Therapeutic agent for ovarian clear cell carcinoma

Abstract

Provided is a therapeutic agent that is effective for the treatment of ovarian clear cell carcinoma. The therapeutic agent for ovarian clear cell carcinoma contains a proteasome inhibitor as an active ingredient. Moreover, said proteasome inhibitor is a substance that reversibly or irreversibly binds to the 20s β5 subunit of a proteasome and inhibits chymotrypsin-like activity. Furthermore, said proteasome is 26s proteasome. The content ratio of the proteasome inhibitor in the therapeutic agent for ovarian clear cell carcinoma is 80 mass% or greater, 90 mass% or greater, or 100 mass%.

Description

Treatment for ovarian clear cell carcinoma

[0001] The present invention relates to a therapeutic agent for ovarian clear cell carcinoma. This application claims priority to Japanese Patent Application No. 2022-125006, filed August 4, 2022, the contents of which are incorporated herein by reference.

[0002] Ovarian cancer has few subjective symptoms, and because the ovaries are organs within the abdominal cavity, it is extremely difficult to detect, and by the time ovarian cancer is discovered, it is often already in an advanced stage. For this reason, the five-year survival rate for ovarian cancer patients is around 40-50%, and the survival rate for Stage III and Stage IV cancer has not improved in the last 40 years.

[0003] Most ovarian cancers are epithelial, and several histological types are known, including serous, endometrioid, clear cell, mucinous, transitional epithelial, and undifferentiated cancers. Among these, ovarian clear cell carcinoma (OCC) is known to have a poor prognosis and tends to be resistant to treatment due to its unclear etiology.

[0004] The primary treatment for ovarian cancer patients involves surgical removal of the tumor, followed by standard chemotherapy such as TC (taclipaxel-carboplatin) therapy. However, because ovarian clear cell carcinoma is heterogeneous, standard chemotherapy alone cannot completely remove the cancer, resulting in many cases of recurrence. For this reason, establishing second-line or later treatments is important for treating ovarian clear cell carcinoma patients.

[0005] As a second-line treatment, the search for effective drugs is being considered through genomic analysis of patient-derived ovarian cancer tissue. However, it is known that the probability of finding a gene mutation through genomic analysis is approximately 50%, and the probability of finding a drug that is expected to be effective from that is approximately 10%. It is expected that drug efficacy is related not only to gene mutations but also to drug metabolism and other gene expression in the cancer tissue. Therefore, it is thought that the probability of finding a potentially effective drug can be increased by examining the drug sensitivity of the cancer tissue using the patient's ovarian tissue or tissue functionally similar to the patient's ovarian tissue in addition to genomic analysis.

[0006] Patent Document 1 and Non-Patent Document 1 investigate drug sensitivity using the ES-2 cell line, which has traditionally been considered to be an ovarian clear cell carcinoma cell line. However, as shown in Non-Patent Documents 2 and 3, immunohistochemical analysis, qPCR analysis, and its mutation profile have revealed that the ES-2 cell line is not an ovarian clear cell carcinoma tissue, but rather the origin of other ovarian cancers such as serous ovarian cancer tissue.

[0007] US Patent Application Publication No. 2014 / 0364375

[0008] PLoS One. 2020 Jan 15;15(1):e0227727. “Structure-function analyzes of candidate small molecule RPN13 inhibitors with antitumor properties“J Clin Pathol. 2014 Oct;67(10):921-2. “Caution over use of ES2 as a model of ovarian clear cell carcinoma”Nat Commun. 2013;4:2126. “Evaluating cell lines as tumor models by comparison of genomic profiles”

[0009] An object of the present invention is to provide a therapeutic agent that is effective in treating ovarian clear cell carcinoma.

[0010] The present invention includes the following aspects: [1] A therapeutic agent for ovarian clear cell carcinoma, comprising a proteasome inhibitor as an active ingredient.

[0011] [2] The therapeutic agent for ovarian clear cell carcinoma according to [1], wherein the proteasome inhibitor is a substance that binds reversibly or irreversibly to the β5 unit of the 20s of the proteasome and inhibits chymotrypsin-like activity.

[0012] [3] The therapeutic agent for ovarian clear cell carcinoma according to [1], wherein the proteasome inhibitor is a substance that irreversibly binds to the β5 unit of the 20s of the proteasome and inhibits chymotrypsin-like activity.

[0013] [4] The therapeutic agent for ovarian clear cell carcinoma according to [2] or [3], wherein the proteasome is a 26s proteasome.

[0014] [5] The therapeutic agent for ovarian clear cell carcinoma according to any one of [1] to [4], wherein the content of the proteasome inhibitor in the active ingredient is 80% by mass or more, 90% by mass or more, or 100% by mass.

[0015] [6] The therapeutic agent for ovarian clear cell carcinoma according to any one of [1] to [5], wherein the ovarian clear cell carcinoma has resistance to cisplatin, carboplatin, or both.

[0016] [7] The therapeutic agent for ovarian clear cell carcinoma according to any one of [1] to [6], wherein the ovarian clear cell carcinoma expresses Calnexin, ero1La, CHOP, PDI, PERK, BiP, or IRE1α.

[0017] [8] The therapeutic agent for ovarian clear cell carcinoma according to [7], wherein the ovarian clear cell carcinoma expresses calnexin and PDI.

