Biomarker and antibody therapy for cancer

Abstract

The present invention addresses the problem of providing: a novel biomarker useful for the prediction of prognosis of a cancer patient or for the detection of cancer; a means capable of specifically recognizing the biomarker; and a novel cancer therapeutic agent.　Provided is a biomarker for cancer, the biomarker comprising claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcript of CLDN16 gene or a nucleic acid fragment thereof. Also provided is an anti-CLDN16 antibody or a fragment thereof, which comprises: a heavy-chain variable region that includes CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; and a light-chain variable region that includes CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6.

Description

Cancer biomarkers and antibody therapy

[0001] The present invention relates to a cancer biomarker, an anti-claudin 16 (CLDN16) antibody or a fragment thereof, a cancer therapeutic agent, and the like.

[0002] Cancer is the leading cause of death in Japan, accounting for approximately 30% of all deaths. Ovarian cancer in particular is one of the gynecological cancers with the poorest prognosis, affecting 11,000 people in Japan and 240,000 people worldwide each year.

[0003] Ovarian cancer is difficult to detect early, and no early diagnostic method has been established. As a result, over 70% of ovarian cancer cases 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 1-3 disclose cancer antigen 125 (CA125), a representative ovarian cancer marker. CA125 is known to be positive in pancreatic cancer, lung cancer, and other cancers in addition to ovarian cancer. 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 4-5 disclose that previous large-scale clinical trials have shown that ovarian cancer diagnosis using CA125 and transvaginal ultrasound does not contribute to extending survival. Non-Patent Document 6 discloses another ovarian cancer marker, human epididymis protein 4 (HE4). While HE4 has higher specificity than CA125, its detection sensitivity is only approximately 50%. Therefore, there is a need for the development of specific and highly sensitive ovarian cancer markers and methods for their specific and sensitive detection.

[0005] Ovarian cancer is also known to have the poorest prognosis among gynecological cancers. Current standard treatment for ovarian cancer involves a combination of surgery and chemotherapy, but more than half of treated patients experience recurrence. The 5-year survival rate for recurrent cases is extremely low, at approximately 20%, making improving the prognosis of ovarian cancer patients a major challenge. However, as with early diagnosis, no effective biomarkers have been identified for prognosis assessment. In particular, there are no prognostic biomarkers for ovarian cancer that are covered by insurance. To improve the prognosis of ovarian cancer patients, the development of biomarkers that can be used to assess prognosis and specific, highly sensitive methods for identifying them is necessary. Furthermore, recently developed ovarian cancer treatments, such as anti-VEGFR (vascular endothelial growth factor receptor) antibodies and PARP (poly ADP-ribose polymerase) inhibitors, have limited efficacy and efficacy, necessitating the development of new therapeutic agents for ovarian cancer.

[0006] Therefore, in order to improve the prognosis of patients with cancers such as ovarian cancer, there is a need for new biomarkers that contribute to the prognosis determination and early diagnosis of cancer, means for recognizing them specifically and with high sensitivity, and new therapeutic agents.

[0007] Bast RCJr., et al., N Engl J Med, 1983, 309(15):883-887.Cwik G., et al., Arch Surg, 2006, 141(10):968-973.Fahrmann JF, et al., JAMA Oncol, 2022, in press. (DOI: 10.1200 / JCO.21.01460)Jacobs IJ, et al., Lancet, 2016, 387(10022):945-956.Menon U., et al., Lancet, 2021, 397(10290):2182-2193.Hellstroem I., et al., Cancer Res, 2003, 63(13):3695-3700.

[0008] An object of the present invention is to provide a new biomarker useful for predicting the prognosis of cancer patients and detecting cancer, a means for specifically recognizing the same, and a new cancer therapeutic agent.

[0009] To solve the above-mentioned problems, the present inventors searched for new cancer biomarkers and found that claudin 16 (CLDN16) protein is highly expressed in ovarian cancer tissues. CLDN16 is expressed in cancer tissues such as ovarian cancer, endometrial cancer, and thyroid cancer, but is barely detectable in normal tissues, including ovarian tissue, making it a highly specific biomarker for cancer. The present inventors further attempted to develop an antibody capable of specifically detecting CLDN16 protein (anti-CLDN16 antibody). From 66 antibody clones obtained by primary screening using ELISA, the present inventors identified one antibody clone capable of detecting CLDN16 protein by immunohistochemical staining of formalin-fixed, paraffin-embedded specimens. The obtained antibody clone did not cross-react with claudin family proteins other than CLDN16 and was found to be capable of specifically and sensitively detecting CLDN16 protein, making it an extremely useful tool for biomarker detection. Furthermore, it was demonstrated that a cancer therapeutic agent utilizing this antibody could be an effective treatment. Based on the above findings, the present invention provides the following:

[0010] (1) A cancer biomarker consisting of claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof. (2) The biomarker according to (1), wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer. (3) The biomarker according to (1) or (2), for predicting the prognosis of a cancer patient suffering from the cancer or for detecting the cancer. (4) An anti-CLDN16 antibody or a fragment thereof, comprising a heavy-chain variable region comprising 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, and a light-chain variable region comprising 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. (5) The anti-CLDN16 antibody or a fragment thereof according to (4), comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. (6) A kit for predicting cancer prognosis or detecting cancer, comprising the anti-CLDN16 antibody or a fragment thereof according to (4) or (5). (7) The kit according to (6), wherein the cancer is ovarian cancer, endometrial cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer. (8) A method for predicting the prognosis of a cancer patient, comprising: a detection step of detecting the biomarker according to (1) in a sample derived from the cancer patient, wherein the sample being positive for the biomarker indicates a poor prognosis for the cancer patient. (9) The method according to (8), wherein the cancer is ovarian cancer, endometrial cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer. (10) The method according to (8) or (9), wherein the biomarker is detected using the anti-CLDN16 antibody or a fragment thereof according to (4).(11) A cancer therapeutic agent comprising an anti-CLDN16 antibody or a fragment thereof as an active ingredient. (12) The cancer therapeutic agent according to (11), wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer. (13) The cancer therapeutic agent according to (11) or (12), wherein the anti-CLDN16 antibody or a fragment thereof comprises a heavy chain variable region comprising 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, and a light chain variable region comprising 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. (14) The cancer therapeutic agent according to (13), wherein the anti-CLDN16 antibody or a fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. This specification incorporates the disclosure of Japanese Patent Application No. 2023-172752, from which the present application claims priority.

[0011] According to the present invention, there are provided new biomarkers useful for predicting the prognosis of cancer patients and detecting cancer, means capable of specifically recognizing the same, and new cancer therapeutic agents.

