Cancer biomarker, antibody for the same, prediction method for prognosis of cancer patient using antibody, and detection method for cancer

Abstract

To identify biomarkers capable of predicting the prognosis of ovarian cancer and to provide a prediction method for prognosis using the biomarkers.SOLUTION: A prediction method for a prognosis of ovarian cancer patients includes a step for measuring the amount of KLHL14 protein or peptide fragments thereof, or transcript thereof, contained in a sample derived from an ovarian cancer patient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cancer biomarker, an antibody against the biomarker, a method for predicting the prognosis of a cancer patient using the antibody, and a method for detecting cancer. [Background technology]

[0002] In Japan, cancer is the leading cause of death, accounting for approximately 30% of all deaths. Ovarian cancer, in particular, is known to be the gynecological cancer with the poorest prognosis. The annual incidence of ovarian cancer in Japan and worldwide is 11,000 and 240,000, respectively, with 150,000 deaths worldwide each year (Non-Patent Document 1).

[0003] Ovarian cancer is difficult to detect early, and no early diagnostic method has been established. As a result, over 70% of ovarian cancers are discovered at an advanced stage, and the 5-year survival rate is currently low at approximately 60%. One reason for the difficulty in early diagnosis of ovarian cancer is that no specific and highly sensitive biomarkers for ovarian cancer or methods that can recognize them specifically and with high sensitivity have been discovered.

[0004] Non-Patent Documents 2 to 4 disclose cancer antigen 125 (CA125), a representative ovarian cancer marker. CA125 is known to be positive not only for ovarian cancer but also for pancreatic cancer, lung cancer, and other cancers. CA125 levels can also be elevated in benign diseases such as endometriosis and uterine fibroids. Therefore, CA125 does not have sufficient specificity for ovarian cancer. Non-Patent Documents 5 and 6 disclose that past large-scale clinical trials have shown that ovarian cancer diagnosis using CA125 and transvaginal ultrasound does not contribute to extending survival. Non-Patent Document 7 reports that in women aged 50 or older with no family history of ovarian cancer, annual screening using CA125 or ultrasound results in more than 30 false positives for every case of ovarian cancer detected. Non-Patent Document 8 discloses another ovarian cancer marker, human epidemiology protein 4 (HE4). HE4 has higher specificity than CA125, but its detection sensitivity remains at approximately 50%. Non-Patent Document 9 suggests the clinical potential of liquid biopsies, such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), cell-free RNA (cfRNA), tumor-associated platelets (TEPs), and exosomes, as a new method for managing ovarian cancer. However, issues remain regarding the sensitivity and specificity of liquid biopsies, as well as technical separation techniques and reproducibility, before they can be applied clinically. Therefore, there is a need for the development of specific and highly sensitive ovarian cancer markers and methods for their specific and highly sensitive recognition.

[0005] Ovarian cancer is also known to have the poorest prognosis of gynecological cancers. The current standard treatment for ovarian cancer involves a combination of surgery and chemotherapy, but more than half of treated patients experience a recurrence. In recurrent cases, the 5-year survival rate is extremely low at approximately 20%, making improving the prognosis of ovarian cancer patients a major challenge. However, as with early diagnosis, no good biomarkers have been found that can be used to determine prognosis. In particular, there are no prognostic biomarkers for ovarian cancer that are covered by insurance. In order to improve the prognosis of ovarian cancer patients, it is necessary to develop biomarkers that can contribute to prognosis determination and methods that can recognize them specifically and with high sensitivity.

[0006] Therefore, in order to dramatically improve the prognosis of ovarian cancer patients, it is essential to develop new prognostic biomarkers, and methods that can recognize them specifically and with high sensitivity are needed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Lheurex et al. Lancet, 2019; Morice et al., N Engl J Med, 2019; Siegel et al., CA Cancer J Clin, 2022 [Non-patent document 2] Bast RC Jr., et al., N Engl J Med, 1983, 309 (15):883-887. [Non-patent document 3] Cwik G., et al., Arch Surg, 2006, 141(10):968-973. [Non-patent document 4] Fahrmann JF, et al., JAMA Oncol, 2022, in press. (DOI:10.1200 / JCO.21.01460) [Non-patent document 5] Jacobs IJ, et al., Lancet, 2016, 387 (10022):945-956. [Non-patent document 6] Menon U., et al., Lancet, 2021, 397 (10290):2182-2193. [Non-Patent Document 7] Carlson KJ, Skates SJ, Singer DE. Screening for ovarian cancer. Ann Intern Med. 1994;121:124-32. [Non-patent document 8] Hellstroem I., et al., Cancer Res, 2003, 63 (13):3695-3700. [Non-Patent Document 9] Zhu JW, Charkhchi P, Akbari MR. Potential clinical utility of liquid biopsies in ovarian cancer. Mol Cancer. 2022;21:114. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to identify biomarkers that can predict the prognosis of ovarian cancer and to provide a method for predicting the prognosis using the same. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors searched for new biomarkers for ovarian cancer and found that kelch-like 14 (KLHL14) protein is highly expressed in ovarian cancer tissues. While KLHL14 is particularly highly expressed in ovarian cancer, it is not detected in normal tissues, including the ovary, except for very slight detection in normal thyroid tissue, making it an extremely specific biomarker for ovarian cancer. The present inventors further found that ovarian cancer patients with high KLHL14 protein expression have a poorer prognosis than ovarian cancer patients with low KLHL14 protein expression. The present invention was completed based on the above findings and includes the following aspects:

[0010] One aspect of the present invention is [1] A method for predicting the prognosis of an ovarian cancer patient, comprising: The present invention relates to a prediction method comprising a step of measuring the amount of KLHL14 protein or a peptide fragment thereof, or a transcription product thereof contained in a sample derived from the ovarian cancer patient. Here, in one embodiment, the prediction method of the present invention includes: [2] The prediction method according to [1] above, When the measured value obtained in the measuring step is equal to or greater than a predetermined value, the prognosis of the ovarian cancer patient is determined to be poor. In one embodiment, the prediction method of the present invention includes: [3] The prediction method according to [1] or [2] above, The sample is characterized in that it contains ovarian tissue. In one embodiment, the prediction method of the present invention includes: [4] The prediction method according to any one of [1] to [3] above, The measuring step is characterized in that it is a step of measuring the amount of KLHL14 protein or a peptide fragment thereof contained in a sample derived from the ovarian cancer patient.

[0011] Another aspect of the present invention is [5] A kit for predicting the prognosis of an ovarian cancer patient, comprising: The kit comprises at least one measuring means selected from the group consisting of an antibody or a fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof.

[0012] Another aspect of the present invention is [6] A kit for detecting ovarian cancer, uterine cancer, or thyroid cancer, comprising: The kit comprises at least one measuring means selected from the group consisting of an antibody or a fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof.

[0013] Another aspect of the present invention is [7] An antibody or a fragment thereof that can be used in the prediction method according to any one of [1] to [4] above, The antibody has a heavy chain complementarity determining region (hereinafter referred to as "CDR") 1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and The present invention relates to an antibody or a fragment thereof that specifically binds to KLHL14. wherein the antibody or fragment thereof of the present invention comprises, in one embodiment: [8] The antibody or fragment thereof according to [7] above, (a) the amino acid sequence of SEQ ID NO: 7; (b) an amino acid sequence having at least 90% identity to the sequence of the framework region other than each CDR sequence in the sequence of (a); and (c) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (a); a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: (d) the amino acid sequence of SEQ ID NO: 8; (e) an amino acid sequence having at least 90% identity to the sequence of the framework region other than each CDR sequence in the sequence of (d); and (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (e); a light chain variable region consisting of an amino acid sequence selected from the group consisting of The present invention is characterized by having the following.

[0014] Another aspect of the present invention is [9] A polynucleotide encoding the antibody or fragment thereof according to [7] or [8] above.

[0015] Another aspect of the present invention is

[10] A vector containing the polynucleotide described in [9] above.

[0016] Another aspect of the present invention is

[11] A host cell transformed with the vector according to

[10] above. [Effects of the Invention]

[0017] According to the method of the present invention for predicting the prognosis of an ovarian cancer patient, the prognosis of ovarian cancer can be predicted by measuring the expression level of KLHL14 in a sample derived from an ovarian cancer patient. [Brief explanation of the drawings]

[0018] [Figure 1]

[0023] Figure 1 shows a method for identifying cell surface proteins of human ovarian cancer cell lines. In the method shown, cell surface proteins are labeled with biotin tags and identified by mass spectrometry. [Figure 2]Figure 2 shows the expression levels of the KLHL14 gene in various human cancer tissues and normal tissues: Figure 2A shows the expression levels of the KLHL14 gene in various cancer tissues; and Figure 2B shows the expression levels of the KLHL14 gene in various normal tissues. [Figure 3] 3A and 3B show the structure and antigenic regions of the KLHL14 protein. Figure 3A shows the domain structure of the KLHL14 protein. Figure 3B shows the three-dimensional structure of the KLHL14 protein. [Figure 4] Figure 1 shows the antigen specificity of anti-KLHL14 monoclonal antibodies. The figure shows the results of immunostaining with 12 types of anti-KLHL14 monoclonal antibodies in HEK293T cells not transfected with a KLHL14 protein expression vector and HEK293T cells transfected with a KLHL14 protein expression vector. The scale bar indicates 100 μm. [Figure 5] Figure showing the antigen specificity of anti-KLHL14 monoclonal antibodies. The figure shows the results of Western blot analysis using five types of anti-KLHL14 monoclonal antibodies on HEK293T cells not transfected with a KLHL14 protein expression vector and HEK293T cells transfected with a KLHL14 protein expression vector. [Figure 6] Figure showing the antigen specificity of anti-KLHL14 monoclonal antibody clone #24. The figure shows the results of fluorescent immunostaining using anti-KLHL14 monoclonal antibody clone #24 in a human ovarian cancer cell line (OVCAR3+KLHL14) into which a vector for KLHL14 protein expression had been introduced. The scale bar indicates 50 μm. [Figure 7] Figure 1 shows the antigen specificity of anti-KLHL14 monoclonal antibody clone #24. HEK293T cells (HEK293T+KLHL14) transfected with a vector for expressing KLHL14 protein were reacted with anti-KLHL14 monoclonal antibody clone #24 or rat IgG, followed by flow cytometry analysis after secondary antibody reaction. [Figure 8]This figure shows fluorescent immunostaining images of KLHL14 in ovarian cancer tissue. Ovarian cancer tissue from three cases was double-immunostained with anti-KLHL14 monoclonal antibody clone #24 and anti-occludin (OCLN) antibody. The arrowheads indicate the cell membrane localization of KLHL14. The scale bar indicates 50 μm. [Figure 9] This figure shows the results of KLHL14 immunohistochemical staining in ovarian cancer tissues (negative cases, weakly positive cases, moderately positive cases, and strongly positive cases). The scale bar indicates 100 μm. [Figure 10] Figure 10 shows semi-quantification of KLHL14 protein expression in 167 ovarian cancer cases. Figure 10A shows signal intensity (SI) and percentage of positive cells (PP). Figure 10B shows immunoreactive score (IRS). Figure 10C shows receiver operating characteristic (ROC) curves for determining IRS cutoff values. [Figure 11] 1 is a graph showing Kaplan-Meier curves of disease-specific survival (DFS) and recurrence-free survival (RFS) in ovarian cancer patients in the low KLHL14 group (60 cases) and in ovarian cancer patients in the high KLHL14 group (107 cases). [Figure 12] FIG. 1 shows the association between clinicopathological factors and KLHL14 expression (low KLHL14 expression / high KLHL14 expression) in ovarian cancer patients. [Figure 13] FIG. 1 shows the results of univariate and multivariate analyses of the prognosis (RFS) of ovarian cancer patients. [Figure 14] 14A and 14B show the migration and invasion abilities of a human ovarian cancer cell line (SKOV3) in which the KLHL14 gene was overexpressed or knocked out. Figure 14A shows the results for migration ability, and Figure 14B shows the results for invasion ability. DETAILED DESCRIPTION OF THE INVENTION

[0019] 1. How to predict the prognosis of ovarian cancer patients Overview A first aspect of the present invention is a method for predicting (or assisting in) the prognosis of an ovarian cancer patient by measuring the amount of KLHL14 protein or its peptide fragment, or its transcription product, contained in a sample from the ovarian cancer patient.