[0018] According to the present invention, a pharmaceutical composition effective for treating ovarian clear cell carcinoma can be provided.

[0019]

[0033] (a) is a photograph showing a micrograph of a cell aggregate in the culture of this example. (b) is a photograph showing HE (Hematoxylin and Eosin) staining and p53 immunostaining of formalin-fixed paraffin-embedded (FFPE) sections of the tumor and organoid of this example. (a) is a diagram showing the results of NGS gene mutation analysis. (b) is a graph showing the results of analyzing the commonality of gene mutations from the results of NGS gene mutation analysis. (b) is a graph showing the results showing the allele frequency of gene mutations. (c) is a graph showing the results showing chromosome copy number changes. (c) is a diagram showing the results of exome sequencing of 11 cases of ovarian clear cell carcinoma organoids. (d) is a graph showing the results of viability in a sensitivity test of ovarian cancer organoids to olaparib or cisplatin. (a) is a photograph of tissue from an immunodeficient mouse transplanted with ovarian cancer organoids. (b) is a photograph showing an HE staining of clear cell carcinoma organoid CCC19-042. (c) A photograph showing HNF1β staining of clear cell carcinoma organoid CCC19-042. (d) A photograph showing HE staining of serous cancer organoid HGSC18-016. (e) A photograph showing p53 staining of serous cancer organoid HGSC18-016. (a) A schematic diagram of a method for constructing an HTDS (High Throughput Drug Screening). (b) A photograph of seeding a cell suspension in constructing an HTDS. A graph of the results of HTDS. A schematic diagram of the protocol for HTDS screening using ovarian clear cell carcinoma organoids. A graph showing a heat map based on the results of HTDS screening using ovarian clear cell carcinoma organoids. A graph showing the results of measuring the viability of ovarian clear cell carcinoma organoids by measuring proteasome inhibitor-sensitive ATP activity. 14 is a graph showing the results of measuring the viability of other samples in FIG. 13. (i) Microscopic images of ovarian clear cell carcinoma organoid CCC19-042. (ii) HE-stained image of ovarian clear cell carcinoma organoid CCC19-042 transplanted into an immunodeficient mouse. (iii) HNF1β immunostained image of ovarian clear cell carcinoma organoid CCC19-042 transplanted into an immunodeficient mouse.(a) A graph showing the change in tumor volume when ovarian clear cell carcinoma organoid-transplanted immunodeficient mice were administered vehicle and bortezomib. (b) A graph showing the results of ssGSEA on gene expression in the IRE1α (inositol-requiring enzyme 1α) pathway, PERK (PKR-like endoplasmic reticulum kinase) pathway, and ATF (activating transcription factor) pathway. FIG. 1 is a graph showing the results of measuring the cell viability for CDK (Cyclin-Dependent Kinase) 1, 2, 5, 9 inhibitors.

[0020] The therapeutic agent according to the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. In this specification, an expression expressing a numerical range, such as "1 to 1000," is synonymous with "1 or more and 1000 or less." Furthermore, an expression including multiple "or more" and "or less," such as "1 to 1000, preferably 10 to 100," is synonymous with "1 or more and 1000 or less, 1 or more and 100 or less, 1 or more and 100 or less, 10 or more and 100 or less."

[0021] [Therapeutic Agent for Ovarian Clear Cell Cancer] The therapeutic agent for ovarian clear cell cancer of this embodiment contains a proteasome inhibitor as an active ingredient.

[0022] Here, proteasome inhibitors broadly refer to substances that inhibit proteasomes. Proteasomes are enzyme complexes involved in protein degradation. Known examples of proteasomes include the 26s proteasome, which is composed of a 20s (20s proteasome, core particle) with protease activity and two 19s (19s particles) bound to it. A form in which an 11s (11s particle) is bound to a 20s is also known. In this embodiment, the term "proteasome" refers to the 26s proteasome, as well as the complexes that constitute them, such as the 20s, 19s, and 11s, and some of these components. Proteasome inhibitors broadly include components that interact with these proteasomes and inhibit their action. Proteasome inhibitors, such as bortezomib, are commonly used in the treatment of multiple myeloma, as described below.

[0023] Proteasome inhibitors include bortezomib (CAS RN (registered trademark): 179324-69-7), MG-132 (CAS RN (registered trademark): 133407-82-6), carfilzomib (CAS RN (registered trademark): 868540-17-4), ixazomib citrate (CAS RN (registered trademark): 1239908-20-3), ixazomib (CAS RN (registered trademark): 1072833-77-2), ONX-0914 (CAS RN (registered trademark): 960374-59-8), and oprozomib (CAS RN (registered trademark): 935888-69-0), Delanzomib (CAS RN (registered trademark): 847499-27-8), Celastrol (CAS RN (registered trademark): 34157-83-0), Epoxomicin (CAS RN (registered trademark): 134381-21-8), Shikonin (CAS RN (registered trademark): 517-89-5), VR23 (CAS RN (registered trademark): 1624602-30-7), Isoginkgetin (CAS RN (registered trademark): 548-19-6), Salinosporamide A (CAS RN (registered trademark): 437742-34-2), RA-190 (CAS RN (registered trademark): 1617495-03-0), PI-1840 (CAS RN (registered trademark): 1401223-22-0), and acetylcorynoline (CAS RN (registered trademark): 18797-80-3), and physiologically acceptable salts thereof.