[0012] Figure 1 shows the expression levels of the CLDN16 gene in various human cancer tissues. Figure 1A shows the expression levels of the CLDN16 gene in various cancer tissues, including ovarian cancer (OV), uterine cancer (UCEC), and thyroid cancer (THCA). Figure 1B shows the expression levels of the CLDN16 gene in ovarian cancer (OV), thyroid cancer (THCA), and other cancer tissues. Figure 2A shows the expression levels of the CLDN16 gene in various normal tissues, including ovary (Ovary), uterus (Uterus), and thyroid (Thyroid). Figure 2B shows the expression levels of the CLDN16 gene in cancer tissues (Cancer (TCGA)) and normal tissues (Normal (GTEx)), including various tissues. Figure 3A shows the location of the antigenic peptide used to generate anti-CLDN16 antibodies. Figure 3B shows an alignment of the amino acid sequences of EC1 of the human claudin family. Amino acid residues identical to those in the CLDN16 protein are shown in bold. Figure 1 shows the antigen specificity of anti-CLDN16 antibodies. The figures show the results of immunohistochemical staining of HEK293T cells ("293T cells") not transfected with a CLDN16 protein expression vector, as well as HEK293T cells expressing the full-length CLDN16 protein ("+CLDN16"), a CLDN16 protein fused with an HA tag at the N-terminus ("+HA-CLDN16"), and a CLDN16 protein fused with an HA tag at the C-terminus ("+CLDN16-HA"), using an antibody that binds to the HA tag (shown as "HA-tag Ab" in the figures) and the anti-CLDN16 antibody of the present invention (shown as "hCLDN16-EC1 Ab" in the figures). The figures show the results of verifying the cross-reactivity of anti-CLDN16 antibodies. 1 shows the results of immunohistochemical staining using an anti-CLDN16 antibody on HEK293T cells expressing CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN8, CLDN9, CLDN12, CLDN19, and CLDN16, respectively. 1 shows the amino acid sequences of the light chain variable region and heavy chain variable region of the anti-CLDN16 antibody.Figure 6A shows the amino acid sequence of the light chain variable region (SEQ ID NO: 8). Figure 6B shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 7). Figure 7A shows the results of Western blotting using an anti-CLDN16 antibody (provided by Dr. Mikio Furuse of the National Institute for Physiological Sciences) to verify that CLDN16 protein is overexpressed in CLDN16-overexpressing ovarian cancer cells (AMOC2 cells). Figure 7B shows the results of evaluating the proliferation ability of the cells. Figure 7B shows the results of evaluating the migration ability of ovarian cancer cells (AMOC2 cells) overexpressing EGFP or CLDN16. Figure 7C shows the results of flow cytometry verifying that the anti-CLDN16 antibody can recognize CLDN16 protein on the surface of live cells. In the figure, "293T" represents HEK293T cells, "293T:EGFP" represents HEK293T cells expressing EGFP, and "293T:CLDN16" represents HEK293T cells expressing CLDN16. "hCLDN16-EC1 Ab" represents the anti-CLDN16 antibody of the present invention. This figure shows the results of verifying that anti-CLDN16 antibodies are taken up into the cells of CLDN16-expressing ovarian cancer cells. In the figure, arrowheads indicate CLDN16 signals on the cell surface, and arrows indicate intracellular CLDN16 signals. "Rat anti-CLDN16-EC1 mAb" represents the anti-CLDN16 antibody of the present invention. This figure shows the cytotoxic effect of anti-CLDN16 antibodies. In the figure, "AMOC:CLDN16" represents CLDN16-expressing ovarian cancer cells, "ADC-rat IgG" represents an anti-rat IgG antibody conjugated with the cytotoxic toxin MMAF (Monomethyl Auristatin F), and "Rat anti-CLDN16-EC1 mAb" represents the anti-CLDN16 antibody of the present invention. Figure 12A shows a representative phase-contrast image. Figure 12B shows the results of measuring absorbance at 490 nm in a cell viability assay. Figure 12B shows representative results of immunohistochemical staining of CLDN16 in various normal tissues.14A shows representative results of immunohistochemical staining of ovarian cancer tissues (CLDN16-negative, CLDN16-weakly positive, CLDN16-moderately positive, and CLDN16-strongly positive cases). Figure 14A shows a negative case. Figure 14B shows a weakly positive case. Figure 14C shows a moderately positive case. Figure 14D shows a strongly positive case. Figure 15A shows the staining intensity (SI), positive rate (PP), and immunoreactivity score (IRS) of 249 ovarian cancer cases. Figure 15B shows the positive rate (PP). Figure 15C shows the immunoreactivity score (IRS). Figure 15B shows the staining intensity (SI). Figure 15C shows the immunoreactivity score (IRS). Figure 15C shows the correlation between clinicopathological factors and CLDN16 expression (low CLDN16 expression / high CLDN16 expression) in ovarian cancer patients. This is a continuation of Figure 16. 18A and 18B are graphs showing Kaplan-Meier curves of recurrence-free survival rates in ovarian cancer patients in the low CLDN16 group (165 cases) and the high CLDN16 group (84 cases). Figure 18A shows overall survival (OS). Figure 18B shows disease-free survival (DFS). Figure 18C shows relapse-free survival (RFS). Figure 18B shows the results of univariate and multivariate analyses of the prognosis (DFS) of ovarian cancer patients based on clinicopathological analysis. Figure 18C shows the results of immunohistochemical staining of frozen mouse kidney tissues using an anti-OCLN antibody and an anti-CLDN16 antibody of the present invention. Figure 18D shows the results of evaluating CLDN16 protein expression in human ovarian cancer cell lines. Figure 21A shows the results of evaluating CLDN16 protein expression in representative human ovarian cancer cell lines, AMOC2 cells, SKOV3 cells, OVCAR3 cells, and OVSAHO cells, using an anti-CLDN16 antibody (provided by Dr. Mikio Furuse of the National Institute for Physiological Sciences). In the figure, "NC" indicates HEK293T cells as a negative control, and "PC" indicates HEK293T cells expressing CLDN16 (positive control). Figure 21B shows the results of Western blotting using an anti-CLDN16 antibody on SKOV3 cells and OVSAHO cells in which the CLDN16 gene was disrupted. This figure shows the cytotoxicity of CLDN16 antibody-drug conjugates.Figure 22A shows the experimental method for evaluating the viability of cells administered with a CLDN16 antibody-drug conjugate using an MTS assay. Figure 22B shows the viability of AMOC2:CLDN16 cells and AMOC2:EGFP cells. Figure 22C shows the viability of OVSAHO:CLDN16 cells. KO Figure 22D shows the viability of SKOV3:CLDN16 cells and control OVSAHO cells. KO The figures show the viability of CLDN16 cells and control SKOV3 cells. In the figure, "Vehicle" indicates a negative control containing no antibody or antibody-drug conjugate, and "IgG" indicates the results of administering rat IgG to the cells. "CLDN16-Ab-MMAE" indicates the results of administering an anti-CLDN16 antibody conjugated with the antitumor agent MMAE to the cells, and "CLDN16-Ab" indicates the results of administering an anti-CLDN16 antibody not conjugated with the antitumor agent MMAE to the cells. This figure shows that the CLDN16 antibody-drug conjugate does not induce nephrotoxicity. MMAE, normal rat IgG, CLDN16 antibody ("CLDN16-Ab" in the figure), and the CLDN16 antibody-drug conjugate ("CLDN16-Ab-MMAE" in the figure) were injected four times into the tail vein of mice, and HE staining of the kidney tissues from these mice is shown.

