of peritoneal tumors in 100% of animals. Figure 22 ).

[0491] In summary, IMAB027-vcMMAE and IMAB027-DM1 were highly effective and showed no toxic side effects within the concentration range tested in vivo. 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 prolonged survival in animals with subcutaneous or peritoneal human xenograft tumors expressing homologous or even heterologous CLDN6. Most impressively, a large proportion of tumor-bearing animals were cured after treatment with the MMAE-conjugated antibody. The excellent antitumor activity of IMAB027-vcMMAE (particularly observed in animals with tumors expressing heterologous CLDN6) demonstrates that the IMAB027-vcMMAE conjugate is suitable for treating tumors with a low percentage of CLDN6 positivity.

[0492] Example 9.3: The endocytosis of CLDN6-specific antibodies depends on their affinity and CLDN6 binding epitopes.

[0493] The cytotoxic potency of toxin-conjugated antibodies depends heavily on their potential to mediate internalization. Therefore, the development of antibodies with high endocytic rates is a crucial factor in the development of toxin-conjugated antibodies.

[0494] The efficiency of endocytosis was tested in vitro by incubating endogenously CLDN6-expressing human cancer cells with a CLDN6-reactive monoclonal chimeric antibody and an anti-human Fab fragment conjugated with the toxin saponin. Internalization of the CLDN6-bound antibody / Fab-saponin complex led to specific cell killing, which could be measured using a cell viability assay. Screening of different CLDN6-reactive antibodies demonstrated that endocytosis depended not only on antibody binding affinity but also on the antigenic epitope. We observed that binding of CLDN6-specific antibodies to the epitope in the first extracellular loop of CLDN6 supported endocytosis in OV-90 and PA-1 human cancer cells. Notably, in this assay, the CLDN6-reactive antibody 5F2D2, with similar or higher affinity but binding to a different epitope, showed lower cytotoxic potential. Figure 23 ).

[0495] Example 10: Materials and methods used in Examples 7 to 9 above

[0496] Cell culture :

[0497] COV362 (Luc) and PA-1 (Luc) cells, which stably express fluorescent reporter genes, were generated by stably transfecting the cell lines COV362 (ECACC, 07071910) and PA-1 (ATCC, CRL-1572) with firefly luciferase.

[0498] NEC14 (JCRB, 0162) and MKN74 (JCRB, 0255) cells were cultured in RPMI 1640 medium (Gibco, 61870-010) supplemented with 10% heat-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% heat-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% heat-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% heat-inactivated FCS. Cells were cultured at 37°C and 5% CO2.

[0499] CLDN6 expression was determined by flow cytometry.

[0500] Cells were harvested 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 incubated with 2 × 10⁻⁶ ppm solution. 6 Cells were resuspended in FACS buffer at a concentration of 100 μl cells / ml. 100 μl of cell suspension was incubated at 4°C for 30 min 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 min 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 FACS Array (BD Biosciences) and FlowJo software (Tree Star Inc.). Dead cells were excluded from the analysis using the live / dead cell dye propidium iodide (Sigma, P4864).

[0501] COV318 cells were treated with platinum derivatives. :

[0502] COV318 cells were cultured at 1.2 × 10⁶ cells per 100 mm 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 culture medium was changed and the treated cells were grown under standard growth conditions. After 3 to 6 days, CLDN6 expression in the cells was analyzed by flow cytometry.

[0503] Antibody-dependent cytotoxicity (ADCC) after treatment with cell growth inhibitors:

[0504] The effects of carboplatin and paclitaxel on IMAB027-mediated ADCC were determined using the stable human ovarian cancer cell line COV362, which is stably transfected with luciferase, a reporter molecule. COV362 (Luc) cells were cultured at 3 × 10⁶ cells per 150 mm cell culture dish. 6 Cells 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 culture medium was changed and the treated cells were allowed to grow again under standard conditions for 3 days (for carboplatin and gemcitabine) or 10 days (for paclitaxel, doxorubicin, or topotecan).

[0505] Cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and the concentration was adjusted to 2 × 10⁻⁶ in DMEM containing 2 mM glutamine (Gibco, 25030-081) and 20 mM HEPES (Gibco, 15630-056). 5 Cells / ml. Place 1×10⁻⁶ cells / ml in each well. 4 One cell was seeded into a white 96-well PP plate and incubated at 37°C and 5% CO2 for 5 hours.

[0506] PBMCs (peripheral blood mononuclear cells) were isolated from human donor blood samples using density gradient centrifugation with Ficoll Hypaque (GE Healthcare, 17144003). Interphase PBMCs were isolated and washed three times with PBS containing 2 mM EDTA. PBMCs were then centrifuged at 1.6 × 10⁻⁶. 7 Cells / mL concentrations were resuspended in X-Vivo 15 medium (Lonza, BE04-418Q) and stored at 37°C and 5% CO2 until assay.