[0024] The proteasome inhibitor of this embodiment is preferably a substance that reversibly or irreversibly binds to the 20S β5 unit of the proteasome. Among the above-mentioned proteasome inhibitors, examples of substances that reversibly or irreversibly bind to the 20S β5 unit of the proteasome include bortezomib, carfilzomib, ixazomib citrate, ixazomib, oprozomib, and salinosporamide A. Among substances that reversibly or irreversibly bind to the 20S β5 unit of the proteasome, bortezomib, carfilzomib, or ixazomib is preferred, and bortezomib or ixazomib is more preferred.

[0025] Furthermore, the proteasome inhibitor of this embodiment is more preferably a substance that irreversibly binds to the β5 unit of the 20s of the proteasome. Examples of substances that irreversibly bind to the β5 unit of the 20s of the proteasome include carfilzomib and oprozomib.

[0026] The reason why substances that bind reversibly or irreversibly to the β5 unit of the 20s proteasome are effective is presumably because only the β1, β2, and β5 units have proteolytic activity, and of these, the β5 unit is involved in the rate-limiting step.

[0027] The proteasome inhibitor of this embodiment may inhibit either β5, which has chymotrypsin-like activity, or β1, which has caspase-like activity, among the 20s subunits. Examples of proteasome inhibitors include those that reversibly or irreversibly bind to and inhibit β5 or β1, but substances that bind to β5 and inhibit chymotrypsin-like activity are preferred because they provide more potent inhibition.

[0028] The proteasome inhibitor of this embodiment is preferably a substance that inhibits chymotrypsin-like activity. Generally, the chymotrypsin-like activity of proteasomes is thought to be specifically mediated by the β5 unit of the β-type subunit, as well as the β1i unit and β5i unit. Therefore, the substance that inhibits chymotrypsin-like activity is preferably a substance that inhibits the function of the β5 unit, β1i unit, and β5i unit. For example, it is also preferable that the substance reversibly or irreversibly binds to the β1i unit and β5i unit in addition to the β5 unit.

[0029] Substances that inhibit chymotrypsin-like activity include, among the above-mentioned proteasome inhibitors, bortezomib, carfilzomib, ixazomib citrate, ixazomib, delanzomib, celastrol, VR23, isoginkgetin, and PI-1840.

[0030] The reason why substances that inhibit chymotrypsin-like activity are effective is presumed to be that the chymotrypsin-like activity of the proteasome β5 subunit (PSMB5) is associated with the rate-limiting step in protein degradation.

[0031] In the therapeutic agent for ovarian clear cell cancer of this embodiment, the proteasome is preferably the 26s proteasome.

[0032] In the therapeutic agent for ovarian clear cell carcinoma of this embodiment, the content of the proteasome inhibitor in the active ingredient may be 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 95% by mass or more, or 100% by mass or more. In this specification, the term "active ingredient" refers to a substance contained in the therapeutic agent for ovarian clear cell carcinoma that exhibits physiological activity.

[0033] The therapeutic agent for ovarian clear cell carcinoma of this embodiment can be used to treat ovarian clear cell carcinoma. Clear cell ovarian cancer generally has low sensitivity to anticancer drugs and is relatively slow to progress, but is difficult to treat. Pathologically, ovarian clear cell carcinoma is characterized by clear cells or Hobnail-like cells, and clear cells have bright, wide cytoplasm containing glycogen. Immunohistochemically, ovarian clear cell carcinoma is positive for glucose metabolism-related transcription factors (hepatocyte nuclear factor-1β, HNF-1β), etc.

[0034] The therapeutic agent for ovarian clear cell carcinoma of this embodiment may be used for treating ovarian clear cell carcinoma that is resistant to cisplatin, carboplatin, or both. Cisplatin and carboplatin are used to treat, prevent, and alleviate symptoms of various types of tumors and cancers, and are also used in chemotherapy for ovarian cancer. Cisplatin and carboplatin are also used to treat ovarian clear cell carcinoma. However, ovarian clear cell carcinoma cells may become resistant to cisplatin or carboplatin, for example, by acquiring resistance through cisplatin treatment. The therapeutic agent for ovarian clear cell carcinoma of this embodiment can be used for treatment even when the ovarian clear cell carcinoma is resistant to cisplatin, carboplatin, or both cisplatin and carboplatin, and is therefore effective for these applications.

[0035] The therapeutic agent for ovarian clear cell carcinoma of this embodiment is preferably used when the ovarian clear cell carcinoma expresses calnexin, ero1La (endoplasmic reticulum oxidoreductase 1α), CHOP (C / EBP homologous protein), PDI (protein disulfide isomerase), PERK (PKR-like endoplasmic reticulum kinase), BiP (binding immunoglobulin protein), IRE1α (inositol-requiring enzyme 1α), or any combination thereof. The factor expressed by the ovarian clear cell carcinoma may be calnexin (NCBI Reference Sequence (RefSeq): XP_031293834.1, etc.), ero1La (RefSeq: NP_001369396.1, etc.), CHOP (RefSeq: XP_047284402.1, etc.), PDI (RefSeq: XP_009308609.1, etc.), PERK (RefSeq: XP_023807016.1, etc.), BiP, or IRE1α (RefSeq: NP_005071.2, etc.). It is particularly preferred that the ovarian clear cell carcinoma expresses calnexin and PDI.