[0013] (Cancer Biomarker) In one aspect, the present invention provides a cancer biomarker. The biomarker of the present invention consists of a claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof. The use of a claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof, which can be expressed in cancer, as a biomarker enables the determination of the malignancy of cancer, the prediction of the prognosis of cancer patients, and detection, such as early diagnosis, of cancer.

[0014] As used herein, the type of "cancer" is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, glioma, neuroblastoma and other childhood tumors, leukemia, lymphoma, and the like. The cancer is preferably ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer, such as ovarian cancer, uterine cancer, or thyroid cancer. In this specification, colorectal cancer includes colon cancer and rectal cancer.

[0015] 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 cancer originating from the surface epithelium, ovarian stroma, sex cord stroma, or germ cells is 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).

[0016] "Uterine cancer" refers to uterine cancer that develops in the uterine corpus, and is also called endometrial cancer because it originates in 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. Uterine cancer can also be classified into stages I (cancer limited to the uterine corpus), 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).

[0017] 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, poorly differentiated carcinoma, medullary carcinoma, undifferentiated carcinoma, malignant lymphoma, etc. Thyroid cancer can be classified into stage I (cancer limited to the uterine body), stage II (tumor limited to the thyroid gland with a maximum diameter of more than 2 cm but not more than 4 cm), stage III, and stage IV.

[0018] As used herein, "prognosis" refers to the reduction in tumor burden, the inhibition of tumor growth, or the course of the disease (e.g., the presence or absence of recurrence, the presence or absence of metastasis, the length of survival after treatment, life or death, etc.) after cancer treatment (e.g., surgery, chemotherapy (drug therapy), or radiation therapy, etc.) for any cancer, such as ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, stomach cancer, colorectal cancer (including colon cancer and rectal cancer), bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer (hereinafter, sometimes referred to as "ovarian cancer, uterine cancer, thyroid cancer, etc." in this specification). "Prognosis prediction" may be prediction of recurrence risk (e.g., recurrence-free survival rate), 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 rate (RFS), or disease-specific survival rate (DFS). In one embodiment, prognosis prediction includes prediction of recurrence risk (e.g., relapse-free survival rate) or metastasis risk. As used herein, recurrence-free survival rate refers to the proportion of patients who do not develop recurrent cancer, such as cancer associated with the primary cancer, and disease-specific survival rate refers to the proportion of patients who do not die from a disease associated with the primary cancer. Prognosis prediction can also be referred to as prognosis determination, evaluation, diagnosis, or assistance therefor.

[0019] As used herein, "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.

[0020] 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 patients" also refer to, for example, a mammal, preferably a primate, more preferably a human.

[0021] As used herein, "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 becomes possible to predict prognosis and select poor prognosis cases with high invasion, metastasis, and recurrence potential. As used herein, determining malignancy also includes predicting prognosis.

[0022] 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).

[0023] In the present invention, in principle, the biomarkers can be claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof (referred to herein as a "CLDN16 protein, etc."). For example, if the patient is human, the biomarkers can be human CLDN16 protein derived from the human CLDN16 gene and a transcription product (mRNA) of the human CLDN16 gene.

[0024] Claudins are a family of over 20 types of molecules that function as intercellular adhesion molecules. Because members of the claudin family share highly homologous amino acid sequences, it is generally difficult to develop monoclonal antibodies that specifically recognize specific claudin molecules.

[0025] Claudin 16 (CLDN16) is a transmembrane protein with an intracellular N-terminus and an extracellular C-terminus. CLDN16 forms tight junctions and is known to control the reabsorption of magnesium ions in normal kidney tissue as an intercellular pore or ion concentration sensor. As used herein, the source of CLDN16 is not limited to specific organisms, but examples include the human CLDN16 protein consisting of the amino acid sequence set forth in SEQ ID NO:9.