[0507] 25 μl of IMAB027 and isotype control antibody were added to the cells at the specified concentration. Subsequently, 25 μl of PBMC suspension was added, and the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0508] After adding 10 μl of 8% Triton X-100 (Sigma, T8787) in PBS to the fully lysed control and 10 μl of PBS to the maximum viable cell control, 50 μl of a luciferin mixture (3.84 mg / ml D-luciferin (Sigma Aldrich, 50227) and 160 mM HEPES in ddH2O) was added to the samples, and the cells were incubated in the dark at room temperature for 90 min. Bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are expressed as integrated digital relative light units.

[0509] The specific cleavage is calculated as follows:

[0510]

[0511] Maximum viable cells: 10 μl PBS, antibody-free

[0512] Total lysis: 10 μL of 8% Triton X-100 in PBS, without antibody.

[0513] Intraperitoneal transplantation of COV318 cells in athymic nude mice:

[0514] The in vivo tumorigenicity of the human ovarian cancer cell line COV318 and the accumulation of CLDN6-positive cells were tested in mice. Therefore, 2 × 10⁻⁶ cells resuspended in PBS were... 7 One COV318 cell was injected intraperitoneally into 6-8 week old female Hsd: athymic naked-Foxn1 nuIn mice, mice were monitored daily. Animals were euthanized once life-threatening symptoms became apparent. Tumor and ascites cells were isolated and cultured for further analysis. Tumors were mechanically dissociated, screened, and washed with DMEM medium (Gibco, 41965-039). To obtain a single-cell suspension, tumor cells were treated with acutase (Life Technologies, A11105-01) at 37°C for 30 min, passed through a 40 μm cell permeabilizer, and washed with DMEM medium. Ascites was collected, and contaminated red blood cells were removed using ACK (ammonium chloride-potassium) lysis buffer (Invitrogen, A10492-01). Tumor and ascites cells were grown under standard conditions using standard COV318 medium supplemented with penicillin / streptomycin (Gibco, 15140). Cells were screened for CLDN6 expression by flow cytometry.

[0515] Treatment of very advanced human NEC14 xenograft tumors:

[0516] For transplantation, to 6-8 week old female Hsd: athymic naked-Foxn1 nu Mice were subcutaneously inoculated in 200 μl PBS at a dose of 2 × 10⁻⁶ mcg. 7 NEC14 cells. In a very late-stage treatment study, the tumor grew to a maximum size of 170 mm in 13 days. 3 Before treatment, the mice were divided into a control group, an IMAB027 group, a PEB (cisplatin, etoposide, bleomycin) group, and an IMAB027 / PEB group (n=14 per group).

[0517] Thirteen days post-transplantation, the following medications were administered: cisplatin 1 mg / kg was rapidly administered intraperitoneally (IP) on days 13, 14, 15, 16, and 17; etoposide 5 mg / kg was rapidly administered intraperitoneally (IP) on days 13, 14, 15, 16, and 17; and bleomycin 10 mg / kg was rapidly administered intraperitoneally (IP) on days 13, 17, and 21. IMAB027 was administered three times weekly via rapid intravenous (IV / IP / IP) administration of 35 mg / kg from day 13 to day 101. As a load control, mice received either drug buffer as an alternative to the antibody or 0.9% NaCl solution as an alternative to PEB.

[0518] Tumor burden and animal health were monitored twice weekly. When the tumor reached a volume of 1400 mm²... 3 Mice were euthanized when the tumors became ulcerative. The Kruskal-Wallis test and post-hoc Dunn multiple comparison test were used to analyze tumor growth inhibition.

[0519] Sphere formation determination :

[0520] For the spheroid formation assay, COV318 cells were stained with 0.5 μg / ml IMAB027 targeting CLDN6 at 4°C for 30 minutes, then incubated with goat anti-human IgG secondary antibody (1:300) at 4°C for 10 minutes. Cells were then sorted for CLDN6 expression using a BD FACSAria cell sorter. Then, 1×10⁻⁶ cells were... 6 CLDN6-positive or negative sorted cells were seeded into the wells of a 6-well ultra-low adhesion plate (Corning) containing 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 spheroids under these stem cell-specific conditions for 21 days. The medium was changed every other day without disturbing the spheroids, and representative images were taken periodically.

[0521] To produce second-generation spheroids, first-generation spheroids of CLDN6-positive COV318 cells (day 22 post-inoculation) were dissociated into single cells and then re-coated into the wells of 6-well ultra-low adhesion plates. Furthermore, the culture medium was changed every other day without disrupting the formed spheroids, and representative images were taken periodically.