[0020] 1-2.Definition As used herein, "ovarian cancer" refers to cancer that develops in the ovaries. Ovarian cancer can be classified based on various cell of origin. For example, ovarian cancers that originate from the surface epithelium, ovarian stroma, sex cord stroma, or luminal cells are known. As used herein, the cell of origin of ovarian cancer is not particularly limited. Furthermore, ovarian cancer can be classified into stage I (tumor localized to the ovary), stage II (tumor present in one or both ovaries, showing pelvic spread), stage III (tumor present in one or both ovaries, showing peritoneal dissemination or lymph node metastasis), and stage IV (excluding peritoneal dissemination, showing distant metastasis).

[0021] As used herein, "uterine cancer" refers to uterine cancer that develops in the uterine body, and is also called endometrial cancer because it develops from the endometrium. Uterine cancer is classified into histological types, such as endometrioid carcinoma, serous carcinoma, and clear cell carcinoma, depending on the state of the cancer tissue. Furthermore, uterine cancer can be classified into stages I (cancer limited to the uterine body), II (cancer invades the cervical stroma but has not spread beyond the uterus), III (cancer has spread outside the uterus but has not spread beyond the small pelvic cavity or has spread to regional lymph nodes), and IV (cancer has spread beyond the small pelvic cavity or has clearly invaded the bladder / intestinal mucosa and / or has distant metastasis).

[0022] As used herein, "thyroid cancer" refers to cancer that develops in the thyroid gland. Thyroid cancer can be classified by histological type into papillary carcinoma, follicular carcinoma, medullary carcinoma, poorly differentiated carcinoma, undifferentiated carcinoma, malignant lymphoma, etc. Thyroid cancer can be classified into stage I (tumors limited to the thyroid gland and measuring 2 cm or less in maximum diameter), stage II (tumors limited to the thyroid gland and measuring more than 2 cm but 4 cm or less in maximum diameter), stage III, and stage IV.

[0023] As used herein, "prognosis" refers to a reduction in tumor burden, suppression of tumor growth, or the course of disease (e.g., presence or absence of recurrence, presence or absence of metastasis, length of survival after treatment, life or death, etc.) after cancer treatment (e.g., surgery, chemotherapy (drug therapy), radiation therapy, etc.) for any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer. In the present invention, it particularly refers to a reduction in tumor burden, suppression of tumor growth, or the course of disease after cancer treatment for ovarian cancer.

[0024] As used herein, "prediction of prognosis" may refer to prediction of recurrence risk (e.g., recurrence-free survival), metastasis risk, survival time, survival rate at a certain time after surgery (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years, or later), relapse-free survival (RFS), or disease-specific survival (DFS). In one embodiment, prediction of prognosis includes prediction of recurrence risk (e.g., relapse-free survival) or prediction of metastasis risk. Note that, as used herein, recurrence-free survival refers to the proportion of patients who do not develop recurrent cancer, such as cancer associated with the primary cancer, and disease-specific survival refers to the proportion of patients who do not die from a disease associated with the primary cancer. Prediction of prognosis can also be referred to as prognosis determination, evaluation, diagnosis, or assistance therefor.

[0025] As used herein, the term "assessment" refers to assessing the malignancy of any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer. In particular, it refers to assessing the malignancy of cancer in a subject (cancer patient) suffering from any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer.

[0026] As used herein, the term "malignancy" refers to the degree of infiltration of surrounding tissues, metastasis to other organs, and / or recurrence of any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer. Specifically, it refers to the proliferation and / or migration ability of cancer cells. By determining the malignancy of cancers such as ovarian cancer, uterine cancer, or thyroid cancer, it is possible to predict the prognosis and select poor prognosis cases with high infiltration, metastasis, and recurrence potential. In this specification, the determination of malignancy also includes prediction of prognosis.

[0027] As used herein, a "cancer patient" refers to, for example, a mammal, preferably a primate, more preferably a human. "Cancer patients" such as "ovarian cancer patients," "uterine cancer patients," and "thyroid cancer" also refer to, for example, a mammal, preferably a primate, more preferably a human. In the present invention, a "cancer patient" particularly refers to an ovarian cancer patient.

[0028] As used herein, the term "sample" refers to a sample collected from a subject or a group of subjects, or a healthy subject or a group of healthy subjects, and used in a method for predicting the prognosis of a cancer patient, such as a tissue or cell. The "tissue" and "cell" used in the prognosis prediction method of the present invention particularly refer to tissues and cells of a subject suffering from ovarian cancer, as well as corresponding tissues and cells in a healthy subject. The sample used in the prognosis prediction method of the present invention is not particularly limited, but may be a specimen collected by biopsy or surgically removed from a subject suffering from cancer. Preferably, the sample is a portion of the cancer (e.g., tissue or cells) collected by biopsy or surgically removed, such as the invasive front. There are no limitations on the stage of cancer suffered by patients who are subjects for the method of the present invention.

[0029] As used herein, the term "subject" refers to a human individual who provides a sample and is subjected to testing. In principle, it refers to an individual, but in this specification it may also include tissues and cells derived from humans. Furthermore, the individual may be not only a healthy individual, but also a patient with a disease (e.g., a malignant tumor), or an individual who may be susceptible to a disease (e.g., a malignant tumor).

[0030] As used herein, the term "healthy individual" refers to a human individual not suffering from a specific cancer, particularly ovarian cancer, preferably a human individual not suffering from any cancer, and more preferably a human individual in a healthy state not suffering from any disease. However, as used herein, healthy human cells are also included in the broad definition of healthy individuals. Therefore, a healthy individual refers not only to an individual level, but also to a cellular level, such as a normal portion of tissue collected from a cancer patient, if the individual is in a healthy state.

[0031] In the present invention, in principle, the biomarkers can be kelch-like 14 (KLHL14) protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof (referred to herein as "KLHL14 protein, etc."). For example, if the patient is human, the biomarkers can be human KLHL14 protein derived from the human KLHL14 gene and the transcription product (mRNA) of the human KLHL14 gene.

[0032] KLHL is a family of proteins with a KELCH domain, consisting of numerous members with various functions in the cytoplasm. Because members of the KLHL family share highly homologous amino acid sequences, it is generally difficult to develop monoclonal antibodies that specifically recognize a particular KLHL molecule.

[0033] "KLHL14" is a 70 kDa protein composed of BTB, KELCH, and BACK domains, and is part of the KLHL family, which consists of 42 species. It is known to negatively regulate tumor progression in diffuse large B-cell lymphoma (Choi J et al., Proc Natl Acad Sci USA, 2020). As used herein, the source of KLHL14 is not limited to specific organisms, but examples include the human KLHL14 protein consisting of the amino acid sequence set forth in SEQ ID NO:9.

[0034] As used herein, the term "peptide fragment" refers to a peptide fragment consisting of a portion of the amino acid sequence constituting the KLHL14 protein, which can be identified as a fragment of the KLHL14 protein from the amino acid sequence constituting the fragment. Typically, the peptide may be a peptide consisting of 10 to 600, 20 to 500, 30 to 400, 40 to 300, 50 to 200, or 60 to 100 consecutive amino acid residues from the full-length amino acid sequence of the KLHL14 protein. For example, a peptide fragment containing an antigenic epitope recognized by the anti-KLHL14 antibody of the present invention described below, such as a peptide fragment containing or comprised in the amino acid sequence set forth in SEQ ID NO: 11 or 20, is preferred.

[0035] "Transcription product of the KLHL14 gene" refers to KLHL14 mRNA. The mRNA may be either a pre-mRNA or a mature mRNA. Normally, pre-mRNA is immediately spliced ​​in the nucleus to become a mature mRNA. Therefore, the transcription product of the KLHL14 gene that essentially serves as a biomarker of the present invention is KLHL14 mature mRNA.

[0036] The "KLHL14 gene" is a gene that encodes the KLHL14 protein. A specific example of the KLHL14 gene is the human KLHL14 gene that encodes the human KLHL14 protein consisting of the amino acid sequence shown in SEQ ID NO: 9. More specifically, the KLHL14 gene is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 10.

[0037] The KLHL14 gene also encompasses KLHL14 variants having activity functionally equivalent to that of the KLHL14 protein encoded by the KLHL14 gene shown in SEQ ID NO: 10, and KLHL14 genes encoding KLHL14 orthologs of other organisms. Specifically, the KLHL14 gene encompasses a nucleotide sequence in which one or more nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 10, or a KLHL14 gene having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 10. Furthermore, the KLHL14 gene encompasses a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions with a nucleic acid fragment consisting of a portion of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10, and that encodes a protein having activity functionally equivalent to that of the KLHL14 protein.

[0038] As used herein, "base identity" refers to the percentage (%) of identical bases between two base sequences relative to the total bases of the KLHL14 gene consisting of the base sequence shown in SEQ ID NO: 10, when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two.

[0039] The nucleotide sequence information of the KLHL14 gene can be searched for in public databases (GenBank, EMBL, DDBJ). For example, based on the known nucleotide sequence information of the KLHL14 gene shown in SEQ ID NO: 10, genes with high nucleotide identity can be searched for and obtained from the database.

[0040] As used herein, "hybridizing under high stringency conditions" refers to hybridization and washing under low salt and / or high temperature conditions. For example, incubation with a probe in 6xSSC, 5xDenhardt's reagent, 0.5% SDS, and 100µg / mL denatured fragmented salmon sperm DNA at 65-68°C is exemplified, followed by washing in a 2xSSC, 0.1% SDS wash solution starting at room temperature, lowering the salt concentration in the wash solution to 0.1xSSC, and raising the temperature to 68°C until no background signal is detected. High stringency hybridization conditions are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and may be used for reference.

[0041] 1-3. Measurement of the amount of KLHL14 protein, its peptide fragments, or its transcripts in samples from ovarian cancer patients In this embodiment, the method for predicting the prognosis of an ovarian cancer patient includes measuring the amount of KLHL14 protein or a peptide fragment thereof, or a transcription product or a nucleic acid fragment thereof contained in a sample derived from an ovarian cancer patient. The measuring step can be performed in vitro.

[0042] In this embodiment, the "measurement step" is a step of measuring the amount of a biomarker for predicting cancer prognosis in a sample derived from a subject suffering from cancer, and determining whether the biomarker is positive or negative based on the measurement value (hereinafter referred to as "determination of positive / negative").

[0043] As used herein, the term "measurement" encompasses qualitative, quantitative, and semi-quantitative measurements.

[0044] The amount of sample required for the prognosis prediction method of the present invention is not particularly limited. For tissues or cells, at least 10 μg, preferably at least 0.1 mg, is desirable, as is biopsy material. Samples can be prepared and treated as needed to detect biomarkers for predicting the prognosis of cancer patients. For example, when detecting biomarkers using immunohistochemical staining, paraffin-embedded sections may be prepared from patient-derived samples. Furthermore, when detecting biomarkers using Western blotting or RT-PCR, protein extracts or mRNA extracts may be prepared from patient-derived samples. Such preparations may include homogenization, cell lysis, removal of contaminants by centrifugation or leakage, addition of protease inhibitors, and the like. Details of these treatments are described in detail in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, and may be used for reference.

[0045] As used herein, the term "measured value of a biomarker for predicting cancer prognosis" specifically refers to the measured amount of a biomarker such as KLHL14 protein in a sample.

[0046] As used herein, a "measured value" refers to a measured value obtained by measuring a biomarker. The measured value may be an absolute value representing the amount of protein in a sample in units such as ng (nanogram) or μg (microgram), or may be a relative value represented by absorbance or fluorescence intensity of a labeling molecule relative to a control value, or may be a score calculated using a certain formula from the spatial distribution of the biomarker in the sample (e.g., a staining pattern). Here, the control value may be a measured value of any biomarker other than the KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof.

[0047] The following describes the steps of the measurement of this embodiment, namely, (1) measurement of a biomarker such as KLHL14 protein, and (2) determination of whether a biomarker such as KLHL14 protein is positive or negative.

[0048] (1) Measurement of biomarkers such as KLHL14 protein The biomarker to be measured by this method may be either the KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof. Measuring the amount (expression level) includes measuring the presence or absence of expression, or the magnitude of the expression level or expression concentration.