[0026] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues in two amino acid sequences being compared. Specifically, the two amino acid sequences are aligned, and gaps are inserted into one or both sequences as needed. In this case, one gap is counted as one amino acid residue in the total number of amino acid residues. Amino acid sequence alignment can be performed using known programs such as Blast, FASTA, and ClustalW (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, S. F. et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, W. R. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). When the total number of amino acid residues differs between the two amino acid sequences being compared, the longer of the two is used as the total number of amino acid residues. Amino acid identity is calculated by dividing the number of identical amino acid residues by the total number of amino acid residues when the degree of amino acid identity between the two amino acid sequences is highest.

[0027] As used herein, "plurality" refers to, for example, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0028] The "CLDN16 gene" is a gene that encodes the CLDN16 protein. A specific example of the CLDN16 gene is the human CLDN16 gene that encodes the human CLDN16 protein consisting of the amino acid sequence shown in SEQ ID NO: 9. More specifically, the CLDN16 gene is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 10.

[0029] The CLDN16 gene also encompasses CLDN16 variants having activity functionally equivalent to that of the CLDN16 protein encoded by the CLDN16 gene shown in SEQ ID NO: 10, and CLDN16 genes encoding CLDN16 orthologs of other organisms. Specifically, the CLDN16 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 CLDN16 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 CLDN16 gene encompasses a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions to 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 CLDN16 protein.

[0030] As used herein, the term "base identity" refers to the percentage (%) of identical bases between two base sequences relative to the total bases of the CLDN16 gene consisting of the base sequence shown in SEQ ID NO: 10, when the two base sequences are aligned and gaps are introduced, if necessary, to maximize the degree of base identity between the two.

[0031] Nucleotide sequence information of the CLDN16 gene can be searched for in public databases (GenBank, EMBL, DDBJ). For example, based on the known nucleotide sequence information of the CLDN16 gene shown in SEQ ID NO: 10, genes with high nucleotide identity can be searched for and obtained from databases.

[0032] The term "transcription product of the CLDN16 gene" refers to CLDN16 mRNA. The mRNA may be either a pre-mRNA or a mature mRNA. Usually, a pre-mRNA is immediately spliced ​​in the nucleus to become a mature mRNA. Therefore, the transcription product of the CLDN16 gene that serves as a biomarker of the present invention is essentially the mature CLDN16 mRNA.

[0033] As used herein, the term "peptide fragment" refers to a peptide fragment consisting of a part of the amino acid sequence constituting the CLDN16 protein, which can be identified as a fragment of the CLDN16 protein from the amino acid sequence constituting the fragment. Typically, the peptide may be a peptide consisting of 10 to 200, 20 to 150, 30 to 100, or 40 to 80 consecutive amino acid residues of the full-length amino acid sequence of the CLDN16 protein. For example, a peptide fragment containing an antigen epitope recognized by the anti-CLDN16 antibody of the present invention described below, such as a peptide fragment containing or comprised in the amino acid sequence shown in SEQ ID NO: 11 or 20, is preferred.

[0034] As used herein, the term "nucleic acid fragment" refers to a nucleic acid fragment consisting of a part of the nucleotide sequence constituting CLDN16 mRNA, which can be identified as a fragment of CLDN16 mRNA from the nucleotide sequence constituting the fragment. Typically, the nucleic acid fragment may be a nucleic acid consisting of 15 to 600, 20 to 500, 25 to 400, 30 to 300, 35 to 200, or 40 to 100 consecutive nucleotides of the full-length nucleotide sequence of CLDN16 mRNA.

[0035] As used herein, the term "subject" refers to a human individual who provides a sample and is subjected to testing. In principle, the term 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).

[0036] As used herein, the term "healthy individual" refers to a human individual not suffering from a specific 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.

[0037] As used herein, "(amino acid) substitution" refers to a substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to alter the properties of peptides.

[0038] As used herein, "hybridizing under highly stringent conditions" refers to hybridization and washing under conditions of low salt concentration and / or high temperature. For example, incubation with a probe in 6xSSC, 5xDenhardt's reagent, 0.5% SDS, and 100 μg / mL denatured, fragmented salmon sperm DNA at 65°C to 68°C can be 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. Highly stringent 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.

[0039] The biomarkers for predicting the prognosis of cancer patients of the present invention can predict the prognosis of patients with any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer, with high accuracy. For example, the risk of recurrence and / or metastasis of cancer patients can be determined. This makes it possible to select cancer cases with a poor prognosis due to a high likelihood of invasion, metastasis, and / or recurrence.