[0522] Using BioMark TM Quantitative real-time RT-PCR analysis using the HD system (Fluidigm):

[0523] Using BioMark TM The HD system (Fluidigm) uses qRT-PCR to target selected ovarian cancer stem cell-specific factors (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1A1, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch1, Pou5F1, CD133, Snail, Sox2). Gene expression assays (Life Technologies) analyzed ovarian cancer samples from 42 individuals. RNA was isolated from ovarian cancer samples using the RNeasy Mini kit (Qiagen) according to the respective manufacturers' instructions, and cDNA was synthesized using the PrimeScript RTReagent kit (Takara Bio Inc.). Advanced Development Protocol 28 - Rapid Gene Expression Analysis GE Assays rev A2 - Sample preparation and analysis. Samples were loaded onto a 96.96 Gene Expression Dynamic Array (IFC) via IFC Controller HX. Analysis was performed using Fluidigm BioMark. TM HD system analysis chip array. TaqMan PreAmp MasterMix was purchased from Applied Biosystems. The dataset was evaluated using the ΔΔCt method. Correlation analysis of CLDN6 with selected ovarian cancer stem cell markers was performed using Spearman's r. The significance of the correlation values ​​was assessed by testing the correlation coefficients. P-values ​​for multiple tests were adjusted using the Benjamini and Hochberg methods, and a adjusted p-value ≤ 0.05 was considered significant.

[0524] Toxin-conjugated CLDN6 antibody

[0525] Toxin conjugation of monoclonal antibodies was performed at Piramal Healthcare (Grangemouth, UK).

[0526] For DM1 conjugation, naked antibodies were modified with SMCC (6 × molarity) reacting with the free NH2 residues of lysine groups by incubation in PBS (pH 7.2) at room temperature for 1 hour. The modified antibody was then dialyzed into 35 mM citrate buffer (pH 5.0) and the linker-to-antibody ratio was determined using a reversed Ellman assay. DM1 (6 × molarity) conjugated to the maleimide moiety of the SMCC linker via its thiol group after incubation at room temperature for 17 hours. The conjugated antibody was dialyzed into formulation buffer (20 mM His, 85 mg / ml sucrose, pH 5.8) and stored at -80°C. The drug-to-antibody ratio was analyzed by UV spectroscopy, monomer content by SEC-HPLC, and free drug content by RP-HPLC.

[0527] For the MMAE conjugate, the naked antibody was dialyzed into PBS (pH 7.2) and modified by thiolation of the free NH2 group of the lysine residue with Traut's reagent (2-iminotetrahydrothiophene) (20 × 10⁻⁶ mcg) for 2 hours at room temperature. Subsequently, the thiolated antibody was dialyzed into 35 mM citrate buffer (pH 5.5) and the linker-to-antibody ratio was determined using a reverse-phase Elman assay. By incubation at room temperature for 15 hours, vcMMAE (6 × 10⁻⁶ mcg) cleaved the valine residue of the linker to the thiol group of the thiolated antibody using cathepsin. The conjugated antibody was dialyzed into formulation buffer (20 mM His, 85 mg / ml sucrose, pH 5.8) and stored at -80°C. The drug-to-antibody ratio was analyzed by UV spectroscopy, the monomer content was analyzed by SEC-HPLC, and the free drug content was analyzed by RP-HPLC.

[0528] Relative binding affinity was determined by flow cytometry. :

[0529] Cells were harvested 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 incubated with 2 × 10⁻⁶ ppm solution. 6 Cells were resuspended in FACS buffer at a concentration of 100 cells / ml. IMAB027, IMAB027-DM1, or IMAB027-vcMMAE (titration series from 0.1 ng / ml to 20 μg / ml) were incubated with 100 μl of cell suspension at 4°C for 30 min. 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 at 4°C for 30 min. Subsequently, the cells were 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.).

[0530] Viability assay using toxin-conjugated IMAB027 :

[0531] The effects of IMAB027-DM1 and IMAB027-vcMMAE on the viability of human tumor cell lines were determined in vitro using a colorimetric assay (Cell Proliferation Kit XTT from AppliChem) to detect cellular metabolic activity.

[0532] OV90 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054), and 2500 cells were seeded into 50 μl of growth medium in 96-well plates. After 24 hours, DM1-conjugated IMAB027 and MMAE-conjugated IMAB027 or allotype control antibodies were added to 50 μl of medium at various concentrations. Cells were cultured for 3 to 7 days until untreated cells reached approximately 80% confluence. Cell viability was analyzed using the AppliChem Cell Proliferation Kit II (AppliChem, A8088-1000) according to the manufacturer's instructions. After incubation with XTT reagent for 3 to 5 hours, 100 μl of cell supernatant was transferred to a new 96-well plate, and absorbance was measured at 480 nm (reference 630 nm) using a spectrophotometer (Tecan). Viability was calculated using the following equation:

[0533]

[0534] Blank: Cell-free culture medium and XTT

[0535] Maximum viable cells: cells, culture medium and XTT

[0536] The EC50 value was determined using nonlinear regression with GraphPad Prism 6 software.