[0036] [Pharmaceutical Composition] The therapeutic agent for ovarian clear cell cancer of this embodiment is suitable for use in treating ovarian clear cell cancer. That is, the therapeutic agent for ovarian clear cell cancer of this embodiment can also be referred to as a pharmaceutical composition used in treating ovarian clear cell cancer. The therapeutic agent for ovarian clear cell cancer can also be a pharmaceutical composition containing other ingredients. The pharmaceutical composition for treating ovarian clear cell cancer may also contain various other ingredients contained in conventionally known pharmaceutical compositions, as appropriate.

[0037] The form of the pharmaceutical composition of this embodiment is not particularly limited, and may be, for example, a solution, a dispersion such as a sol or gel, or a powder. The pharmaceutical composition may be administered orally in the form of, for example, a tablet, capsule, or elixir, or parenterally in the form of an enema.

[0038] As the pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without any particular limitation. More specifically, examples thereof include binders such as gelatin, corn starch, gum tragacanth, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, etc.; solvents such as water, ethanol, glycerin, etc.

[0039] The pharmaceutical composition of this embodiment may contain additives, such as lubricants such as calcium stearate and magnesium stearate, sweeteners such as sucrose, lactose, saccharin and maltitol, flavorings such as peppermint and rhizome oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, and preservatives.

[0040] The pharmaceutical composition of this embodiment can be formulated by appropriately combining the above-mentioned proteasome inhibitor, the above-mentioned pharmaceutically acceptable carrier, and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

[0041] The dosage of the pharmaceutical composition varies depending on the type of drug, method of use, dosage, symptoms, weight, age, sex, etc. of the patient, and cannot be determined in general. However, the dosage is usually 0.3 mg / m2 of the proteasome inhibitor in the active ingredient contained in the pharmaceutical composition per day. ２ Body surface area ~550mg / m ２ Body surface area.

[0042] For bortezomib, in the case of intravenous administration, for example, 0.3 mg / m per day ２ Body surface area ~2.5mg / m ２ body surface area, preferably e.g. 0.7 mg / m ２ Body surface area ~2.3mg / m ２ body surface area, more preferably 1 mg / m ２ Body surface area ~2mg / m ２ It is believed that administering the active ingredient to the body surface area is appropriate.

[0043] For ixazomib, in the case of intravenous administration, for example, 1 mg / m per day２ Body surface area ~550mg / m ２ body surface area, preferably e.g. 0.5 mg / m ２ Body surface area ~270mg / m ２ body surface area, more preferably 1 mg / m ２ Body surface area ~3mg / m ２ It is believed that administering the active ingredient to the body surface area is appropriate.

[0044] Carfilzomib is administered intravenously at a dose of, for example, 10 mg / m per day. ２ Body surface area ~100mg / m ２ , preferably 15 mg / m ２ ~80 mg / m ２ , more preferably 20 mg / m ２ Body surface area ~56mg / m ２ It is believed that administering the active ingredient to the body surface area is appropriate.

[0045] (Effects of this embodiment) According to the therapeutic agent for ovarian clear cell cancer of this embodiment, by containing a proteasome inhibitor as an active ingredient, a therapeutic agent effective for treating ovarian clear cell cancer can be obtained.

[0046] As shown in the Examples, the present inventors established a cell line and culture method for ovarian cancer organoids. Furthermore, they performed drug screening using ovarian clear cell carcinoma cell lines (ovarian clear cell carcinoma organoids) to examine the cell viability of candidate drugs. As a result, they found that proteasome inhibitors are effective against clear cell carcinoma organoids.

[0047] Previous studies have reported that cell lines considered to represent high-grade serous carcinoma, such as Kuramochi, OVSAHO, and SNU119, were transplanted into immunodeficient mice, but no tumor formation was observed (Mitra AK et al., Gynecol Oncol. 2015). Therefore, there was a need for ovarian cancer cell lines suitable for in vivo tumor research. As shown in the Examples below, the present inventors established ovarian cell carcinoma organoids, transplanted them into immunodeficient mice, and confirmed tumor formation, pathological characteristics, and HNF1-β positivity.

[0048] Regarding the effectiveness of proteasome inhibitors in the treatment of clear cell ovarian cancer, which is a cancer characterized by heterogeneous tumors and a wide variety of mutations, proteasomes play an essential role in all cells as housekeeping molecules for maintaining protein homeostasis. Therefore, proteasomes can function as targets even in cases with a wide variety of mutations, such as those in clear cell ovarian cancer. This may explain why proteasome inhibitors are effective in the treatment of clear cell ovarian cancer.

[0049] [Method for Treating Ovarian Clear Cell Carcinoma] The proteasome inhibitor of this embodiment can be used in a method for treating ovarian clear cell carcinoma, which comprises administering an effective amount of the proteasome inhibitor to a patient.

[0050] The method for treating ovarian clear cell carcinoma broadly includes not only a method for treating a subject suffering from ovarian clear cell carcinoma, but also a method for preventing ovarian clear cell carcinoma, a method for treating the prognosis of ovarian clear cell carcinoma, etc. Patients broadly include humans and other animals suffering from the above-mentioned ovarian clear cell carcinoma, and subjects for which preventive or prognostic treatment is to be performed.