[0049] When the biomarker to be measured is KLHL14 protein or a peptide fragment thereof, the measurement method may be any known protein quantification method, and includes, but is not limited to, immunological detection, aptamer analysis, and mass spectrometry.

[0050] An "immunological detection method" is a method for quantifying a target molecule using an antibody or a binding fragment thereof that specifically binds to the target molecule. Known immunological detection methods include enzyme-linked immunosorbent assays (including ELISA and EIA), fluorescent immunoassays, radioimmunoassays (RIA), luminescence immunoassays, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), immunoturbidimetry, latex agglutination immunoassays, latex turbidimetry, hemagglutination, particle agglutination, gold colloid assays, capillary electrophoresis, Western blotting, and immunohistochemistry (immunostaining). Any of these detection methods may be used in this method. Although not limited to these, immunohistochemistry is preferred.

[0051] When immunohistochemistry is used, the method for quantifying the KLHL14 protein or its peptide fragments is not limited. For example, the amount may be quantified as a score calculated using a certain formula based on the observation of the staining pattern of a tissue section.

[0052] The antibody used for immunological detection may be either a monoclonal antibody or a polyclonal antibody, and the immunoglobulin constituting the antibody may be of any class or subclass, may be derived from any animal, including mammals and birds, or may be an artificially produced antibody, such as a recombinant antibody, a synthetic antibody, or an antibody fragment. The forms of these antibodies are as described above, and a detailed description thereof will be omitted here. In one embodiment, the antibody used for immunological detection in this step is the above-mentioned anti-KLHL14 antibody or a fragment thereof.

[0053] "Aptamer analysis" is a method for quantifying a target molecule, a biomarker protein for prognosis prediction, using an aptamer that binds tightly and specifically to a target substance due to its three-dimensional structure. Aptamers can be broadly classified into nucleic acid aptamers and peptide aptamers depending on the type of molecule, but either type of aptamer may be used.

[0054] "Nucleic acid aptamer" refers to an aptamer composed of nucleic acids. The nucleic acid constituting the nucleic acid aptamer may be DNA, RNA, or a combination thereof. If necessary, it may also contain chemically modified nucleic acids such as PNA, LNA / BNA, methylphosphonate-type DNA, phosphorothioate-type DNA, and 2'-O-methylated RNA. In the present invention, examples of the aptamer include an anti-KLHL14 RNA aptamer and an anti-KLHL14 DNA aptamer.

[0055] Nucleic acid aptamers can be prepared using methods known in the art, such as the systematic evolution of ligands by exponential enrichment (SELEX) method, with the KLHL14 protein or a portion thereof as the target molecule. SELEX is a well-known method, and a specific method may be performed, for example, according to Pan et al. (Proc. Natl. Acad. Sci. USA, 1995, 92: 11509-11513).

[0056] A peptide aptamer is an aptamer composed of amino acids, and like an antibody, is a 16 kDa peptide molecule that recognizes and specifically binds to the surface structure of a specific target molecule. In the present invention, an example is an anti-KLHL14 peptide aptamer. Peptide aptamers may be produced based on production methods known in the art. For example, see Whaley, SR, et al., Nature, 2000, 405, 665-668. They can usually be produced using phage display or cell surface display.

[0057] The above-mentioned antibody or aptamer may be labeled as necessary. Labeling may be performed using labeling substances known in the art. In the case of antibodies and peptide aptamers, for example, fluorescent dyes (fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (e.g., PE, APC, GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), radioisotopes (e.g., 3 H, 14 C. 35 S), biotin, or (strept)avidin. In the case of nucleic acid aptamers, they can be labeled with, for example, radioisotopes (e.g., 32 P, 3 H, 14 Examples of labeling agents include fluorescent dyes (e.g., FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, and TAMRA), and luminescent substances (e.g., acridinium ester). Antibodies and aptamers labeled with labeling agents can be useful tools for detecting aptamers bound to target proteins.

[0058] "Mass spectrometry" includes high performance liquid chromatography mass spectrometry (LC-MS), high performance liquid chromatography tandem mass spectrometry (LC-MS / MS), gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS / MS), capillary electrophoresis mass spectrometry (CE-MS), and inductively coupled plasma (ICP-MS) mass spectrometry.

[0059] The immunological detection method, aptamer analysis method, and mass spectrometry are all well-known techniques in the art, and may be carried out in accordance with these methods. For example, this can be performed according to the methods described in Green, MR and Sambrook J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Christopher J., et al., 2005, Chemical Review, 105: 1103-1169; Iijima Y. et al., 2008, The Plant Journal, 54, 949-962; Hirai M. et al., 2004, Proc Natl Acad Sci USA, 101 (27) 10205-10210; Sato S, et al., 2004, The Plant Journal, 40 (1) 151-163; Shimizu M. et al., 2005, Proteomics, 5, 3919-3931. Furthermore, peptide quantification kits are commercially available from various manufacturers, and these can also be used.

[0060] Furthermore, when the biomarker to be measured is a KLHL14 gene transcription product or a nucleic acid fragment thereof, the measurement method may be any known nucleic acid quantification method, and is not particularly limited, but examples include nucleic acid amplification methods using primers, hybridization methods using probes, and RNA sequencing (RNA-Seq) analysis methods.

[0061] "Nucleic acid amplification" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer set. Examples of nucleic acid amplification methods include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction).

[0062] The "hybridization method" is a method in which a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of a target nucleic acid to be detected is used as a probe, and a target nucleic acid or a fragment thereof is detected and quantified by utilizing base pairing between the nucleic acid and the probe. Several hybridization methods using different detection means are known, including, for example, Northern hybridization (Northern blot hybridization), in situ hybridization, and microarray.

[0063] "RNA sequencing (RNA-Seq) analysis" refers to a method of converting RNA into cDNA by reverse transcription and then measuring gene expression levels by counting the number of reads using a next-generation sequencer (such as, but not limited to, the HiSeq series (Illumina) and the Ion Proton system (Thermo Fisher)). All of these methods are well known and are described in appropriate protocols in the relevant technical field, so please refer to these for details.

[0064] Nucleic acid strands such as primers and probes can be appropriately designed by methods known to those skilled in the art based on known biomarker sequence information, and can be obtained by known production methods such as chemical synthesis.

[0065] The above-mentioned measurement methods are all well known in the art. Therefore, specific measurement methods may be performed in accordance with known methods. For example, the method described in Green, MR and Sambrook J., 2012 (mentioned above) can be used as a reference.

[0066] (2) Determining whether biomarkers such as KLHL14 protein are positive or negative Next, based on the measurement values ​​obtained in (1), the positive / negative state of a biomarker such as KLHL14 protein in the sample is determined.

[0067] The method for determining positivity / negative based on a measured value is not limited. For example, a cutoff value for the measured value of a biomarker such as KLHL14 protein is set, and positivity / negative is determined based on the cutoff value. That is, a predetermined value is set as the cutoff value, and if the measured value is equal to or greater than this value, the biomarker such as KLHL14 protein is determined to be positive, and conversely, if the measured value is less than the cutoff value, the biomarker is determined to be negative.

[0068] The cutoff value refers to a boundary value for classifying a measurement value as positive or negative. The cutoff value can usually be calculated based on the disease incidence rate and the sensitivity and specificity calculated from an ROC curve (receiver operating characteristic curve). The method for setting the cutoff value is not particularly limited. For example, the cutoff value can be set to a value at which the sensitivity and specificity are maximized.

[0069] For example, the cutoff value can be the measurement value of a sample derived from a healthy subject not suffering from any cancer such as ovarian cancer, uterine cancer, or thyroid cancer, or the average value of the measurement values ​​of samples derived from a group of healthy subjects, and when the measurement value of a subject is higher than the cutoff value, the subject can be determined to be positive.

[0070] Alternatively, a cutoff value may be set to 1.5 times or more, 2.0 times or more, 3.0 times or more, 4 times or more, 5 times or more, or 6 times or more the average value of the measurement values ​​of samples derived from healthy individuals not suffering from any cancer such as ovarian cancer, uterine cancer, or thyroid cancer, or the average value of the measurement values ​​of samples derived from a group of healthy individuals, and a subject's measurement value may be determined to be positive if it is higher than the cutoff value.

[0071] Alternatively, the measured values ​​obtained from the control group can be classified by percentile, and the percentile value used for the classification can be used as the cutoff value. For example, if the 95th percentile of the measured values ​​obtained from the control subjects is used as the cutoff value, and values ​​above that value are considered positive and values ​​below that value are considered negative, then a subject's measured value of 95th percentile or above can be determined to be positive.

[0072] Unlike the measurements of the subject, the measurements of the control healthy subjects do not necessarily need to be performed each time. For example, if the amount of sample used in the measurement, the measurement method for the cancer prognosis predicting biomarker, and the measurement conditions are kept constant, the measurements of the control healthy subjects previously measured can be reused.

[0073] When immunohistochemical staining is used, for example, if the number of stained tumor cells relative to the total number of tumor cells exceeds a certain percentage (e.g., 10%, 15%, or 20%), the result may be determined as positive, and if the number of stained tumor cells relative to the total number of tumor cells is equal to or less than this certain percentage, the result may be determined as negative.

[0074] In the prognosis prediction method of the present invention, the prognosis of a cancer patient is predicted or indicated based on the detection results of a biomarker such as KLHL14 protein obtained in the detection step. Specifically, if a sample derived from a patient with cancer such as ovarian cancer, endometrial cancer, or thyroid cancer is positive for a biomarker such as KLHL14 protein, it indicates that the cancer patient has a poor prognosis or is likely to have a poor prognosis. Conversely, if a sample derived from a cancer patient is negative for a biomarker such as KLHL14 protein, it indicates that the cancer patient has a good prognosis or is likely to have a good prognosis.

[0075] As used herein, "poor prognosis" refers to poor clinical outcomes (e.g., after surgical resection) (e.g., high risk or rate of cancer recurrence, high risk or rate of cancer metastasis, low recurrence-free survival, low disease (cancer)-specific survival, or low overall survival). In the case of a poor prognosis, the 5-year recurrence-free survival or disease-specific survival rate may be 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, or 45% or less. In the present invention, survival rate refers to cumulative survival rate.

[0076] As used herein, the term "good prognosis" refers to a good clinical outcome. A good prognosis may mean that the recurrence-free survival rate or survival rate 5 years after cancer resection surgery is 50% or more, 55% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%.

[0077] In one embodiment, a biomarker for predicting the prognosis of cancer patients other than the biomarker such as KLHL14 protein may be detected in a sample derived from a cancer patient, and the prognosis of the cancer patient may be predicted based on the detection results and the detection results of the biomarker such as KLHL14 protein. Examples of biomarkers for predicting the prognosis of cancer patients other than the biomarker such as KLHL14 protein and antibodies for detecting them are as described above.

[0078] In addition, in the prognosis prediction method of this embodiment, one or more of cellular atypia, structural atypia, invasion, and metastasis may be detected in a sample derived from a cancer patient, and the prognosis of the cancer patient may be predicted based on the detection results and the detection results of a biomarker such as KLHL14 protein. Here, "cellular atypia" refers to a deviation from normal cellular structure, specifically, an increased nucleoplasmic ratio, cell or nucleus size, irregular nuclear shape, increased nuclear chromatin, increased or increased nucleolus size, increased mitotic figures, and / or the appearance of abnormal mitotic figures. Furthermore, "structural atypia" refers to a deviation from normal tissue structure, i.e., irregular tissue structure. In this embodiment, the method for detecting cellular or structural atypia is not limited. For example, it can be visualized using hematoxylin-eosin staining. "Invasion" refers to the continuous progression of a malignant tumor while destroying surrounding normal tissues and organs, and can be determined by the blurring of the border with the surrounding tissue. "Metastasis" refers to the discontinuous spread of a malignant tumor to an organ distant from the primary focus.