[0537] Treatment of advanced subcutaneous OV90 xenograft tumors :

[0538] Human ovarian cancer cell line OV90 was cultured under standard conditions. For transplantation, the cells were introduced into 6-8 week old female Hsd: thymic naked-Foxn1 cells. nu Mice were subcutaneously inoculated in 200 μl PBS with 1 × 10⁻⁶ saturates. 7 One OV90 cell. In late-stage treatment studies, prior to treatment, tumors were allowed to grow for 10 days and had established a 50mm diameter. 3 Up to 150mm 3 Mice with tumor volume were randomly assigned to either the loading group or the antibody group (n=10). Tumor volume (TV = (length × width)) was monitored every two weeks. 2 ) / 2). TV is represented as mm 3 This allows the tumor to develop a growth curve over time.

[0539] In the initial dose range determination study, animals were administered a single intravenous injection of the loading agent, IMAB027-DM1 (1.78 mg / kg, 5.33 mg / kg, or 16 mg / kg), and necropsy was performed on day 35 post-transplantation. In the second dose range determination study, animals were administered a single intravenous injection of the loading agent, 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 weekly via rapid bolus injection of 35 mg / kg via alternating intravenous / intravenous / intravenous administration. When the tumor reached a size greater than 1400 mm... 3 Mice were euthanized when tumor volume increased or ulceration occurred. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and post-hoc Dunn multiple comparison test. Survival was analyzed using the Mantel-Cox test.

[0540] Treatment of advanced subcutaneous PA-1 xenograft tumors and immunohistochemistry of tumor sections :

[0541] Human ovarian cancer cell line PA-1 was cultured under standard conditions. For transplantation, the cells were introduced into 6-8 week old female Hsd: thymic naked-Foxn1 cells. nu Mice were subcutaneously inoculated in 200 μl PBS with 1 × 10⁻⁶ saturates. 7 PA-1 cells. In the advanced treatment study, prior to treatment, the tumor was allowed to grow for 15 days and had established a 40mm diameter. 3 Up to 120mm 3 Mice with tumor volume were randomly assigned to either the loading group or the antibody group (n=10). Tumor volume (TV = (length × width)) was monitored every two weeks. 2 ) / 2). TV is represented as mm 3 This allows the tumor to develop a growth curve over time.

[0542] Animals were administered a single intravenous injection of the loading agent, 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 then administered three weekly via rapid bolus injections of 35 mg / kg via alternating intravenous / intra- ... 3 Mice were euthanized when tumor volume increased or ulceration occurred. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and post-hoc Dunn multiple comparison test. Survival was analyzed using the Mantel-Cox test.

[0543] To analyze CLDN6 expression during tumor establishment and progression, PA-1 tumors from untreated mice were dissected on days 7, 14, and 56, fixed in formalin, and embedded in paraffin. 4 μm tissue sections were prepared from each FFPE (formalin-fixed paraffin-embedded) block, mounted on adhesive slides (SuperFrost Ultra Plus, Thermo Fisher Scientific), and baked at 60°C for 60 min. The FFPE tissue sections were deparaffinized before staining. The sections were boiled at 120°C in 10M citric acid (pH 6.0) supplemented with 0.05% Tween-20 for 10 min. Endogenous peroxidase was quenched by incubation in 0.3% H2O2 in PBS for 15 min. After washing with PBS, the nonspecific antibody binding sites were blocked for 30 minutes at room temperature with blocking buffer (10% goat serum in PBS), followed by overnight incubation with a first rabbit anti-dimin-6 antibody (IBL-America, 18865) diluted in blocking buffer at 0.2 μg / ml. The samples were then washed three times with PBS and incubated for 30 minutes at room temperature with their respective second ready-to-use antibodies (Power Vision HRP goat anti-rabbit; Immunologic). Developing was performed using a chromogenic substrate solution (VectorRed; Vector Laboratories) for 4:30 minutes. After counterstaining with hematoxylin, dehydration, and mounting, the sections were analyzed using a Leica DM2000 microscope.

[0544] Treatment of advanced subcutaneous MKN74 xenograft tumors and immunohistochemistry of tumor sections :

[0545] Human gastric cancer cell line MKN74 was cultured under standard conditions. For transplantation, 6-8 week old female Hsd: thymic naked-Foxn1 cells were cultured. nu Mice were subcutaneously inoculated in 200 μl PBS with 1 × 10⁻⁶ saturates. 7 MKN74 cells. In the advanced treatment study, tumors were allowed to grow for 7 days prior to treatment and had reached a size of 200±30 mm. 3 Mice with tumor volume were randomly assigned to either the loading group or the antibody group (n=10). Tumor volume (TV = (length × width)) was monitored every two weeks. 2 ) / 2). TV is represented as mm 3 This allows the tumor to develop a growth curve over time.