[0051] The effective amount of the proteasome inhibitor administered is the same as that described for the dosage of the pharmaceutical composition.

[0052] Another aspect of this embodiment is a proteasome inhibitor for use in treating ovarian clear cell carcinoma. The composition and method of use of the proteasome inhibitor can be selected from those described above.

[0053] Examples are shown below. However, the present invention is not limited to these examples. All experiments were performed using pathological specimens from ovarian cancer patients who had given informed consent, and were conducted in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.

[0054] (Establishment of Ovarian Cancer Organoids) [Experimental Example 1: Preparation of Cell Aggregates] Ovarian clear cell carcinoma organoids (CCCO) were established from ovarian clear cell carcinoma specimens (CCC). A pathological specimen, lesion tissue named CA18-015, was collected from an ovarian cancer patient. The histological type of CA18-015 was clear cell carcinoma. Under aseptic procedures, the ovarian cancer tissue was placed in a centrifuge tube containing a cell preservation solution (product name "Advanced DMEM / F-12", manufactured by ThermoFisher Scientific), and transported to the laboratory while cooled on ice. In a safety cabinet, the cell preservation solution used during transportation was removed, and the ovarian cancer tissues were washed three times with 20 ml of ice-cold washing solution (product name "HBSS, no calcium, no magnesium", manufactured by ThermoFisher Scientific) by inversion.

[0055] The ovarian cancer tissue was transferred to a 9 cm tissue culture dish (product name: "Cell Culture Petri Dish 90Φ", manufactured by Sumitomo Bakelite Co., Ltd.). This culture dish was placed on ice, and necrotic tissue was removed from the dish using ophthalmic scissors. The ovarian cancer tissue fragments from which the necrotic tissue had been removed were then minced into approximately 0.5-1 mm cubes using ophthalmic scissors. The minced ovarian cancer tissue fragments and 45 mL of washing solution were transferred to a 50 mL centrifuge tube and centrifuged. The supernatant was discarded, and 45 mL of washing solution was added and mixed by inversion.

[0056] 10 mL of cell dispersion solution was added per 600 mg of minced ovarian cancer tissue fragments and mixed. The container containing the minced ovarian cancer tissue fragments was shaken in a shaking incubator at 37°C and 160 rpm for 30 minutes, and the contents of the container were passed through a 100 μm mesh cell strainer. Next, 40 mL of washing solution was added, centrifuged, and the supernatant was discarded. Next, 40 mL of washing solution was added, centrifuged, and the supernatant was discarded.

[0057] The cell dispersion solution used was prepared by adding 1.1 mL of collagenase (final concentration 1 mg / mL), 0.55 mL of dispase II (final concentration 3.6 mg / mL), Y-27136 (final concentration 10 μM), and 100 U of DNase I (Roche, 100 Units / 10 mL) to 8.5 mL of washing solution.

[0058] The cell aggregates after the dispersion treatment were mixed with a hemolytic agent (product name "ACK Lysing Buffer" manufactured by ThermoFisher Scientific) and allowed to stand for 5 minutes to perform a hemolysis treatment. Next, 40 mL of washing solution was added, followed by centrifugation (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), the supernatant was discarded, 40 mL of washing solution was added, followed by centrifugation (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), the supernatant was discarded, a cell preservation solution was added, followed by centrifugation (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), the supernatant was discarded, and cell aggregates with a cell diameter of 11 μm or more were obtained.

[0059] Cell aggregates were obtained from ovarian cancer patients by similar procedures from lesion tissues CA18-148, CA19-001, CA19-010, CA19-042, CA19-044, CA19-055, CA19-076, CA19-078, and CA19-079 as pathological specimens of ovarian clear cell carcinoma, lesion tissues CA18-016, CA18-036, and CA18-064 as pathological specimens of serous ovarian cancer, and CA18-053, CA18-055, and CA18-065 as pathological specimens of endometrioid ovarian cancer.

[0060] Experimental Example 2: Establishment of ovarian clear cell carcinoma cell line (ovarian clear cell carcinoma organoid) 3,000 cell aggregates obtained from CA18-015 were seeded onto a 48-well plate together with 25 μL of Matrigel (registered trademark, Corning) thawed under ice-cooling. ２ Incubator (37°C, 5% CO ２ The plates were then placed in a 10-well plate containing Matrigel (ThermoFisher Scientific) for 10 minutes to allow the Matrigel to gel. 200-300 μL of medium containing B27 serum-free supplement (ThermoFisher Scientific), Wnt3A, EGF, R-spondin, and Noggin was added to each well, and the plates were incubated in CO ２ Incubator (37°C, 5% CO ２ ) for 14 days. During the culture, the medium was changed every 2 or 3 days after seeding. The resulting cultures were capable of being passaged, and ovarian clear cell carcinoma organoids were established. These ovarian clear cell carcinoma organoids were designated CCC18-015.