[0040] Furthermore, the biomarker for predicting cancer prognosis of the present invention provides the use of the CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof as a biomarker for predicting the prognosis of cancer patients.

[0041] The biomarkers for cancer detection 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 use of the CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof as a biomarker for detecting any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer.

[0042] (Anti-CLDN16 Antibody or Fragment Thereof) In one aspect, the present invention provides an anti-CLDN16 antibody or a fragment thereof. Also provided are an anti-CLDN16 antibody or a fragment thereof for predicting the prognosis of a patient with any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer, and an anti-CLDN16 antibody or a fragment thereof for detecting any cancer, such as ovarian cancer, endometrial cancer, or thyroid cancer. The anti-CLDN16 antibody or a fragment thereof of the present invention can predict the prognosis of cancer in a subject by detecting the CLDN16 protein or a peptide fragment thereof that can be expressed in highly malignant cancers, such as ovarian cancer, endometrial cancer, or thyroid cancer. Furthermore, the anti-CLDN16 antibody or a fragment thereof of the present invention can detect cancer by detecting the CLDN16 protein or a peptide fragment thereof that can be expressed in cancers such as ovarian cancer, endometrial cancer, or thyroid cancer.

[0043] (1) Anti-CLDN16 Antibody The term "anti-CLDN16 antibody" refers to an antibody that exhibits immunoreactivity against the CLDN16 protein or a peptide fragment thereof.

[0044] The species from which the anti-CLDN16 antibody of the present invention is derived is not particularly limited. Antibodies derived from birds and mammals are preferred. Examples include chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, and human.

[0045] The anti-CLDN16 antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody, as long as it recognizes the CLDN16 protein or a peptide fragment thereof and exhibits immunoreactivity. Preferably, it is a monoclonal antibody with a stable antibody titer.

[0046] As used herein, the term "polyclonal antibody" refers to a group of multiple different immunoglobulins that can specifically bind to and recognize an antigen.

[0047] 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 a complementarity determining region (hereinafter referred to as "CDR") 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 refers to an antibody that contains a specific region.

[0048] When the anti-CLDN16 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).

[0049] The position of the epitope in the CLDN16 protein or a peptide fragment thereof recognized by the anti-CLDN16 antibody of the present invention is not particularly limited. Examples of epitopes recognized by the anti-CLDN16 antibody of the present invention include peptide sequences contained in the amino acid sequence shown in SEQ ID NO: 11 or 20.

[0050] A specific example of an anti-CLDN16 antibody that recognizes the above epitope is the rat anti-CLDN16 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.

[0051] Amino acid sequence of heavy chain variable region: DRQMGLLLCLVTFPRCVLSQVQLEESGPGLVQPSQTLSLTCTVSGFSLTSNGVSWVRQPPGKGLEWIAAISSAGNTYYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYFCNTLYYGYNRGYFDYWGQGVMVTVSSAETTAPSVYP (SEQ ID NO: 7) Amino acid sequence of light chain variable region: MTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVSNRFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPLTFGSGTKLEIKRADAAPTVSIFPPSMEHEMVSWTVLLIRTPKTARTA (SEQ ID NO: 8) Amino acid sequence of heavy chain CDR1: GFSLTSN (SEQ ID NO: 1) Amino acid sequence of heavy chain CDR2: SSAGN (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: LYYGYNRGYFDY (SEQ ID NO: 3) Light chain CDR1 amino acid sequence: RSSQSLVHNNGNTYLS (SEQ ID NO: 4) Light chain CDR2 amino acid sequence: KVSNRFS (SEQ ID NO: 5) Light chain CDR3 amino acid sequence: GQGTQYPLT (SEQ ID NO: 6)

[0052] 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-CLDN16 antibody obtained in Example 2, 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-CLDN16 antibody obtained in Example 2, is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 19. 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.

[0053] "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-CLDN16 antibody clone obtained in Example 2, 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 mammal other than human. 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 obtained by substituting 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, which are derived from the heavy chain of the rat-derived anti-CLDN16 antibody clone obtained in Example 2, with human antibody heavy chain CDR1, CDR2, and CDR3, respectively, and by substituting 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, which are derived from the light chain of the antibody clone, with human antibody light chain CDR1, CDR2, and CDR3, respectively. Such humanized antibodies are derived from human antibodies except for the CDRs, and therefore can reduce the immune response against the antibody in the human body more than chimeric antibodies.