[0546] Animals received either the load-controlled buffer or 16 mg / kg IMAB027-vcMMAE via a single intravenous rapid bolus on day 8. Tumors larger than 1400 mm were treated. 3Mice were euthanized when tumor volume increased or ulcers formed. The Kruskal-Wallis test and post-hoc Dunn multiple comparison test were used to analyze tumor growth inhibition. The Mann-Whithney test and post-hoc Dunn multiple comparison test were used to analyze tumor growth inhibition. The Mantel-Cox test was used to analyze survival.

[0547] CLDN6-targeted expression in MKN74 cells was analyzed by flow cytometry before transplantation and by histochemistry on dissected untreated MKN74 xenografts on day 31.

[0548] For immunohistochemistry, 3 μm thick tissue sections were prepared, mounted on glass slides, and air-dried at room temperature for 90 minutes. All tissue sections were fixed in acetone for 10 minutes at -20°C 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 for 30 minutes at room temperature with blocking buffer (10% goat serum in PBS), followed by incubation with 5 μg / ml IMAB027-FITC for 60 minutes at room temperature. The samples were then washed three times with PBS and incubated with their respective second ready-to-use antibodies (Bright Vision polyHRP anti-rabbit IgG, Immunologic, DPVR-110HRP) for 30 minutes at room temperature. Development was performed using a chromogenic substrate solution (VectorRed; Vector Laboratories) for 2:30 minutes. After counterstaining with hematoxylin, dehydration, and mounting, the sections were analyzed using a Leica DM2000 microscope.

[0549] Treatment of advanced intraperitoneal PA-1 (Luc) xenograft tumors :

[0550] The human ovarian teratogenic cancer cell line PA-1(Luc), which stably expresses firefly luciferase as a luminescent reporter gene, was used as an intraperitoneal xenograft tumor model to investigate the antitumor activity of the toxin-conjugated IMAB027 antibody in vivo. Previous transplantation experiments have shown that intraperitoneal inoculation of PA-1(Luc) cells generates intraperitoneal tumor nodules.

[0551] 1×10⁻⁶ resuspended in PBS 7 PA-1 (Luc) cells were injected intraperitoneally into female Hsd: athymic naked-Foxn1 nuBioluminescence imaging began on day 14 post-tumor cell inoculation and continued weekly until the end of the study. D-luciferin (PerkinElmer, 122796) was dissolved in sterile water and injected intraperitoneally 5 minutes before imaging with the IVIS Lumina imaging system (Advanced Molecular Vision) (150 mg / kg, injection volume 200 μl). Mice were anesthetized with isoflurane and placed in the dark chamber of the IVIS Lumina system, and the emitted photons were quantified over a 1-minute integration time. The intensity of transmitted light from PA-1 cells expressing luciferase within the animals was represented as a pseudocolor image, where blue represents the lowest intensity bioluminescent signal and red represents the highest intensity. Grayscale images of mice were obtained under low LED illumination. These images were overlaid using Living Image software (Xenogen). Comparable 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 per second (p / s). Background bioluminescence values ​​obtained from non-signal emission regions on the animal were subtracted from the individual bioluminescence values ​​of each animal.

[0552] On day 14, mice were randomly assigned to either IMAB027-DM1 or IMAB027-vcMMAE administered intraperitoneally. Control animals received a carrier buffer. Tumor growth was monitored weekly by subsequent analysis of total flux (photons / second) from bioluminescent imaging from a ventral view and covering the region of interest in the mouse abdomen.

[0553] Endocytosis :

[0554] Cytotoxicity-based endocytosis assays were used to determine the endocytosis of the CLDN6-binding antibody. These assays relied on the co-internalization of the target-binding antibody and the saponin-conjugated anti-human IgG Fab fragment (Fab-ZAP human, Advanced Targeting Systems, IT-51). Saponins are ribosome-inactivating proteins that, upon internalization, inhibit protein biosynthesis, thus leading to cell death.