[0061] Furthermore, from the cell aggregates obtained from CA18-148, CA19-001, CA19-010, CA19-042, CA19-044, CA19-055, CA19-076, CA19-078, and CA19-079 by the same procedure, ovarian clear cell carcinoma organoids were obtained, respectively, as CCC18-148, CCC19-001, CCC19-010, CCC19-042, CCC19-044, CCC19-055, CCC19-076, CCC19-078, and CCC19-079. and CCC19-079; from the cell aggregates obtained from CA18-016, CA18-036, and CA18-064, serous ovarian cancer organoids were obtained, respectively, HGSC18-016, HGSC18-036, and HGSC18-064; from the cell aggregates obtained from CA18-053, CA18-055, and CA18-065, endometrioid ovarian cancer organoids were obtained, respectively, EM18-053, EM18-055, and EM18-065.

[0062] Figure 1 shows micrographs of cell aggregates in culture. Photographs of EM18-053, EM18-055, and EM18-065 are shown on days 4, 7, and 14, respectively. The scale in the figure is 100 μm. Observations were performed using an all-in-one fluorescence microscope BZ-X700 (Keyence Corporation). Figure 2 shows images of thin-section slides prepared from FFPE sections of tumors and organoids, stained with HE and immunostained with p53. Images of tumors and organoids 18-015 (CCC18-015), tumors and organoids 18-036 (HGS18-036), and tumors and organoids 18-053 (END18-053) are shown. Of these, 18-015 is a clear cell carcinoma and its organoid (CCCO). The scale in the figure is 100 μm, and observations were made using an all-in-one fluorescence microscope BZ-X700 (Keyence Corporation).

[0063] Experimental Example 3: Evaluation of ovarian clear cell carcinoma organoids DNA was extracted from pathological specimens and the ovarian cancer organoids obtained therefrom, and exon sequences were enriched using an exome capture kit to prepare a library (1053 gene panel). Gene mutation analysis was performed using NGS (Illumina). Analysis of tumors (T) and organoids (O) was performed for high-grade serous carcinomas HGSC18-016, HGSC18-064, and HGSC18-036, clear cell carcinoma CCC18-015, and endometrioid carcinomas EM18-053, EM18-055, and EM18-065, respectively.

[0064] Figure 3 shows the results of gene mutation analysis. Figure 3(a) shows the results of gene mutation analysis by NGS, and Figure 3(b) shows the commonality of gene mutations between T and O for each cell line. In all cell lines, the number of mutations (N) was approximately 20-30 between T and O. In particular, EM18-053 had a common number of mutations (N) of 100 or more.

[0065] Figure 4 is a graph showing the results showing the allele frequencies of gene mutations. Figure 4(a) shows the results for CCC18-015, Figure 4(b) shows the results for HGSC18-016, Figure 4(c) shows the results for HGSC18-036, Figure 4(d) shows the results for EM18-053, Figure 4(e) shows the results for EM18-055, Figure 4(f) shows the results for HGSC18-064, and Figure 4(g) shows the results for EM18-065.

[0066] Figure 5 is a graph showing the results of changes in chromosome copy number. Figure 5(a) shows the results for CCC18-015, Figure 5(b) shows the results for HGSC18-016, Figure 5(c) shows the results for HGSC18-036, Figure 5(d) shows the results for EM18-065, Figure 5(e) shows the results for EM18-053, Figure 5(f) shows the results for EM18-055, and Figure 5(g) shows the results for EM18-064.

[0067] Figure 6 shows the results of exome sequencing of 11 ovarian clear cell carcinoma organoids. Of the 11 ovarian clear cell carcinoma organoids, ARID1A mutations were observed in seven cases (63.6%), PIK3CA mutations in six cases (54.5%), and ARID1B mutations in two cases (18.2%). Similar to the previously reported genetic mutation analysis of ovarian clear cell carcinoma (Itamochi et al. Br J Cancer, 2017), the 11 ovarian clear cell carcinoma organoids comprehensively covered the genetic mutations of ovarian clear cell carcinoma, including clear cell carcinomas with both ARID1A and PIK3CA mutations, clear cell carcinomas with either mutation, and clear cell carcinomas with neither mutation.

[0068] [Experimental Example 4: PARP inhibitor sensitivity test of ovarian cancer organoids] Sensitivity test of ovarian cancer organoids to olaparib was carried out. Olaparib, a type of PARP inhibitor, was prepared to a final concentration of 0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM, and ovarian cancer organoids were exposed to olaparib at each concentration for 3 days, and the viability was measured by measuring ATP activity. Figure 7 shows a graph of viability. The vertical axis shows the viability as a ratio to the control, and the horizontal axis shows the final concentration (μM) of olaparib.

[0069] As shown in Figure 7, the overall trend was that the viability of each cell line decreased depending on the concentration of olaparib. Furthermore, the results showed that the viability of HGSC18-016 changed significantly. CA18-016 is a high-grade serous ovarian cancer with a pathogenic variant (p.L63X) in BRCA1, and HGSC18-016 is also a high-grade serous ovarian cancer organoid with a pathogenic variant in BRCA1. Furthermore, other pathological specimens do not have a pathogenic variant in BRCA1. Figure 7 reflects the clinical results that olaparib is effective against pathogens with pathogenic variants in BRCA1, and shows that the established ovarian cancer organoids reflect the characteristics of the pathological specimens.