[0054] "Synthetic antibody" refers to an antibody synthesized chemically or by using recombinant DNA technology. For example, it includes antibodies newly synthesized using recombinant DNA technology. 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 are included. L and heavy chain variable region V H ) are located on separate polypeptide chains, known as light and heavy chains. scFvs are immunoglobulin molecules with V L and V HA synthetic antibody with a molecular weight of approximately 35 kDa or less has a structure in which two variable regions are linked by a flexible linker of sufficient length and incorporated into a single polypeptide chain. Within an scFv, a pair of variable regions 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 length 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 disassembled. L is the V of the other scFv H The scFv fragments can aggregate 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, the anti-CLDN16 antibody of the present invention is 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.

[0055] The anti-CLDN16 antibody of the present invention can also be modified. As used herein, "modification" includes functional modifications necessary for antigen-specific binding activity, such as glycosylation, and labeling modifications necessary for antibody detection.

[0056] Glycosylation modifications on anti-CLDN16 antibodies are performed to adjust the affinity of the anti-CLDN16 antibody for the target CLDN16 protein or a peptide fragment thereof. Specific examples include modifications such as introducing substitutions into amino acid residues that constitute glycosylation in the FR of the anti-CLDN16 antibody to remove the glycosylation site, thereby eliminating glycosylation at that site.

[0057] The anti-CLDN16 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 such labels include those labeled with ATP (S) or biotin or (strept)avidin.

[0058] The anti-CLDN16 antibody of the present invention has a dissociation constant with the CLDN16 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 as high as 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.

[0059] (2) Fragment thereof As used herein, the term "fragment thereof" refers to an antibody fragment that consists of a part of an anti-CLDN16 antibody and exhibits immunoreactivity to the CLDN16 protein or a fragment thereof, similar to the anti-CLDN16 antibody. Examples of such fragments include Fab, F(ab')2, and Fab'.

[0060] Fab is an antibody fragment generated by cleavage of an IgG molecule with papain at the N-terminal side of the hinge disulfide bond, and contains the heavy chain constant region (heavy chain constant region: hereinafter referred to as C H The three domains that make up the H 1. C H 2. C H 3) V H C adjacent to H 1 and V H , and a full-length L chain.

[0061] 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.

[0062] (3) Preparation of Anti-CLDN16 Antibody The anti-CLDN16 antibody 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, the monoclonal antibody can also be obtained from a hybridoma that produces the antibody.

[0063] An antigenic peptide that can be used as an immunogen for the anti-CLDN16 antibody of the present invention is any part of the CLDN16 protein (hereinafter referred to as a "CLDN16 antigenic peptide"). For example, an antigenic peptide that can be used as an immunogen for the anti-CLDN16 antibody of the present invention includes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 11 or 20. The CLDN16 antigenic peptide can be prepared, for example, by chemical synthesis or DNA recombinant technology.

[0064] (Cancer prognosis prediction kit and cancer detection kit) In one aspect, the present invention provides a cancer prognosis prediction kit and a cancer detection kit. The cancer prognosis prediction kit or cancer detection kit of the present invention comprises an anti-CLDN16 antibody or an immunoreactive fragment thereof as an essential component, and is capable of detecting a biomarker for predicting the prognosis of a cancer patient or a biomarker for detecting cancer. The cancer prognosis prediction kit or cancer detection kit of the present invention comprises, as a selected component, an antibody (hereinafter referred to as "other prognosis prediction antibody") against a cancer prognosis prediction biomarker other than the CLDN16 protein or a peptide fragment thereof (hereinafter referred to as "other prognosis prediction biomarker").

[0065] (1) Essential Components The cancer prognosis prediction kit or cancer detection kit of the present invention contains the above-mentioned anti-CLDN16 antibody or a fragment thereof as an essential component. The cancer prognosis prediction kit or cancer detection kit of the present invention may contain a single type of anti-CLDN16 antibody or multiple types of anti-CLDN16 antibodies.

[0066] (2) Selected Components The cancer prognosis kit or cancer detection kit of the present invention may further comprise one or more other prognosis prediction antibodies or other detection antibodies, or immunoreactive fragments thereof, as selected components. The other prognosis prediction antibodies or other detection antibodies may be any antibodies that, when used in combination with the anti-CLDN16 antibody, can improve the accuracy of prognosis prediction or detection in patients with cancers such as ovarian cancer, endometrial cancer, or thyroid cancer. Antibodies against any cancer prognosis prediction or cancer 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).

[0067] In addition to the above-mentioned essential components, the cancer prognosis prediction kit or cancer detection kit of the present invention may also include other reagents necessary for predicting the prognosis of cancer patients or detecting cancer, such as buffers and secondary antibodies, and instructions for use in detection and determining the results.