[0555] PA-1 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and 2.5 × 10⁻⁶ cells were added. 3Cells were seeded per well in 50 μl of growth medium in 96-well plates. After 24 hours, 25 μl of Fab-ZAP was added to each well, followed by IMAB027 or an allotype control antibody. CLDN6 antibody was administered in 6- or 8-step dilution series, simultaneously with a constant concentration of Fab-ZAP (FAB-ZAP:antibody ratio of 3:1 to 6561:1). Cells were then cultured for another 72 hours in a humidified CO2 incubator at 37°C. Cell viability was then analyzed using the AppliChem Cell Proliferation Kit II (AppliChem, A8088-1000) according to the manufacturer's instructions. Absorbance was measured at 480 nm (reference 630 nm) using a spectrophotometer (Tecan). sequence list <110> BioNTech AG et al. <120> Diagnosis and treatment of cancers involving cancer stem cells <130> 342-79 PCT <150> PCT / EP2013 / 002272 <151> 2013-07-31 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 2 <211> 220 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Val Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 3 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 3 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 4 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 4 Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu His 20 25 30 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Val Tyr Ser 35 40 45 Thr Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Gly Gly Ser Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 117 <212> PRT <213]] Artificial sequence <220> <223> Antibody fragment <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 6 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 6 Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His 20 25 30 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr Ser 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 117 <212> PRT <213> Artificial sequence <220> <223> antibody fragments <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ile Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Arg Asp Phe Gly Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 8 <211> 106 <212> PRT <213> Artificial sequence <220> <223> antibody fragments <400> 8 Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His 20 25 30 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr Ser 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 9 Glu Val Gln Leu Gln Gln Ser Arg Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Leu Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 10 <211> 106 <212> PRT <213> artificial sequence <220> <223> antibody fragment <400> 10 Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met His 20 25 30 Trp Phe Gln Leu Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr Ser 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Asn Asn Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 11 Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His 20 25 30 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Gly Ile Tyr Ser 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Antibody fragment <400> 12 Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His 20 25 30 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr Ser 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. Use of an antibody capable of binding to CLDN6 or one or more nucleic acids encoding said antibody in a method for preparing a medicament for treating cancer, said cancer being characterized by cancer cells expressing CLDN6. The method includes inhibiting and / or eliminating cancer stem cells by administering the antibody capable of binding CLDN6 or one or more nucleic acids encoding the antibody to a cancer patient. The method also includes the administration of chemotherapy. in (i) The antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (ii) The antibody comprises (a) A VH comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 5, wherein the amino acid sequence of the VH has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and (b) A VL comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 4, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or A VL comprising the following amino acid sequence, wherein the amino acid sequence includes the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 12, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; or (iii) The antibody comprises (a) complementation-determining regions CDR1, CDR2, and CDR3 of VH containing the amino acid sequence shown in SEQ ID NO: 5, and (b) complementation-determining regions CDR1, CDR2, and CDR3 of VL containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (iv) The antibody is a monoclonal antibody, chimeric antibody, or humanized antibody of (i), (ii), or (iii). The chemotherapy described therein increases CLDN6 expression in cancer cells, and the chemotherapy includes... (a) Chemotherapy agents selected from carboplatin and its salts, gemcitabine and its salts, paclitaxel and its salts, doxorubicin and its salts, topotecan and its salts, cisplatin and its salts, or (b) Multichemotherapy for PEB containing cisplatin or a salt thereof, etoposide or a salt thereof, and bleomycin or a salt thereof.

2. The use according to claim 1, wherein the cancer stem cells express CLDN6.

3. The use according to claim 1, further comprising administering radiotherapy.

4. The use according to claim 1 or 3, wherein the inhibition and / or elimination of the anticancer effects of cancer stem cell-enhanced chemotherapy and / or radiotherapy.

5. The use according to claim 4, wherein the enhanced anticancer effect of said chemotherapy and / or radiotherapy includes an extension of lifespan in cancer patients undergoing chemotherapy and / or radiotherapy.

6. Use of an antibody capable of binding to CLDN6 or one or more nucleic acids encoding said antibody in a method for preparing a medicament for treating cancer, said cancer being characterized in that cancer cells express CLDN6. The method comprises administering to a cancer patient (i) the antibody capable of binding CLDN6 or one or more nucleic acids encoding the antibody and (ii) chemotherapy, wherein (i) The antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (ii) The antibody comprises (a) A VH comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 5, wherein the amino acid sequence of the VH has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and (b) A VL comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 4, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or A VL comprising the following amino acid sequence, wherein the amino acid sequence includes the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 12, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; or (iii) The antibody comprises (a) complementation-determining regions CDR1, CDR2, and CDR3 of VH containing the amino acid sequence shown in SEQ ID NO: 5, and (b) complementation-determining regions CDR1, CDR2, and CDR3 of VL containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (iv) The antibody is a monoclonal antibody, chimeric antibody, or humanized antibody of (i), (ii), or (iii). The chemotherapy described therein increases CLDN6 expression in cancer cells, and the chemotherapy includes... (a) Chemotherapy agents selected from carboplatin and its salts, gemcitabine and its salts, paclitaxel and its salts, doxorubicin and its salts, topotecan and its salts, cisplatin and its salts, or (b) Multichemotherapy for PEB containing cisplatin or a salt thereof, etoposide or a salt thereof, and bleomycin or a salt thereof.

7. The use according to claim 6, wherein the cancer involves cancer stem cells expressing CLDN6.

8. The use according to claim 6, wherein administration of an antibody capable of binding CLDN6 or one or more nucleic acids encoding said antibody results in the inhibition or elimination of cancer stem cells expressing CLDN6.

9. The use according to claim 6, wherein the administration of an antibody capable of binding CLDN6 or one or more nucleic acids encoding said antibody enhances the anticancer effect of chemotherapy.

10. The use according to claim 9, wherein the enhanced anticancer effect of the chemotherapy includes an extension of the lifespan of the cancer patient undergoing chemotherapy.

11. The use according to any one of claims 1, 2 and 6 to 10, wherein the elimination of cancer stem cells leads to a cure of cancer.