[0070] Experimental Example 5: Transplantation of ovarian cancer organoids into immunodeficient mice. Ovarian cancer organoids were transplanted into immunodeficient mice, and tumorigenicity was evaluated. Figure 8 shows histological images of ovarian cancer organoids transplanted into immunodeficient mice, as well as images of thin-section slides prepared from FFPE sections and stained with HE, p53 immunostaining, and HNF1β. Figure 8(a) shows a photograph of the tissue of an immunodeficient mouse transplanted with ovarian cancer organoids. Figure 8(b) shows an HE stained image of the clear cell carcinoma organoid CCC19-042, and Figure 8(c) shows an HNF1β stained image. Figure 8(d) shows an HE stained image of the serous cancer organoid HGSC18-016, and Figure 8(e) shows a p53 stained image. Figure 8 shows tumor formation in both clear cell carcinoma organoids and serous cancer organoids. The results of HE staining and immunohistochemical staining also showed that the tumor retained the pathological and immunohistochemical characteristics of clear cell carcinoma and serous carcinoma.

[0071] (Drug Screening Using Ovarian Cancer Organoids) [Experimental Example 6: Assay System Construction] Figure 9 shows the construction method of an assay system for drug screening (High-Throughput Drug Screening, HTDS). Figure 9(a) shows a schematic diagram of the construction method, and Figure 9(b) shows a photograph of the cell suspension seeding. After dispersing ovarian cancer organoids into single cells, they were suspended in Matrigel (ThermoFisher Scientific) at 2000 cells / 10 μl while cooling on ice. The suspension was seeded into a 384-well plate (product name: Ultra-Low Attachment Surface 384-Well Low Flange Clear Bottom Plate, Black, with Flat Bottom Lid, Corning Inc.) at 10 μl / well, and then the medium described in Experimental Example 2 was layered at 40 μl / well to prepare the assay system.

[0072] [Experimental Example 7: HTDS using a compound library] After constructing the assay system, on the third day, drugs (1 μM) from the library (Selleck Bioactive compound library (4650 cpds), Ministry of Education, Culture, Sports, Science and Technology Molecular Profiling Support Standard Inhibitor Kit (384 cpds)) were added to the screening system, and ATP activity was detected on the fifth day. Figure 10 shows a graph of the HTDS results. The coefficient of determination R ２ was 0.7673, indicating that this was a valid screening system.

[0073] Figure 11 shows a schematic diagram of the HTDS protocol using ovarian clear cell carcinoma organoids. HTDS was performed on six clear cell carcinoma organoids using a 384-compound library of standard inhibitors. Similarly, HTDS was performed on two clear cell carcinoma organoids using a 4,650-compound library. As a result, nine compounds were identified as common hits. Figure 12 shows a heat map based on the results of HTDS (0.1 μM).

[0074] (Drug sensitivity test of ovarian clear cell carcinoma organoids with HTDS hit compounds) [Experimental Example 8: Proteasome inhibitor sensitivity test of ovarian clear cell carcinoma organoids] Based on the above-mentioned HTDS results, we focused on proteasome inhibitors that were multiple hits among the nine hit compounds. Proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), microtubule inhibitors (paquiritaxel), and platinum agents (cisplatin, carboplatin) were prepared to final concentrations of 0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM, respectively. Ovarian clear cell carcinoma organoids were exposed to each concentration for 3 days, and the viability was measured by measuring ATP activity. Figures 13 and 14 show graphs of the measured cell viability. Figure 13(a) shows the cell viability for cisplatin, Figure 13(b) shows the cell viability for carboplatin, Figure 13(c) shows the cell viability for paclitaxel, Figure 13(d) shows the cell viability for bortezomib, Figure 14(a) shows the cell viability for carfilzomib, and Figure 14(b) shows the cell viability for ixazomib. Figures 13 and 14 show that administration of the microtubule inhibitor paclitaxel, and the proteasome inhibitors bortezomib, carfilzomib, and ixazomib significantly reduced the cell viability of all ovarian clear cell carcinoma organoids in a concentration-dependent manner.

[0075] Experimental Example 9: Transplantation of ovarian clear cell carcinoma organoids into immunodeficient mice and evaluation of tumorigenicity Ovarian clear cell carcinoma organoids CCC19-042 were cultured until they reached a diameter of approximately 50-100 μm. The organoids were recovered by removing the medium from the plate, suspending them in Cell Recovery Solution (Corning, 354235), and then collecting them in a 50 mL tube. The solution containing the recovered organoids was placed on ice to depolymerize the Matrigel. After the reaction was completed, the organoids were washed with medium and 1 x 10 cells were collected in 100 μL. ６ The cells were resuspended in Matrigel so that the number of cells was equivalent to 1000.

[0076] 1 x 10 ６The organoids equivalent to ovarian clear cell carcinoma cells were transplanted subcutaneously (left shoulder) into immunodeficient mice, and tumorigenicity was evaluated 7 days after transplantation. Thin-section slides were also prepared from FFPE sections of the tumor, and HE staining and HNF1β immunostaining were performed. Figure 15A shows a microscopic image of ovarian clear cell carcinoma organoid CCC19-042. Observation was performed as in Experimental Example 2, and the scale in the figure is 100 μm. Figure 15B (i) shows a visual histological image of the tumor one week after transplantation of ovarian clear cell carcinoma organoid CCC19-042 into an immunodeficient mouse. The area surrounded by a line in the photograph is the tissue at the transplant site. Figure 15B (ii) shows an HE-stained image of the tumor, and Figure 15B (iii) shows an HNF1β immunostained image. HE staining and HNF1β staining were performed using the same procedures as in Experimental Example 2. 15A and 15B, tumor formation was observed in ovarian clear cell carcinoma organoids. Results of HE staining and immunohistochemical staining also showed that the organoids retained the pathological and immunohistochemical characteristics of clear cell carcinoma.