[0079] According to the prognosis prediction method of the present invention, by examining a biological sample removed by biopsy or surgery, the prognosis of the subject who provided the sample can be accurately predicted. The prognosis prediction method of this embodiment, which has a high accuracy rate, can determine the risk of recurrence or metastasis, and based on the results, a treatment plan (e.g., type of anticancer drug, dosage, administration interval, etc.) or the interval between tests for cancer recurrence and metastasis can be determined. If a cancer patient's prognosis is indicated to be poor by the present invention, the patient may be subjected to surgery or anticancer drug therapy (e.g., treatment methods including the administration of anticancer drugs such as paclitaxel, carboplatin, etc.) to prevent cancer recurrence, improve prognosis, or improve survival rate. Therefore, a method for preventing cancer recurrence, improving prognosis, or improving survival rate is also provided, which includes performing at least one of the above procedures on a cancer patient indicated to have a poor prognosis by the method of the present invention. Furthermore, if a cancer patient's prognosis is predicted to be poor, the frequency of tests can be increased to detect cancer recurrence early.

[0080] The prognosis prediction method of the present invention also provides a method for assisting in predicting cancer prognosis, a method for determining the malignancy of cancer in a subject, and a method for assisting in determining the malignancy of cancer in a subject.

[0081] The method of the present invention can be used in combination with other methods (e.g., X-ray photography; ultrasound (echo) examinations such as transluminal ultrasound; endoscopy; mammography; palpation; pelvic examination; rectal examination; CT examination; MRI examinations such as pelvic MRI examinations; imaging examinations such as PET-CT examinations; blood tests; pathological examinations such as cytology and histology; tumor marker tests; and / or genetic diagnosis) and other factors (e.g., stage classification, tumor size, presence or absence of lymph node metastasis, histological grade, etc.). Combining with other methods can improve the accuracy of the prognosis prediction method of the present invention.

[0082] Furthermore, there are provided methods for treating cancer and methods for improving the prognosis of cancer patients, which comprise a step of predicting the prognosis of cancer using the method of the present invention.

[0083] 2. Methods for detecting ovarian cancer, uterine cancer, or thyroid cancer 2-1. Overview In one aspect, the present invention provides a method for detecting ovarian cancer, endometrial cancer, or thyroid cancer. The cancer detection method of the present invention includes a detection step of detecting a biomarker consisting of KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof (hereinafter referred to as a "biomarker such as KLHL14 protein") in a sample such as a body fluid derived from a subject, and the subject is indicated to be likely to have cancer based on the positive / negative determination result of the biomarker. The detection step can be carried out in vitro. The cancer detection method of the present invention enables early diagnosis of cancer.

[0084] 2-2.Definition In this embodiment, the "detection step" is a step of measuring the amount of a biomarker for detecting ovarian cancer, endometrial cancer, or thyroid cancer in a sample derived from a subject, and determining whether the biomarker is positive or negative based on the measurement value (hereinafter referred to as "determination of positive / negative").

[0085] In the cancer detection method of the present invention, a "sample" refers to a sample collected from a subject or a group of subjects, or a healthy subject or a group of healthy subjects, and subjected to the cancer detection method of this embodiment, such as a body fluid. Examples of body fluids include blood (including serum, plasma, and interstitial fluid), lymph, tissue or cell extracts, ascites, pleural effusion, saliva, and urine. Blood may be serum or plasma prepared from blood.

[0086] As used herein, the term "measured value of a biomarker for detecting cancer" specifically refers to the measured amount of a biomarker such as KLHL14 protein in a sample such as a body fluid.

[0087] 2-3. Detection process of KLHL14 in ovarian cancer, uterine cancer, or thyroid cancer The detection step of this embodiment, which comprises (1) measuring a biomarker such as KLHL14 protein, and (2) determining whether a biomarker such as KLHL14 protein is positive or negative, will be described below.

[0088] (1) Measurement of biomarkers such as KLHL14 protein The biomarker to be measured by this method may be either the KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof. Measuring the amount (expression level) includes measuring the presence or absence of expression, or the magnitude of the expression level or expression concentration.

[0089] When the biomarker to be measured is KLHL14 protein or a peptide fragment thereof, the measurement method may be any known protein quantification method, and is not particularly limited, but examples thereof include the above-mentioned immunological detection method, aptamer analysis method, and mass spectrometry method.

[0090] Furthermore, when the biomarker to be measured is a KLHL14 gene transcription product or a nucleic acid fragment thereof, the measurement method may be any known nucleic acid quantification method, and is not particularly limited, but examples thereof include the above-mentioned nucleic acid amplification method using a primer, the hybridization method using a probe, or the RNA sequencing (RNA-Seq) analysis method.

[0091] (2) Determining whether biomarkers such as KLHL14 protein are positive or negative Next, the positive / negative state of a biomarker such as KLHL14 protein in the sample is determined based on the measurement value obtained in (1). The positive / negative state determination in this method is in accordance with "(2) Determination of positive / negative state of a biomarker such as KLHL14 protein" described in the prognosis prediction method above, and therefore will not be described here.

[0092] In the method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention, if a sample is positive for a biomarker such as KLHL14 protein based on the detection result obtained in the detection step, it indicates that the subject is highly likely to have ovarian cancer, endometrial cancer, or thyroid cancer. Conversely, if a sample derived from a subject is negative for a biomarker such as KLHL14 protein, it indicates that the subject is low likely to have cancer.

[0093] In one embodiment, a biomarker for detecting cancer other than the biomarker such as KLHL14 protein may be detected in a sample derived from a subject, and cancer may be detected based on the detection results and the detection results of the biomarker such as KLHL14 protein. Examples of the biomarker for detecting cancer other than the biomarker such as KLHL14 protein and the antibodies for detecting it are as described above.

[0094] According to the method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention, the possibility of a subject having cancer can be determined by examining a sample of body fluids (such as ascites or serum) isolated from the subject. Early diagnosis of cancer is possible based on the results of the method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention, allowing treatment to be initiated at an early stage. If the results of the method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention indicate a high possibility of cancer, the patient may be treated with drug therapy and / or radiation therapy to treat the cancer. Therefore, a method for treating cancer is also provided, comprising administering at least one of drug therapy and radiation therapy to a cancer patient who has been shown to have a high possibility of cancer by the method of the present invention.

[0095] The method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention also provides a method for assisting in cancer detection. The method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention can be used in combination with other methods (e.g., X-ray photography; ultrasound (echo) examinations such as transluminal ultrasound; endoscopy; mammography; palpation; pelvic examination; rectal examination; CT examination; MRI examinations such as pelvic MRI examinations, imaging examinations such as PET-CT examinations; blood tests; pathological examinations such as cytology and histology; tumor marker tests; and / or genetic diagnosis) or other factors (e.g., stage classification, tumor size, presence or absence of lymph node metastasis, histological grade, etc.). Combining with other methods can improve the accuracy of the method for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention.

[0096] 3. A kit for predicting the prognosis of ovarian cancer patients and a kit for detecting ovarian cancer, uterine cancer, or thyroid cancer Overview One aspect of the present invention provides a kit for predicting the prognosis of an ovarian cancer patient and a kit for detecting ovarian cancer, endometrial cancer, or thyroid cancer. The kit for predicting the prognosis of an ovarian cancer patient and the kit for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention comprise, as an essential component, at least one measurement means selected from the group consisting of an antibody or fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof, and are capable of detecting a biomarker for predicting the prognosis of an ovarian cancer patient or a biomarker for detecting ovarian cancer, endometrial cancer, or thyroid cancer. The kit for predicting the prognosis of ovarian cancer of the present invention comprises, as a selected component, an antibody (hereinafter referred to as an "other prognosis prediction antibody") against a cancer prognosis prediction biomarker other than the KLHL14 protein or a peptide fragment thereof (hereinafter referred to as an "other prognosis prediction biomarker").

[0097] 3-2.Required components The kit for predicting the prognosis of ovarian cancer or the kit for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention comprises at least one measurement means selected from the group consisting of an antibody or fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof. Examples of the antibody or fragment thereof include the above-mentioned anti-KLHL14 antibody or fragment thereof. The anti-KLHL14 antibody contained in the kit for predicting the prognosis of ovarian cancer or the kit for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention may be a single type or multiple types.

[0098] 3-3.Selection components The ovarian cancer prognosis kit or ovarian cancer, endometrial cancer, or thyroid cancer detection kit of the present invention may further comprise, as an optional component, one or more other prognostic or detection antibodies, or immunoreactive fragments thereof, primers, probes, or labeled versions thereof. The other prognostic or detection antibodies may be any antibody that, when used in combination with the anti-KLHL14 antibody, can improve the accuracy of prognosis or detection in patients with cancers such as ovarian cancer or endometrial cancer. Antibodies against any cancer prognostic or detection biomarkers can be used. Such biomarkers can be selected from known cancer biomarkers. Examples of known ovarian cancer markers include Cancer Antigen 125 (CA125) and Human Epidemiology Protein 4 (HE4). An example of an antibody against CA125 is CA125 (Leica Biosystems, PA0539). An example of an antibody against HE4 is Recombinant Anti-HE4 antibody (Abcam, ab200828).

[0099] The kit for predicting the prognosis of ovarian cancer or the kit for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention may include, in addition to the above-mentioned essential components, other reagents necessary for predicting the prognosis of ovarian cancer patients or detecting cancer, such as buffers and secondary antibodies, and instructions for use in detection and determining the results.

[0100] 4. Biomarkers for predicting the prognosis of ovarian cancer and biomarkers for detecting ovarian cancer, uterine cancer, or thyroid cancer Overview One aspect of the present invention provides a biomarker for predicting the prognosis of an ovarian cancer patient and a biomarker for detecting ovarian cancer, endometrial cancer, or thyroid cancer. The biomarker for predicting the prognosis of an ovarian cancer patient and the biomarker for detecting ovarian cancer, endometrial cancer, or thyroid cancer of the present invention may include the KLHL14 protein, a peptide fragment thereof, or a transcription product thereof.

[0101] 4-2.Definition As used herein, the term "cancer biomarker" refers to a biomarker that can predict or indicate the prognosis of a cancer patient (a biomarker for predicting cancer prognosis), or a biomarker that can detect cancer (a biomarker for detecting cancer).

[0102] 4-3. Biomarkers for predicting prognosis and detecting cancer in ovarian cancer According to the biomarker for predicting the prognosis of ovarian cancer of the present invention, use of the KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof as a biomarker for predicting the prognosis of ovarian cancer patients is provided.

[0103] The cancer detection biomarkers of the present invention enable highly accurate detection of any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer. For example, early diagnosis or early detection of ovarian cancer, endometrial cancer, or thyroid cancer becomes possible. The present invention also provides the use of the KLHL14 protein or a peptide fragment thereof, or a transcription product of the KLHL14 gene or a nucleic acid fragment thereof as a biomarker for detecting any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer.

[0104] 5. Anti-KLHK14 antibody or fragment thereof Overview In one aspect, the present invention provides an anti-KLHL14 antibody or a fragment thereof. Also provided are an anti-KLHL14 antibody or a fragment thereof for predicting the prognosis of an ovarian cancer patient, and an anti-KLHL14 antibody or a fragment thereof for detecting ovarian cancer, endometrial cancer, or thyroid cancer. The anti-KLHL14 antibody or a fragment thereof of the present invention can predict the prognosis of cancer in a subject by detecting the KLHL14 protein or a peptide fragment thereof that can be expressed in highly malignant ovarian cancer, endometrial cancer, or thyroid cancer. Furthermore, the anti-KLHL14 antibody or a fragment thereof of the present invention can detect cancer by detecting the KLHL14 protein or a peptide fragment thereof that can be expressed in ovarian cancer, endometrial cancer, or thyroid cancer.

[0105] 5-2.Definition The term "anti-KLHL14 antibody" refers to an antibody that is immunoreactive with the KLHL14 protein or a peptide fragment thereof.

[0106] The species from which the anti-KLHL14 antibody of the present invention is derived is not particularly limited. Preferably, the antibody is derived from birds or mammals, such as chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, ropa, sheep, camel, horse, or human.

[0107] The anti-KLHL14 antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody, as long as it recognizes the KLHL14 protein or a peptide fragment thereof and exhibits immunoreactivity. Preferably, it is a monoclonal antibody with a stable antibody titer.

[0108] As used herein, the term "polyclonal antibody" refers to a group of multiple different immunoglobulins that can specifically bind to and recognize an antigen.