12. The use according to any one of claims 1, 2 and 6 to 10, wherein the antibody capable of binding CLDN6 or one or more nucleic acids encoding the antibody and the chemotherapy are administered in a synergistically effective amount.

13. The use according to any one of claims 1, 2 and 6 to 10, wherein the chemotherapy is administered at a dose below the maximum tolerated dose.

14. The use according to any one of claims 1, 2 and 6 to 10, wherein the cancer stem cells are located at the tumor site of the cancer patient.

15. The use according to any one of claims 1, 2 and 6 to 10, wherein the cancer is resistant to chemotherapy.

16. The use according to any one of claims 1, 2 and 6 to 10, wherein the antibody capable of binding CLDN6 has an inhibitory and / or cytotoxic effect on cancer stem cells.

17. The use according to claim 16, wherein the antibody capable of binding CLDN6 exerts its inhibitory and / or cytotoxic effects on cancer stem cells by mediating one or more of the following: complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation.

18. The use according to any one of claims 1, 2 and 6 to 10, wherein the applied antibody capable of binding CLDN6 is coupled to the therapeutic portion.

19. The use according to claim 18, wherein the therapeutic component is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide.

20. The use according to claim 18, wherein the therapeutic portion acts on slowly growing cells.

21. The use according to any one of claims 1, 2 and 6 to 10, wherein the antibody capable of binding CLDN6 binds to the first extracellular loop of CLDN6.

22. Use of antibody-drug conjugates (ADCs) in the preparation of medicaments for treating cancer, wherein the cancer is characterized by cancer cells expressing CLDN6. The method described therein includes administering the ADC to a cancer patient. The ADC comprises an antibody capable of binding CLDN6, covalently linked to at least one drug moiety via a linker. The drug component is or contains N 2' -Deacetylated-N 2' -(3-mercapto-1-oxopropyl)-matansine (DM1) or monomethyl guanetine E (MMAE), wherein (i) The antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (ii) The antibody comprises (a) A VH comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 5, wherein the amino acid sequence of the VH has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and (b) A VL comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 4, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or A VL comprising the following amino acid sequence, wherein the amino acid sequence includes the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 12, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; or (iii) The antibody comprises (a) complementation-determining regions CDR1, CDR2, and CDR3 of VH containing the amino acid sequence shown in SEQ ID NO: 5, and (b) complementation-determining regions CDR1, CDR2, and CDR3 of VL containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (iv) The antibody is a monoclonal antibody, chimeric antibody or humanized antibody of the antibody described in (i), (ii) or (iii).

23. The use according to claim 22, wherein the drug portion is permeable to the cell membrane.

24. The use according to claim 22 or 23, wherein the connector is a cuttable connector.

25. The use according to claim 22 or 23, wherein the adapter is a cathepsin-cleavable adapter.

26. The use according to claim 22 or 23, wherein the antibody is connected to the linker via a cysteine ​​thiol group of the antibody.

27. The use according to claim 22 or 23, wherein the cancer involves cancer stem cells expressing CLDN6.

28. The use according to claim 22 or 23, wherein administration of the antibody-drug conjugate results in the inhibition or elimination of cancer stem cells expressing CLDN6.

29. The use according to claim 22, further comprising administering chemotherapy and / or radiation therapy.

30. The use according to claim 22 or 29, wherein the administration of the antibody-drug conjugate enhances the anticancer effect of chemotherapy and / or radiotherapy.

31. The use according to claim 22 or 23, wherein the antibody capable of binding CLDN6 in the antibody-drug conjugate binds to the first extracellular loop of the CLDN6.

32. The use according to any one of claims 1, 2, 6 to 10, 22 and 23, wherein CLDN6 has an amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:

2.

33. The use according to any one of claims 1, 2, 6 to 10, 22 and 23, wherein the cancer includes primary cancer, advanced cancer, metastatic cancer, recurrent cancer, or a combination thereof.

34. Use of cancer therapeutic agents targeting cancer stem cells in the preparation of a medicament for treating cancer, said cancer being characterized by cancer cells expressing CLDN6, said method comprising: (i) Identifying cancer stem cells in cancer patients, including detecting cells expressing CLDN6, wherein the detection of said CLDN6-expressing cells or determination of their quantity is performed using an antibody capable of binding to CLDN6, and (ii) administering cancer treatment targeting cancer stem cells to the cancer patient, wherein the cancer treatment targeting cancer stem cells comprises performing a method for treating cancer according to any one of claims 1 to 33. The cancer therapeutic agent targeting cancer stem cells described herein comprises an antibody as defined in claim 1 capable of binding CLDN6 or one or more nucleic acids encoding said antibody. 35.(i) The antibody capable of binding CLDN6 as defined in claim 1, or (ii) One or more nucleic acids encoding an antibody capable of binding CLDN6 as defined in claim 1 Use in the preparation of medicaments for methods of preventing cancer chemoresistance, cancer recurrence, or cancer metastasis. The method includes treating cancer by administering the antibody capable of binding CLDN6 or one or more nucleic acids encoding the antibody to a cancer patient, wherein the cancer is characterized by cancer cells expressing CLDN6. The method also includes the administration of chemotherapy. The chemotherapy described therein increases CLDN6 expression in cancer cells, and the chemotherapy includes... (a) Chemotherapy agents selected from carboplatin and its salts, gemcitabine and its salts, paclitaxel and its salts, doxorubicin and its salts, topotecan and its salts, cisplatin and its salts, or (b) Multichemotherapy for PEB containing cisplatin or a salt thereof, etoposide or a salt thereof, and bleomycin or a salt thereof.