[0077] [Experimental Example 10: Drug sensitivity test on immunodeficient mice transplanted with ovarian clear cell carcinoma organoids] For the immunodeficient mice transplanted with ovarian clear cell carcinoma organoids shown in Experimental Example 9, medication was administered, body weight was measured once a week, and tumor volume was calculated starting from day 7 after transplantation. The mice were intraperitoneally administered DMSO (vehicle) or bortezomib at 1 mg / kg twice a week with a three-day interval. Tumor volume was calculated using the following formula. Tumor volume (mm ３ ) = major axis (mm) × {minor axis (mm)} ２ ×1 / 2.

[0078] Figure 15C shows the weekly tumor volume of the mice treated with vehicle and bortezomib. Figure 15C shows that administration of the proteasome inhibitor bortezomib also reduced the tumor volume of ovarian clear cell carcinoma organoids transplanted into immunodeficient mice.

[0079] Proteasome inhibitors, such as bortezomib, are drugs used to treat multiple myeloma. Tumor cells typically actively synthesize proteins to maintain biological functions, and the proteasome-ubiquitin pathway is well developed to process unnecessary proteins within the cell. While the mechanisms of action of proteasome inhibitors are diverse, it is believed that proteasome inhibition disrupts the balance of endoplasmic reticulum-associated protein degradation (ERAD), leading to the accumulation of misfolded proteins and resulting in apoptosis. Multiple myeloma is a tumor of plasma cells that secrete large amounts of abnormal immunoglobulins, and its cytoplasm is rich in endoplasmic reticulum. Clear cell carcinoma also has an abundance of endoplasmic reticulum as well as glycogen within the cells (Silverberg SG. Ultrastructure and histogenesis of clear cell carcinoma of the ovary. Am J Obstet Gynecol 1973; 115: 394-400.), and both multiple myeloma and clear cell carcinoma are characterized by a high incidence of thrombosis. This gives clear cell carcinoma and multiple myeloma common characteristics.

[0080] The unfolded protein response (UPR) in the endoplasmic reticulum includes the IRE1α pathway, the PERK pathway, and the ATF6 pathway. Under normal circumstances, these pathways process proteins, but persistent endoplasmic reticulum stress can induce cell death.

[0081] Single-sample Gene Set Enrichment Analysis (ssGSEA) was used to compare clear cell carcinoma organoids, multiple myeloma (MMRF CoMMpass Study (TCGA)), and normal ovaries (GTEx Analysis V8). Figure 16(a) shows an outline of the ssGSEA. Figure 16(b) shows a graph of gene expression in the IRE1α pathway, PERK pathway, and ATF pathway. The figure shows that PERK pathway-related gene expression was significantly higher in clear cell organoids, followed by multiple myeloma and normal ovaries. The ATF6 pathway was also more elevated in clear cell carcinoma organoids than in multiple myeloma and normal ovaries. This suggests that UPR is elevated in clear cell carcinoma, and that proteasome inhibitors are effective, as in multiple myeloma.

[0082] Experimental Example 11: CDK1,2,5,9 inhibitor sensitivity test of ovarian clear cell carcinoma organoids For the CDK1,2,5,9 inhibitor (dinaciclib) that was found among the nine compounds hit by HTDS, the viability of cells was measured by measuring ATP activity in the same manner as in Experimental Example 8. The results are shown in Figure 17.

[0083] As shown in Figure 17, the results showed that the cell viability decreased for each cell line as a whole depending on the concentration of dinaciclib. Dinaciclib is also known to inhibit the UPR, which is presumably one of the reasons why it is effective against clear cell carcinoma.

[0084] According to the present invention, a pharmaceutical composition effective for treating ovarian clear cell carcinoma is provided.

Claims

1. A therapeutic agent for ovarian clear cell carcinoma, comprising a proteasome inhibitor as an active ingredient.

2. The therapeutic agent for ovarian clear cell carcinoma according to claim 1, wherein the proteasome inhibitor is a substance that reversibly or irreversibly binds to the β5 subunit of 20s of the proteasome and inhibits chymotrypsin-like activity.

3. The therapeutic agent for ovarian clear cell carcinoma according to claim 1, wherein the proteasome inhibitor is a substance that irreversibly binds to the β5 subunit of 20s of the proteasome and inhibits chymotrypsin-like activity.

4. The method of claim 2, wherein the proteasome is the 26s proteasome.

5. The therapeutic agent for ovarian clear cell carcinoma according to claim 1 , wherein the content of the proteasome inhibitor in the active ingredient is 80% by mass or more, 90% by mass or more, or 100% by mass.

6. The method of claim 1 , wherein the ovarian clear cell carcinoma has resistance to cisplatin, carboplatin, or both.

7. The method of claim 1, wherein the ovarian clear cell carcinoma expresses Calnexin, ero1La, CHOP, PDI, PERK, BiP, or IRE1α.

8. The method of claim 7, wherein the ovarian clear cell carcinoma expresses calnexin and PDI.

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