[0109] Furthermore, as used herein, the term "monoclonal antibody" refers to a single type of immunoglobulin that contains a framework region (hereinafter referred to as "FR") and CDRs and is capable of specifically binding to and recognizing an antigen, or at least one set of light chain variable regions (V) contained in an immunoglobulin. L region) and heavy chain variable region (V H A recombinant or synthetic antibody includes a target region.

[0110] 5-3. Anti-KLHL14 antibody When the anti-KLHL14 antibody is composed of an immunoglobulin molecule, the immunoglobulin can be of any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0111] The position of the epitope in the KLHL14 protein or a peptide fragment thereof recognized by the anti-KLHL14 antibody of the present invention is not particularly limited. Examples of epitopes recognized by the anti-KLHL14 antibody of the present invention include peptide sequences contained in the amino acid sequence shown in SEQ ID NO: 11 or 20.

[0112] A specific example of an anti-KLHL14 antibody that recognizes the above epitope is the rat anti-KLHL14 monoclonal antibody clone obtained in Example 2, which will be described later. The heavy chain variable region of this antibody clone consists of the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 8. According to Kabat's rules (Kabat EA, et al., 1991, Sequences of proteins of immunological interest, Vol. 1, eds. 5, NIH publication), in the heavy chain variable region of the antibody clone, CDR1 consists of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 3. In addition, in the light chain variable region of the antibody clone, CDR1 consists of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consists of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 consists of the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequences of SEQ ID NOs: 1 to 8 are shown below.

[0113] Amino acid sequence of the heavy chain variable region: MDIRLSLGVLVLFIKGVQCEVQLVESGGGLVQPGRSLKLSCAASGFTFSNYGMHWIRQAPTKGLEWVTSISRSGGFTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCATGISLGGVMDAWGQGASVTVSSAQTTAPSVY (SEQ ID NO: 7) Amino acid sequence of the light chain variable region: MRVQIQFLGLLLLWTSGAQCVVQMTQSPSYLAASPGESVSISCKASKSISNNLAWYQEKPGKANKLLIHSGSALQSGTPSRFSGSGSGTDFTLTIRSLESEDFAVYYCQQYNEYPLTFGSGTKLEIKRADAAPTVSIFPPSME (SEQ ID NO: 8) Amino acid sequence of heavy chain CDR1: NYGMH (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: SISRSGGFTYYRDSVKG (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: GISLGGVMDA (SEQ ID NO: 3) Light chain CDR1 amino acid sequence: KASKSISNNLA (SEQ ID NO: 4) Light chain CDR2 amino acid sequence: SGSALQS (SEQ ID NO: 5) Light chain CDR3 amino acid sequence: QQYNEYPLT (SEQ ID NO: 6)

[0114] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 7, which corresponds to the heavy chain variable region of the anti-KLHL14 antibody obtained in Example 2 below, is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 18. An example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 8, which corresponds to the light chain variable region of the anti-KLHL14 antibody obtained in Example 2, is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 19. Furthermore, examples of nucleotide sequences encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the above antibody clone include nucleic acids consisting of the nucleotide sequences shown in SEQ ID NOs: 12, 13, and 14, respectively. Examples of nucleotide sequences encoding CDR1, CDR2, and CDR3 of the light chain variable region of the above antibody clone include nucleic acids consisting of the nucleotide sequences shown in SEQ ID NOs: 15, 16, and 17, respectively.

[0115] "Recombinant antibody" refers to a chimeric antibody or a humanized antibody. A "chimeric antibody" is an antibody created by combining the amino acid sequences of antibodies derived from different animals, in which the constant region (C region) of one antibody is replaced with the C region of another antibody. For example, an antibody in which the C region of a rat monoclonal antibody is replaced with the C region of a human antibody falls into this category. A specific example is an antibody in which the heavy chain variable region of a human antibody against a given antigen is replaced with the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 of the anti-KLHL14 antibody clone obtained in Example 2 above, and the light chain variable region of the human antibody is replaced with the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. This can reduce the immune response against the antibody in the human body. A "humanized antibody" is a mosaic antibody in which the CDRs of a human antibody are replaced with the CDRs of an antibody derived from a non-human mammal. The variable region (V region) of an immunoglobulin molecule is composed of four FRs (FR1, FR2, FR3, and FR4) and three CDRs (CDR1, CDR2, and CDR3) linked in the following order from the N-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The FRs are relatively conserved regions that form the framework of the variable region, while the CDRs directly contribute to the antigen-binding specificity of the antibody. Humanized antibodies can be constructed as human antibodies that inherit the antigen-binding specificity of a rat antibody clone, for example, by replacing a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a rat-derived antibody clone with a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a human antibody against any antigen. Specific examples include antibodies in which CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, derived from the heavy chain of the rat-derived anti-KLHL14 antibody clone obtained in Example 2 above, are substituted with heavy chain CDR1, CDR2, and CDR3 of a human antibody, respectively, and also antibodies in which CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, derived from the light chain of the aforementioned antibody clone, are substituted with light chain CDR1, CDR2, and CDR3 of a human antibody, respectively.Such humanized antibodies are derived from human antibodies except for the CDRs, and therefore can reduce immune responses against the antibodies in the human body more than chimeric antibodies.

[0116] The term "synthetic antibody" refers to an antibody synthesized chemically or by using recombinant DNA techniques. For example, it includes antibodies newly synthesized using recombinant DNA techniques. Specific examples include scFv (single chain fragment of variable region), diabody, triabody, tetrabody, etc. In an immunoglobulin molecule, a set of variable regions (light chain variable region V) that form a functional antigen-binding site is called a synthetic antibody. L and heavy chain variable region V H ) are located on separate polypeptide chains, known as light and heavy chains. scFv is a type of immunoglobulin molecule that has V L and V H A scFv is a synthetic antibody with a molecular weight of approximately 35 kDa or less, in which two variable regions are linked by a flexible linker of sufficient length and incorporated into a single polypeptide chain. A pair of variable regions within an scFv can self-assemble to form a functional antigen-binding site. An scFv can be obtained by incorporating recombinant DNA encoding it into a vector using known techniques and expressing it. A diabody is a molecule based on the dimeric structure of scFvs (Holliger et al., 1993, Proc. Natl. Acad Sci. USA 90: 6444-6448). For example, if the linker is shorter than approximately 12 amino acid residues, the two variable regions within the scFv cannot self-assemble, but by allowing two scFvs to interact to form a diabody, the V of one scFv can be bound to the V of the other scFv. L is the V of the other scFv HThe scFv fragments can assemble with each other to form two functional antigen-binding sites. Furthermore, adding a cysteine ​​residue to the C-terminus of an scFv allows disulfide bonding between two scFvs, resulting in the formation of a stable diabody. Thus, diabodies are bivalent antibody fragments. Triabodies and tetrabodies, like diabodies, are trivalent and tetravalent antibodies based on the scFv structure, possessing trimer and tetramer structures, respectively. Diabodies, triabodies, and tetrabodies may also be multispecific antibodies. A "multispecific antibody" refers to a multivalent antibody, i.e., an antibody that has multiple antigen-binding sites within a single molecule, each of which binds to a different epitope. For example, a diabody may be a bispecific antibody, in which each antigen-binding site binds to a different epitope. Specifically, for example, in the case of the anti-KLHL14 antibody of the present invention, a diabody in which one antigen-binding site binds to an epitope contained in the amino acid sequence shown in SEQ ID NO: 11 or 20 and the other antigen-binding site binds to an epitope other than the above epitopes is included.

[0117] The anti-KLHL14 antibody of the present invention can also be modified. The term "modification" as used herein includes functional modifications necessary for antigen-specific binding activity, such as glycosylation, and labeling modifications necessary for antibody detection.

[0118] Glycosylation modifications on anti-KLHL14 antibodies are performed to adjust the affinity of the anti-KLHL14 antibody for the target KLHL14 protein or a peptide fragment thereof. Specific examples include modifications such as introducing substitutions into the amino acid residues that make up the glycosylation in the FR of the anti-KLHL14 antibody to remove the glycosylation site, thereby eliminating glycosylation at that site.

[0119] The anti-KLHL14 antibody can be labeled with, for example, fluorescent dyes (FITC, rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (e.g., PE, APC, GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), radioisotopes (e.g., 3 H, 14 C. 35 Examples of labeling include labeling with biotin or (strept)avidin.

[0120] The anti-KLHL14 antibody of the present invention has a dissociation constant with the KLHL14 protein of 10 -7 It is preferable that the value is equal to or less than M, for example, 10 -8 It is preferable that the affinity is high, ie, 10 M or less, and more preferably 10 -9 M or less, particularly preferably 10 -10 The dissociation constant is equal to or less than M. The dissociation constant can be measured using techniques known in the art. For example, it may be measured using a Biacore system (GE Healthcare) with rate evaluation kit software.

[0121] 5-4.The fragment As used herein, the term "fragment thereof" refers to an antibody fragment that consists of a portion of an anti-KLHL14 antibody and exhibits immunoreactivity to the KLHL14 protein or a fragment thereof, similar to the anti-KLHL14 antibody. Examples include Fab, F(ab'), Fab, etc.

[0122] Fab is an antibody fragment generated by cleavage of an IgG molecule with papain at the N-terminal side of the disulfide bond in the hinge region, and contains the heavy chain constant region (hereinafter referred to as C H The three domains (C H 1. C H 2. C H 3) V H C adjacent to H l and V H , and a full-length L chain.

[0123] F(ab')2 is a dimer of Fab' produced by pepsin cleavage of an IgG molecule at the C-terminal side of the disulfide bond in the hinge region. Fab' has a slightly longer heavy chain than Fab due to the inclusion of the hinge region, but has essentially the same structure as Fab. Fab' can be obtained by reducing F(ab')2 under mild conditions to cleave the disulfide bond in the hinge region. Since all of these antibody fragments contain an antigen-binding site, they have the ability to specifically bind to an antigen epitope.

[0124] 5-5. Preparation of anti-KLHL14 antibody The anti-KLHL14 antibodies of the present invention can be obtained by conventional methods in the art. Furthermore, if the amino acid sequence of a monoclonal antibody is known, it can also be prepared by chemical synthesis or recombinant DNA technology based on the amino acid sequence. Furthermore, monoclonal antibodies can also be obtained from hybridomas that produce the antibodies.

[0125] The antigenic peptide that can be used as an immunogen for the anti-KLHL14 antibody of the present invention is any part of the KLHL14 protein (hereinafter referred to as a "KLHL14 antigenic peptide"). For example, an example of an antigenic peptide that can be used as an immunogen for the anti-KLHL14 antibody of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 11 or 20. The KLHL14 antigenic peptide can be prepared, for example, using chemical synthesis or DNA recombinant technology.

[0126] 6. Polynucleotide encoding anti-KLHK14 antibody or fragment thereof, vector containing same, and host cell One aspect of the present invention provides a polynucleotide encoding the antibody or fragment thereof according to the present invention. Another aspect of the present invention provides a vector containing the polynucleotide according to the present invention. Another aspect of the present invention provides a host cell containing the vector according to the present invention.