36. Use of a pharmaceutical product in the preparation of a medicament for treating cancer, said cancer being characterized by cancer cells expressing CLDN6, wherein said pharmaceutical product comprises (a) An antibody capable of binding to CLDN6 or one or more nucleic acids encoding said antibody, and (b) Chemotherapy agents, in: (i) The antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (ii) The antibody comprises (a) A VH comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 5, wherein the amino acid sequence of the VH has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and (b) A VL comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 4, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or A VL comprising the following amino acid sequence, wherein the amino acid sequence includes the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 12, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; or (iii) The antibody comprises (a) complementation-determining regions CDR1, CDR2, and CDR3 of VH containing the amino acid sequence shown in SEQ ID NO: 5, and (b) complementation-determining regions CDR1, CDR2, and CDR3 of VL containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (iv) The antibody is a monoclonal antibody, chimeric antibody, or humanized antibody of (i), (ii), or (iii). The chemotherapeutic agent increases CLDN6 expression in cancer cells, and the chemotherapeutic agent contains... (1) Medications selected from carboplatin and its salts, gemcitabine and its salts, paclitaxel and its salts, doxorubicin and its salts, topotecan and its salts, cisplatin and its salts, or (2) Multichemotherapy for PEB containing cisplatin or its salts, etoposide or its salts and bleomycin or its salts.

37. The use according to claim 36, wherein the pharmaceutical product is in the form of a cassette, the cassette comprising a first container containing the antibody capable of binding CLDN6 or one or more nucleic acids encoding the antibody and a second container containing the chemotherapeutic agent.

38. The use according to claim 36 or 37, wherein the pharmaceutical product further includes a printed instruction manual for treating cancer with the product.

39. An antibody-drug conjugate comprising an antibody capable of binding CLDN6, covalently linked to at least one drug moiety via a linker. The drug component is or contains N 2' -Deacetylated-N 2' -(3-mercapto-1-oxopropyl)-matansine (DM1) or monomethyl guanetine E (MMAE), wherein (i) The antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (ii) The antibody comprises (a) A VH comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 5, wherein the amino acid sequence of the VH has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and (b) A VL comprising the following amino acid sequence, wherein the amino acid sequence comprises the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 4, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, or A VL comprising the following amino acid sequence, wherein the amino acid sequence includes the same complementarity-determining regions CDR1, CDR2, and CDR3 as those in SEQ ID NO: 12, wherein the amino acid sequence of the VL has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; or (iii) The antibody comprises (a) complementation-determining regions CDR1, CDR2, and CDR3 of VH containing the amino acid sequence shown in SEQ ID NO: 5, and (b) complementation-determining regions CDR1, CDR2, and CDR3 of VL containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; or (iv) The antibody is a monoclonal antibody, chimeric antibody, or humanized antibody of (i), (ii), or (iii). The antibody has an affinity and / or specificity for CLDN6, enabling endocytosis of the antibody-drug conjugate.

40. The antibody-drug conjugate of claim 39, wherein the drug portion is permeable to a cell membrane.

41. The antibody-drug conjugate according to claim 39 or 40, wherein the connector is a cuttable connector.

42. The antibody-drug conjugate according to claim 39 or 40, wherein the linker is a cathepsin-cleavable linker.

43. The antibody-drug conjugate according to claim 39 or 40, wherein the antibody is linked to the linker via a cysteine ​​thiol group of the antibody.

44. The antibody-drug conjugate according to claim 39 or 40, wherein the antibody in the antibody-drug conjugate capable of binding CLDN6 binds to the first extracellular loop of CLDN6.

45. A pharmaceutical formulation comprising an antibody-drug conjugate according to any one of claims 39 to 44, and a pharmaceutically acceptable diluent, carrier, or excipient.

46. ​​A pharmaceutical product comprising an antibody drug conjugate according to any one of claims 39 to 44, and a chemotherapeutic agent.

47. The pharmaceutical product of claim 46, which is in the form of a medicine box, the medicine box comprising a first container containing the antibody drug conjugate and a second container containing the chemotherapeutic agent.

48. The pharmaceutical product according to claim 46 or 47, further comprising a printed instruction manual for treating cancer with the product.

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