[0127] The polynucleotides of the present invention encode antibodies or fragments thereof comprising heavy chain CDRs identified as SEQ ID NOs: 1-3 or light chain CDRs identified as SEQ ID NOs: 4-6. Nucleotide sequence encoding the amino acid sequence of heavy chain CDR1: AACTATGGCATGCAC (SEQ ID NO: 12) Nucleotide sequence encoding the amino acid sequence of heavy chain CDR2: TCCATTAGTCGTAGTGGTGGTTTCACTTACTATCGAGACTCCGTGAAGGGC (SEQ ID NO: 13) Nucleotide sequence encoding the amino acid sequence of heavy chain CDR3: GGAATAAGCCTAGGGGGTGTTATGGATGCC (SEQ ID NO: 14) Nucleotide sequence encoding the amino acid sequence of the light chain CDR1: AAGGCAAGTAAGAGCATTAGCAATAATTTAGCC (SEQ ID NO: 15) Nucleotide sequence encoding the amino acid sequence of light chain CDR2: TCTGGGTCAGCTTTGCAATCT (SEQ ID NO: 16) Nucleotide sequence encoding the amino acid sequence of light chain CDR3: CAACAGTATAATGAATACCCGCTCACG (SEQ ID NO: 17)

[0128] In one embodiment, the polynucleotide of the present invention is a polynucleotide encoding the heavy chain or light chain of an IgG antibody, wherein the polynucleotide encoding the heavy chain is SEQ ID NO: 12, or the polynucleotide encoding the light chain is SEQ ID NO: 13. Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region: ATGGACATCAGGCTCAGCTTGGGTGTCCTTGTCCTTTTCATAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTAACTATGGCATGCACTGGATCCGCCAGGCTCCAACGAAGGGGCTGGAGTGGGTCACATCCATTAGTCGTAGTGGTGGTTTCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGATAATGCAAAAAGCACCCTATACCTGCAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTGCAACAGGAATAAGCCTAGGGGGTGTTATGGATGCCTGGGGTCAAGGAGCTTCAGTCACTGTCTCCTCAGCCCAAACAACAGCCCCATCTGTCTATCC (SEQ ID NO: 18) Nucleotide sequence encoding the amino acid sequence of the light chain variable region: ATGAGGGTCCAGATTCAGTTTCTGGGGCTCCTTCTGCTCTGGACATCAGGTGCCCAGTGTGTTGTCCAGATGACCCAGTCTCCATCTTATCTTGCTGCGTCTCCTGGAGAAAGTGTTTCCATCAGTTGCAAGGCAAGTAAGAGCATTAGCAATAATTTAGCCTGGTATCAGGAGAAACCTGGGAAAGCAAATAAGCTTCTTATTCACTCTGGGTCAGCTTTGCAATCTGGAACTCCATCGAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACGCTCACCATCAGAAGCCTGGAGTCTGAAGATTTTGCAGTCTATTACTGTCAACAGTATAATGAATACCCGCTCACGTTCGGTTCTGGGACCAAGCTGGAGATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCATGGAAC (SEQ ID NO: 19)

[0129] A transformant can be produced by introducing this polynucleotide or a vector containing the polypeptide into a host cell. The polynucleotide or vector may be constructed to be capable of expressing an anti-KLHL14 antibody. The polynucleotide or vector may contain components necessary for protein expression, such as a promoter, enhancer, and replication origin, and / or a screening gene, such as an antibiotic resistance gene. The polynucleotide or vector may also contain a heterologous nucleotide sequence. The heterologous nucleotide sequence may contain, for example, nucleotide sequences derived from two or more organisms selected from the group consisting of humans and organisms other than humans (e.g., bacteria, archaea, yeast, insects, birds, viruses, or mammals other than humans).

[0130] The above-mentioned vector is not limited as long as it is capable of expressing a protein in a host cell, and examples that can be used include plasmids derived from Escherichia coli (e.g., pET-Blue), Bacillus subtilis (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5 / His-TOPO, pcDNA 3.1(-) Mammalian Expression Vector, pcDNA 3.1 / Hygro(-) Mammalian Expression Vector), bacteriophages such as λ phage, and virus-derived vectors. The vector may be an expression vector or may be circular.

[0131] Another aspect of the present invention provides a host cell transformed with the expression vector according to the present invention. The "host cell" may be a cell of a human or a non-human mammal (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human embryonic kidney cells (e.g., HEK293 cells), and mammalian cells Expi293. The host cell may also be Escherichia bacteria, yeast, etc. Methods for introducing the above-mentioned polynucleotides or vectors into cells are known, and can be, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, microinjection, etc. Methods for producing antibodies using cells are also known, and involve culturing cells under culture conditions suitable for the host cells to produce and recover antibodies.

[0132] Another aspect of the present invention provides a method for producing an antibody, comprising the steps of culturing the host cell according to the present invention and collecting the antibody of interest from the culture obtained in the step. In one embodiment, the method for producing the antibody may also include the step of preparing a cell culture medium and the step of purifying the anti-KLHL14 antibody.

[0133] Methods for purifying antibodies are known, and include, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, protein L, gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography.

[0134] 6. Cancer treatment Overview In one aspect, the present invention provides a cancer therapeutic agent. The cancer therapeutic agent of the present invention contains, as an active ingredient, an anti-KLHL14 antibody or a fragment thereof conjugated to an antitumor agent, and can treat cancers such as ovarian cancer, endometrial cancer, and thyroid cancer in cancer patients. The anti-CLDN16 antibody or a fragment thereof in this aspect is similar to the descriptions above in "(1) Anti-KLHL14 antibody" and "(2) Fragment thereof," and detailed description here is omitted.

[0135] The antibody can be bound to the antitumor agent via a spacer having a group reactive with an amino group, a carboxyl group, a hydroxyl group, a thiol group, etc. (e.g., a succinimidyl group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, a hydroxyl group, etc.).

[0136] Antitumor agents include known antitumor agents and pharmaceutically acceptable salts or derivatives thereof. Examples of known antitumor agents include paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and the like. ramide), trimethylolomelamine, bullatacin, bullatacinone, camptothecin, bryostatin, callystatin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estradiol Mustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin ), dynemycin, clodronate, esvelamycin, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, adriamycin, epirubicin,Esorubicin, idanorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate imetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens, calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, florinic acid acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, lentinan, lonidamine, maytansine, ansamitocin, mitoguazone, mitoxantrone,Mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbacine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, Triaziquone, roridine A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, oxaliplatin, galactosamine ... Examples of such anti-cancer drugs include boplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitors, difluoromethylolnitine (DMFO), retinoic acid, and capecitabine.

[0137] The present invention also provides a composition for treating cancer, which contains, in addition to the above-mentioned cancer therapeutic agent, other cancer therapeutic agents and a pharmaceutically acceptable carrier.

[0138] The term "pharmaceutically acceptable carrier" refers to additives commonly used in the technical field of formulation, such as solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, excipients, vehicles, preservatives, binders, diluents, isotonicity adjusters, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0139] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0140] The above-mentioned carriers are used to avoid or suppress the degradation of the active ingredient, anti-KLHL14 antibody or its fragment, by enzymes, etc. in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and efficacy, and may be used appropriately as needed.

[0141] The subjects to which the cancer therapeutic agent or composition of the present invention is administered are mammals, including, for example, primates, pet animals, livestock, and sport animals, with humans being particularly preferred.

[0142] There are no particular limitations on the preferred administration form of the cancer therapeutic agent or cancer therapeutic composition of the present invention. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, and administration via blood transfusion. The administration form can be appropriately selected depending on the age, weight, sex, symptoms, etc. of the patient.

[0143] The dosage of the antibody or a fragment thereof can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration, or from the range of 0.001 mg / body to 100,000 mg / body per patient, but is not necessarily limited to these values.

[0144] The cancer therapeutic agent or composition of the present invention may be administered in a single dose or multiple doses. In the case of multiple doses, the agent or composition may be administered daily or at appropriate intervals (e.g., every 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the antibody or fragment thereof in the cancer therapeutic agent or cancer therapeutic composition can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be, for example, any amount within the range of 0.01 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 The dose may be selected appropriately from the range of 1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg.

[0145] The present invention will be specifically described below with reference to examples, but these examples are merely illustrative and the present invention is not limited to the scope described in the examples. [Example]

[0146] Example 1: Identification of proteins specifically expressed on the surface of ovarian cancer cells (the purpose) To discover new biomarkers for ovarian cancer, we will perform a comprehensive analysis of proteins expressed throughout the cells, including the cell surface, of ovarian cancer cell lines, and identify proteins detected only on the cell surface as candidate biomarkers.

[0147] (Methods and Results) We identified proteins expressed throughout the cell, including the cell surface, in the human ovarian cancer cell lines OVCAR3 and SKOV3. Two ovarian serous carcinoma-derived cell lines, OVCAR3 (RIKEN BioResource Research Center, RCB2135) and SKOV3 (American Type Culture Collection, HTB-77), were used for analysis. Cell surface proteins were identified according to the method described previously (Taguchi A. et al., Cancer Cell, 2011, 20(3), 289-299) (Figure 1). Specifically, cell surface proteins were first biotinylated using EZ-Link® Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) diluted to 0.25 mg / mL in phosphate buffered saline (PBS). The cells were then suspended in PBS containing 3% n-octyl-β-D-glucoside (Dojin Chemical Laboratory) (3% OG solution), sonicated, and centrifuged at 15,000 rpm for 30 minutes at 4°C to extract the protein. Streptavidin-conjugated Sepharose beads (Cytiva) were then suspended in the resulting protein and incubated at 4°C for 4 hours with gentle agitation. The precipitated Sepharose beads were then centrifuged at 2,000 rpm at 4°C and washed twice with 3% OG solution. Finally, 1 mL of reducing solution (3% OG solution containing 100 mM Tris-HCl, pH 8.0, and 7 mM dithiothreitol) was added and gently mixed overnight at 4°C to purify the biotin-labeled protein (hereafter referred to as "cell surface protein"). On the other hand, ovarian cancer cell lines were sonicated with an extractant (PBS containing 4 M urea and 3% OG), and then centrifuged at 15,000 rpm at 4°C for 30 minutes to extract all proteins contained in the ovarian cancer cell lines (hereafter referred to as "total cellular proteins"). The proteins contained in each cell line were digested into peptides consisting of several dozen amino acids using trypsin after cleavage of disulfide bonds, and then comprehensively identified using a mass spectrometer.

[0148] The results of mass spectrometry are shown in Table 1 below, which shows the total number of types of whole cell proteins and cell surface proteins identified from each of OVCAR3 and SKOV3. [Table 1]

[0149] We found KLHL14 (KELCH-LIKE 14) protein, which is common to 5,762 cell surface proteins of OVCAR3 and 5,494 cell surface proteins of SKOV3, but was not identified as a whole-cell protein in either cell line.

[0150] The results of the KLHL14 protein in the above mass spectrometry are shown in Table 2 below. In both OVCAR3 and SKOV3, KLHL14 protein was specifically detected in cell surface proteins. On the other hand, KLHL14 protein was not detected in total cellular proteins, even though total cellular proteins may include cell surface proteins. This result suggests that it is difficult to detect trace proteins in total cellular proteins, and that detection of KLHL14 protein can only be achieved by concentrating and purifying cell surface proteins.

[0151] [Table 2] These results identify KLHL14 protein as a new candidate biomarker for ovarian cancer.

[0152] <Example 2: Examination of KLHL14 gene expression in cancer tissues and normal tissues> (the purpose) To verify that KLHL14 can be a specific biomarker for ovarian cancer, we will examine the expression of the KLHL14 gene in various cancer tissues and normal tissues.

[0153] (Methods and Results) The expression data for the KLHL14 gene in various cancer tissues and normal tissues were collected from The Cancer Genome Atlas database and The Genotype-Tissue Expression database.

[0154] Figure 2(A) shows the expression levels of the KLHL14 gene in various cancer tissues (ovarian cancer, thyroid cancer, endometrial cancer, uterine carcinosarcoma, cervical adenocarcinoma, esophageal cancer, gastric adenocarcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, bile duct carcinoma, pancreatic adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, malignant mesothelioma, head and neck squamous cell carcinoma, thymoma, renal cell carcinoma, adrenal carcinoma, bladder cancer, breast cancer, prostate cancer, glioblastoma, low-grade glioma, testicular germ cell tumor, cutaneous melanoma, uveal melanoma, bone and soft tissue sarcoma, B-cell lymphoma, and acute myeloid leukemia). Strong expression of the KLHL14 gene was detected in ovarian cancer, and mild to moderate expression was observed in thyroid cancer and endometrial cancer. However, its expression was barely detected in other cancers. Therefore, it was demonstrated that the KLHL14 gene can be specifically expressed in ovarian cancer among various cancer types.

[0155] Figure 2(B) shows the expression levels of the KLHL14 gene in various adult normal tissues (lung, bladder, ovary, endometrium, uterine body, cervix, mammary gland, small intestine, prostate, esophagus, large intestine, stomach, adrenal gland, brain, heart, spleen, salivary gland, thyroid, kidney, testis, spleen, and liver). KLHL14 gene expression was detected at very low levels in normal thyroid tissue, but was not detected in various other normal tissues. These results indicate that KLHL14 can be a marker capable of detecting ovarian cancer specifically and with high sensitivity.

[0156] <Example 3: Preparation of anti-KLHL14 monoclonal antibody> (the purpose) Develop a monoclonal antibody (anti-KLHL14 monoclonal antibody) that can specifically and sensitively detect KLHL14 protein.

[0157] (Methods and Results) The monoclonal antibodies were produced according to the following procedure based on the method described in Kishiro Y, et al., 1995, Cell Struct Funct, 20(2):151-6.

[0158] (1) Design of antigen peptides The antigen peptide was selected from a region of the BTB domain of KLHL14 that shows low homology within the KLHL14 family (Figure 3). Specifically, a peptide consisting of an amino acid sequence (CPDDKLLTSPRAINNL: SEQ ID NO: 20) in which a cysteine ​​was added to the N-terminus of the amino acid sequence (PDDKLLTSPRAINNL: SEQ ID NO: 11) corresponding to positions 111 to 125 of the human KLHL protein (initial methionine is at position 1) shown in SEQ ID NO: 9 was used.

[0159] (2) Preparation of antigen peptides A 10 mg / mL KLH solution was prepared by dissolving 2 mg of Imject™ Maleimide Activated mcKLH (Thermo Fisher Scientific) in 200 μL of ultrapure water. The antigen peptide was dissolved in ultrapure water to prepare a 5 mg / mL antigen peptide solution. 200 μL of each KLH solution and antigen peptide solution were mixed and left to stand at room temperature for 2 hours. To remove the EDTA from the KLH solution, the mixture was transferred to a boiled dialysis membrane and dialyzed against PBS as the external solution. The resulting solution was used as the antigen solution. 500 μL of the antigen solution was mixed with 1 mL of Freund's complete adjuvant (Sigma-Aldrich) using a 2 mL Luer-lock glass syringe and emulsified to prepare an antigen emulsion.

[0160] (3) Immunity Anesthetized 8-week-old female rats (Wistar strain) were immunized by injecting 100 μL of the antigen emanation into both hind legs.

[0161] (4) Cell fusion and cell culture Iliac lymph nodes were excised from rats 14 days after immunization and placed in a sterile dish with 1 mL of DMEM. The lymph nodes were cut into small pieces with scissors and strained with a 100 μm centrifugal strainer (BD Falcon). Approximately 10 7 100 μL of mouse multiple myeloma cell line SP2 was added to the cells, mixed thoroughly with a pipette, and centrifuged at 1,200 rpm / min for 5 minutes. The supernatant was then aspirated. A 37°C PEG solution (50% solution, Merck Millipore) was slowly added dropwise over approximately 1 minute, then allowed to stand for 2 minutes. 9 mL of DMEM medium was then slowly added dropwise over 5 minutes. The cells were centrifuged at 900 rpm / min for 5 minutes, and the supernatant was aspirated. 40 mL of hybridoma medium (78% GIT medium [Wako Fujifilm Industries Co., Ltd.], 2% HAT Supplement [Thermo Fisher Scientific], 10% BM Condimmed H1 Hybridoma Cloning Supplement [Roche], 10% fetal bovine serum) was added, and 100 μL of the mixture was seeded into four 96-well culture dishes and cultured in a CO2 incubator at 37°C.

[0162] (6) Screening After 7 days of culture, the medium was replaced with 100 μL of hybridoma medium. Two days after the medium replacement, 50 μL of the culture supernatant was used to screen for positive clones by ELISA. Screening was performed as follows.

[0163] First, a peptide solution containing the aforementioned antigen peptide solution was adjusted to 3 μg / mL, and 50 μL was added to each well of a 96-well ELISA plate and allowed to stand overnight at 4°C. The antigen peptide solution was then removed from each well, and the plate was washed once with 200 μL of 0.1% Tween 20-containing Tris-HCl buffer (TBS-T). 200 μL of blocking solution (1% bovine serum albumin / TBS) was added, and the plate was allowed to stand at 37°C for 1 hour. The blocking solution was then removed from each well, and the plate was washed once with 200 μL of TBS-T. 50 μL of culture supernatant was added, and the plate was allowed to stand at 37°C for 1 hour. After removing the culture supernatant from each well, the plate was washed three times with 200 μL of TBS-T. Next, 50 μL of ECL™ Rat IgG, HRP linked whole antibody (Cytiva) diluted 2,000-fold with blocking solution was added to each well as a secondary antibody, and the reaction was allowed to proceed at 37°C for 1 hour. After that, the secondary antibody solution was removed from each well, and the wells were washed three times with TBS-T. After that, color development was carried out using TMB Substrate Set (BioLegend) according to the method recommended by the manufacturer, and the absorbance (OD ) at a wavelength of 490 nm was measured. 490 ) was measured.

[0164] Positive clones were subcultured in 6-well culture dishes, and then transferred to 10-cm culture dishes when the confluency reached approximately 50% for further proliferation. As a result of screening, positive signals were detected in 27 of 111 clones.

[0165] <Example 4: Verification of antigen specificity of anti-KLHL14 monoclonal antibody> (the purpose) The antigen specificity of the anti-KLHL14 monoclonal antibody obtained in Example 3 will be verified by immunohistochemical staining.

[0166] (Methods and Results) A vector expressing the human wild-type KLHL14 protein consisting of the amino acid sequence shown in SEQ ID NO:9 was transiently transfected into HEK293T cells using polyethyleneimine "MAX" (24765-1, Cosmo Bio). Two days later, the cells were harvested by scraping (referred to as HEK293T+KLHL14 cells). The cells were fixed with 10% neutral formalin at 4°C for 16 hours and washed once with 1 mL of PBS. The cell mass was gently suspended in 1 mL of 1% sodium alginate solution using a 1 mL tip with the tip cut off, and the cells were pelleted by centrifugation at 2,000 rpm for 5 minutes. The supernatant was then removed, and 1 mL of 1 M calcium chloride solution was added to gel the cell pellet. The gelled pellet was then placed in a cassette and formalin-fixed, paraffin-embedded blocks were prepared using an automated fixation and embedding system (Tissue-Tek VIP® 5 Jr, Sakura Finetech Japan) and a paraffin-embedded block preparation system (Tissue-Tek® TEC™, Sakura Finetech Japan). The blocks were sectioned to a thickness of 3 μm using a sliding microtome, mounted on anti-peeling coated glass slides (CRE-12, Matsunami Glass Industry Co., Ltd.), and allowed to dry at room temperature.

[0167] The specimens were deparaffinized in xylene for 10 minutes, followed by dexylene in 100% ethanol. Endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide / methanol for 10 minutes, followed by a 5-minute wash in phosphate-buffered saline (PBS). Antigen retrieval was performed for 10 minutes in a microwave-controlled 10 mM Tris / 1 mM ethylenediaminetetraacetic acid solution (pH 9.0). After returning to room temperature, the specimens were washed in PBS for 5 minutes. The specimens were then incubated with 0.5% casein (C3400, Merck Millipore) diluted in Tris-HCl-buffered saline (TBS) for 10 minutes at room temperature to prevent nonspecific reactivity of the primary antibody. The specimens were then incubated overnight at 4°C with anti-KLHL14 monoclonal antibody clone #24 as the primary antibody. The next day, the specimens were washed three times with PBS for 5 minutes each and incubated with an anti-rat polymer-conjugated secondary antibody (Histofine Simple Stain MAX-PO (Rat), Nichirei Biosciences) for 1 hour at room temperature. After washing three times with PBS for 5 minutes, the specimens were incubated in a color development solution (DAB Substrate Kit, Nichirei Biosciences) until appropriate staining was achieved. For nuclear staining, the specimens were shaken in Tissue-Tek® Hematoxylin 3G (Sakura Finetech Japan Co., Ltd.) solution for 5 seconds and then washed in running water for 10 minutes. Excess hematoxylin was then removed by shaking in a 0.5% hydrochloric acid / 70% ethanol solution for 5 seconds. The specimens were dehydrated in 100% ethanol, cleared with xylene, and then prepared in an automatic coverslipper (Shiraimatsu Kikai Co., Ltd.). Using a similar method, immunostaining was also performed on HEK293T cells without the KLHL14 protein expression vector.

[0168] The results of immunostaining using the 12 established anti-KLHL14 monoclonal antibody clones are shown in Figure 4. No KLHL14-positive signal was observed in HEK293T cells that had not been transfected with a KLHL14 protein expression vector. In contrast, a strong KLHL14-positive signal was observed in HEK293T+KLHL14 cells that had been transfected with a KLHL14 protein expression vector. When these 12 clones were tested for their potential for Western blotting, five clones, clones #1, #14, #24, #29, and #52, specifically detected the KLHL14 protein (Figure 5).

[0169] Based on the above results, clone #24 was selected as the main subject of analysis in the following examples as a clone with significant advantages, namely high reactivity in ELISA and the ability to specifically and sensitively detect KLHL14 protein in immunostaining and Western blotting.

[0170] We also performed studies using the ovarian cancer cell line OVCAR3. OVCAR3 cells were transiently transfected with a vector expressing a protein (HA-KLHL14) consisting of the human wild-type KLHL14 protein (SEQ ID NO: 9) with an HA tag added to its N-terminus (polyethyleneimine "MAX" (24765-1, Cosmo Bio)). These cells are referred to as OVCAR3+KLHL14 cells. Two days later, the cells were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries) for 10 minutes. After washing three times with PBS, the cell membrane was permeabilized for 10 minutes with 0.1% Triton X-100 diluted in PBS. The cells were then washed three times with PBS and incubated with 5% skim milk diluted in PBS for 30 minutes to prevent nonspecific antibody adsorption. The primary antibody reaction was performed overnight at 4°C using anti-KLHL14 antibody clone #24 and anti-hemagglutinin (HA) tag antibody (clone HA-7, Abcam). The cells were washed three times with PBS and then incubated with Alexa488-labeled anti-rat IgG antibody and Cy3-labeled anti-mouse IgG antibody for 1 hour. After washing three times with PBS, the cells were mounted using a water-soluble mounting medium and the fluorescent signals from both antibodies were observed using a confocal laser scanning biological microscope (FV-1000, Olympus). The results are shown in Figure 6. GFP signals derived from the vector and positive immunostaining signals using the anti-HA tag antibody and anti-KLHL14 antibody clone #24 were observed in the same OVCAR3+KLHL14 cells. This indicates that the KLHL14 antibody reacts only with cells transfected with the vector, demonstrating that anti-KLHL14 antibody clone #24 can be used for fluorescent immunostaining and can specifically detect overexpressed KLHL14 protein.

[0171] Furthermore, flow cytometry analysis was performed using HEK293T+KLHL14 cells. A vector expressing human wild-type KLHL14 protein consisting of the amino acid sequence shown in SEQ ID NO:9 was transiently transfected into HEK293T cells using polyethyleneimine "MAX" (24765-1, Cosmo Bio). Two days later, the cells were scraped and harvested using trypsin (Fujifilm Wako Pure Chemical Industries). First, the cells were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries) for 10 minutes. Next, the cells were washed three times with PBS and then permeabilized with 0.1% Triton X-100 diluted in PBS for 10 minutes. The cells were then washed three times with PBS and incubated with 5% skim milk diluted in PBS for 30 minutes to prevent nonspecific adsorption of the antibody. The primary antibody reaction was performed using anti-KLHL14 antibody clone #24 and a rat isotype control IgG antibody (Fujifilm Wako Pure Chemical Industries) as a negative control, and the reaction was carried out at room temperature for 1 hour. The cells were washed three times with PBS and then incubated with Alexa488-labeled anti-rat IgG antibody for 1 hour. After washing the cells three times with PBS, the cells were analyzed using a flow cytometer (On-Chip Biotechnologies). The results are shown in Figure 7. HEK293T+KLHL14 cells showed a significant change in signal response to the anti-KLHL14 antibody clone #24 compared to the rat isotype control IgG antibody, and flow cytometry analysis also demonstrated that this antibody can specifically detect the KLHL14 protein expressed in HEK293T cells.

[0172] <Example 5: CDR sequencing of anti-KLHL14 monoclonal antibody clone #24> (the purpose) The CDR sequences of anti-KLHL14 monoclonal antibody clone #24 are determined.

[0173] (Methods and Results) The sequences of the heavy and light chain variable regions and CDRs of anti-KLHL14 monoclonal antibody clone #24 were determined. Specifically, the hybridoma was first mixed with 1 mL of TRIzol® reagent (Thermo Fisher Scientific) and 200 μL of chloroform and centrifuged at 15,000 rpm at 4°C for 15 minutes. Next, an equal volume of isopropanol was mixed with the aqueous phase containing the mRNA and centrifuged at 15,000 rpm at 4°C for 15 minutes to extract the mRNA. Finally, 1 mL of 70% ethanol was added to desalt the mRNA, which was then dissolved in 50 μL of water. The mRNA concentration was then quantified using a NanoDrop® 2000.

[0174] The 5'-Full RACE Core Set (Takara Bio Inc.) was used to determine the CDRs. All experiments were performed using the instructions and accompanying reagents. The resulting DNA fragments were cloned into the pGEM®-T Easy Vector (Promega) and then subjected to DNA sequencing. DNA sequencing was performed by Macrogen Japan Co., Ltd., and CDRs were identified according to the Kabat antibody numbering system.

[0175] The sequences of the heavy and light chain variable regions and CDRs of anti-KLHL14 monoclonal antibody clone #24 were determined, and the results are shown below. Amino acid sequence of the heavy chain variable region: EVQLVETGGDLVQPGKSLKLTCATSGFTFTAAWMHWLRQSPDKRLEWIARIKDKSNNYATDYVESVKGRFTISRDDSKSCVYLQMNSLKEEDTATYYCTSGGFAYWGQGTLVTVSSAQTT (SEQ ID NO: 7) Amino acid sequence of the light chain variable region: DVVMTQTPPSLSVAIGQSVSISCKSSQSLVYRDGKTYLHWLLQSPDRSPKRLIYQVSNLGSGVPDRFSGTGSQKDFTLKISRVEAEDLGVYYCAQTTHLYTFGAGTKLELKRADAAPTNH (SEQ ID NO: 8) Heavy chain CDR1 amino acid sequence: ATSGFTFTAAWMH (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: RIKDKSNNYATD (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: TSGGFAY (SEQ ID NO: 3) Light chain CDR1 amino acid sequence: KSSQSLVYRDGKTYLH (SEQ ID NO: 4) Light chain CDR2 amino acid sequence: YQVSNLGS (SEQ ID NO: 5) Light chain CDR3 amino acid sequence: AQTTHLYT (SEQ ID NO: 6)

[0176] <Example 6: Immunohistochemistry of ovarian cancer tissue, and recurrence-free survival rate analysis and clinicopathological analysis based on the immunohistochemistry> (the purpose) Using anti-KLHL14 monoclonal antibody clone #24, KLHL14 protein will be detected in ovarian cancer tissues, and the relationship between KLHL14 expression and prognosis will be clarified.

[0177] (Methods and Results) (1) Collection of tissue specimens Tissue specimens were collected from three patients who underwent surgery for ovarian cancer at Fukushima Medical University Hospital in 2021, and fresh-frozen tissue specimens were prepared to evaluate KLHL14 expression. The collection of tissue materials was approved by the Fukushima Medical University Ethics Committee (approval number 2021-057) and was carried out in compliance with ethical guidelines for clinical research. Furthermore, tissue specimens were collected from 167 patients who underwent surgery for ovarian cancer at Fukushima Medical University Hospital between 2003 and 2015, and KLHL14 expression was evaluated using formalin-fixed, paraffin-embedded specimens. Only patients with known survival information for 3 to 5 years after diagnosis were included, and deaths unrelated to the underlying disease were excluded. The collection of tissue samples was approved by the Fukushima Medical University Ethics Committee (approval number 2019-0311) and was conducted in compliance with ethical guidelines for clinical research.

[0178] (2) Evaluation of KLHL14 localization by immunofluorescence staining Ovarian cancer tissue samples collected from patients diagnosed with ovarian cancer and undergoing surgery in 2021 were immediately minced into 1 mm cubes using a surgical scalpel, embedded in OCT compound (Sakura Finetech Japan), and then sliced ​​into 4 μm slices using a Cryostar NX70 (Thermo Fisher) to prepare fresh-frozen tissue specimens. Fluorescent immunostaining was performed using a modified version of the method described in Example 4, "Evaluating the specificity of anti-KLHL14 antibody clone #24 using OVCAR3." Specifically, anti-KLHL14 clone #24 and anti-occludin antibody (clone 3F10, Zymed) were used as primary antibodies. The results are shown in Figure 8. KLHL14 signals were observed at the cell boundaries of ovarian cancer tissue in all three cases examined, consistent with occludin signals. This indicates that KLHL14 expression is concentrated on the cell surface or cell membrane in ovarian cancer tissue.

[0179] (3) Immunohistochemical staining and evaluation of KLHL14 expression All ovarian cancer tissues collected between 2003 and 2015 were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin eosin (HE) and immunohistochemically as described in Example 4.

[0180] After masking patient characteristics such as outcome, two pathologists semiquantitatively evaluated staining using a modified immunoreactive score (IRS; Remmele et al., 1986, Virchows Arch, 409: 127-147). Specifically, the percentage of positive cells was classified as follows: Percentage of Positive Cells (PP) = 0 (<1%), PP = 1 (1%-10%), PP = 2 (11%-30%), PP = 3 (31%-50%), and PP = 4 (≥ 51%). Staining intensity was then classified as Signal Intensity (SI) = 0 (none), SI = 1 (weak), SI = 2 (moderate), and SI = 3 (strong). The IRS was calculated by multiplying PP and SI. Furthermore, based on the receiver operating characteristic (ROC) curve, IRS 0 / 1 / 2 / 3 / 4 were classified as the low KLHL14 group, and IRS 6 / 8 / 9 / 12 were classified as the high KLHL14 group. Representative examples of KLHL14 staining results (negative, weakly positive, moderately positive, and strongly positive) in ovarian cancer tissues are shown in Figure 9. Figure 10 shows the results of evaluation of IS, PP, and IRS.

[0181] (4) Analysis of recurrence-free survival rate Next, disease-specific survival rates and relapse-free survival rates were calculated for each of the low KLHL14 (60 cases) and high KLHL14 (107 cases) groups using the Kaplan-Meier method. Furthermore, survival rates between the two groups were compared using the log-rank test. All statistical analyses were two-sided and performed using Graphpad Prism and SPSS Statistics. All P values ​​were two-sided, and a P value of less than 0.05 was considered statistically significant. The results of the analysis of disease-specific survival and recurrence-free survival are shown in Figure 11. Compared with ovarian cancer patients in the low KLHL14 group, those in the high KLHL14 group showed significantly lower disease-specific survival and recurrence-free survival rates (DFS, P = 0.019; RFS, P = 0.019). These results indicate that ovarian cancer patients in the low KLHL14 group tend to have a good prognosis after ovarian cancer resection surgery, whereas ovarian cancer patients in the high KLHL14 group tend to have a poor prognosis after ovarian cancer resection surgery.

[0182] (5) Clinicopathological analysis Clinicopathological analysis was performed on 167 ovarian cancer patients. KLHL14 was evaluated based on the IRS score as described above (2), and analysis was performed using StatFlex Ver. 7.0.11 (Artec, Osaka). First, the results of analyzing the association between each clinicopathological factor and KLHL14 expression (low or high KLHL14 expression) are shown in Figure 12. High KLHL14 expression showed a significant correlation with stage classification, histological type (clear cell carcinoma), peritoneal dissemination, high levels of cancer antigen 125 (CA125), and recurrence. Next, univariate analysis of recurrence-free survival (DFS) using a logistic regression model showed that high KLHL14 expression was significantly correlated with ovarian cancer recurrence (Figure 13). Furthermore, multivariate analysis using the Cox proportional hazards model showed that high KLHL14 expression was an independent poor prognostic factor.

[0183] <Example 7: Analysis of the mechanism by which KLHL14 affects the malignant traits of ovarian cancer cells> (the purpose) We will evaluate the migration and metastasis of ovarian cancer cells and ovarian cancer cells that overexpress KLHL14 protein or lack KLHL14 protein expression, with the aim of elucidating the mechanism by which KLHL14 promotes ovarian cancer.

[0184] (Methods and Results) A vector expressing a protein (HA-KLHL14) consisting of the human wild-type KLHL14 protein (amino acid sequence shown in SEQ ID NO:9) with an HA tag added to its N-terminus was packaged into lentivirus using HEK293T cells. The vector was then transfected into SKOV3 cells using a polybrene solution (Nacalai Tesque), and two clones of SKOV3 cells stably expressing the KLHL14 protein (referred to as SKOV3:KLHL14) were established (denoted as OE1 and OE2 in Figure 14). As a control experiment, a similar experiment was performed using the expression vector alone, and SKOV3:EGFP cells were established (denoted as WT in Figure 14). On the other hand, we used the CRISPR-Cas9 system to transform SKOV3 cells (SKOV3:KLHL14) in which the expression of human wild-type KLHL14 protein was deleted. - / - Two clones of this mutant (referred to as KO1 and KO2 in Figure 12) were also established. To assess migration, equal numbers of each established cell line were seeded inside a Transwell migration chamber (Corning), and the number of cells that migrated to the outside of the chamber was counted after 24 hours. To assess invasion, equal numbers of each established cell line were seeded inside a Corning™ BioCoat™ Matrigel invasion chamber, and the number of cells that migrated to the outside of the chamber was counted after 24 hours. Cell counts were performed using Image J software with the Transwell counter plugin (J. Vis. Exp. (117), e54719, doi:10.3791 / 54719 (2016)). The results of assessing migration ability are shown in Figure 14A. The number of migrating cells was significantly increased in OE1 and OE2 cells overexpressing KLHL14 protein compared to the WT control, whereas a significant decrease was observed when KLHL14 protein expression was lost. The results of assessing invasion ability are shown in Figure 14B. The number of invasive cells was significantly increased in OE1 and OE2 cells overexpressing KLHL14 protein compared to the WT control, whereas a significant decrease was observed when KLHL14 protein expression was lost. These findings suggest that KLHL14 protein expression plays a central role in promoting the progression of ovarian cancer.

Claims

1. A method for predicting the prognosis of an ovarian cancer patient, comprising: measuring the amount of KLHL14 protein or a peptide fragment thereof, or a transcription product thereof contained in a sample derived from the ovarian cancer patient. A prediction method, including:

2. 2. The prediction method of claim 1, A prediction method in which the prognosis of the ovarian cancer patient is judged to be poor if the measurement value obtained in the measurement step is equal to or greater than a predetermined value.

3. 2. The prediction method of claim 1, A prediction method wherein the sample comprises ovarian tissue.

4. 2. The prediction method of claim 1, A prediction method, wherein the measuring step is a step of measuring the amount of KLHL14 protein or a peptide fragment thereof contained in a sample derived from the ovarian cancer patient.

5. A kit for predicting the prognosis of an ovarian cancer patient, comprising: A kit comprising at least one measuring means selected from the group consisting of an antibody or a fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof.

6. A kit for detecting ovarian cancer, uterine cancer, or thyroid cancer, comprising: A kit comprising at least one measuring means selected from the group consisting of an antibody or a fragment thereof against the KLHL14 protein or a peptide fragment thereof, or a transcription product thereof, a primer, a probe, or a labeled version thereof.

7. An antibody or a fragment thereof that can be used in the prediction method according to claim 1, The antibody has a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and An antibody or a fragment thereof that specifically binds to KLHL14.

8. The antibody or fragment thereof according to claim 7, (a) the amino acid sequence of SEQ ID NO: 7; (b) an amino acid sequence having at least 90% identity to the sequence of the framework region other than each CDR sequence in the sequence of (a); and (c) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (a); a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: (d) the amino acid sequence of SEQ ID NO: 8; (e) an amino acid sequence having at least 90% identity to the sequence of the framework region other than each CDR sequence in the sequence of (d); and (f) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (e); a light chain variable region consisting of an amino acid sequence selected from the group consisting of An antibody or fragment thereof having the formula:

9. A polynucleotide encoding the antibody or fragment thereof according to claim 7 or 8.

10. A vector containing the polynucleotide of claim 9.

11. A host cell transformed with the vector of claim 10.

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