[0068] (Cancer prognosis prediction method) In one aspect, the present invention provides a cancer prognosis prediction method. The cancer prognosis prediction method of the present invention includes a detection step of detecting a biomarker consisting of CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof (hereinafter referred to as a "biomarker such as CLDN16 protein") in a sample derived from a cancer patient, and the prognosis of the cancer patient is indicated based on the positive / negative determination result of the biomarker. The detection step can be performed in vitro.

[0069] In this embodiment, the "detection 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 measured value (hereinafter referred to as "determination of positive / negative").

[0070] As used herein, the term "detection" encompasses measurement, qualitative, quantitative, and semi-quantitative.

[0071] In the prognosis prediction method of this embodiment, 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 prognosis prediction method for cancer patients of this embodiment, such as tissue or cells. The "tissue" and "cells" used in the prognosis prediction method of the present invention include, for example, tissues and cells of a subject suffering from any cancer, such as ovarian cancer, endometrial cancer, or thyroid 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 obtained by biopsy or surgically resected from a subject suffering from cancer. Preferably, the sample is a portion of the cancer (e.g., tissue or cells), such as the invasive front, obtained by biopsy or surgically resected. The stage of cancer suffered by patients who are subjects of the method of the present invention is not limited.

[0072] 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 the case with biopsy materials. 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 filtration, 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.

[0073] 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 CLDN16 protein in a sample.

[0074] As used herein, the term "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 CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof.

[0075] The following describes (1) the measurement of a biomarker such as CLDN16 protein, and (2) the determination of whether a biomarker such as CLDN16 protein is positive or negative, which constitute the detection step of this embodiment.

[0076] (1) Measurement of biomarkers such as CLDN16 protein The biomarker to be measured by this method may be either the CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 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. As used herein, the term "measurement" includes detection, qualitative, quantitative, and semi-quantitative measurements.

[0077] When the biomarker to be measured is the CLDN16 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.

[0078] 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. Examples of 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. Immunohistochemistry is preferred, but is not limited thereto.

[0079] When immunohistochemistry is used, the method for quantifying the CLDN16 protein or a peptide fragment thereof is not limited. For example, the CLDN16 protein or a peptide fragment thereof may be quantified as a score calculated using a certain formula based on the observation of the staining pattern of a tissue section.

[0080] The antibody used for immunological detection in this step 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-described anti-CLDN16 antibody or a fragment thereof.

[0081] "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.

[0082] "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 DNA, phosphorothioate DNA, and 2'-O-methyl RNA. In the present invention, examples of the aptamer include an anti-CLDN16 RNA aptamer and an anti-CLDN16 DNA aptamer.

[0083] 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 CLDN16 protein or a portion thereof as the target molecule. The SELEX method 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).

[0084] Peptide aptamers are aptamers composed of amino acids, and, like antibodies, are peptide molecules of 1 to 6 kDa that recognize and specifically bind to the surface structure of a specific target molecule. In the present invention, examples include anti-CLDN16 peptide aptamers. Peptide aptamers may be produced using production methods known in the art. For example, see Whaley, SR, et al., Nature, 2000, 405, 665-668. Typically, they can be produced using phage display or cell surface display.

[0085] 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 fluorophores (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.

[0086] "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.

[0087] The immunological detection method, aptamer analysis method, and mass spectrometry are all techniques known in the art, and may be carried out in accordance with these methods. For example, 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. Peptide quantification kits are also commercially available from various manufacturers, and these can also be used.

[0088] Furthermore, when the biomarker to be measured is a CLDN16 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 a nucleic acid amplification method using a primer or a hybridization method using a probe.

[0089] "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).

[0090] 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.

[0091] 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.

[0092] 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.

[0093] (2) Determining whether a biomarker such as CLDN16 protein is positive or negative Next, based on the measurement value obtained in (1), it is determined whether a biomarker such as CLDN16 protein in the sample is positive or negative.

[0094] 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 CLDN16 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 CLDN16 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.

[0095] The cutoff value refers to the 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). There are no particular limitations on the method for setting the cutoff value.

[0096] For example, a cutoff value can be set to 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 a subject's measurement value can be determined to be positive when it is higher than the cutoff value.

[0097] 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, endometrial 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] In the prognosis prediction method of the present invention, the prognosis of a cancer patient is predicted or indicated based on the detection result of a biomarker such as CLDN16 protein obtained in the detection step. Specifically, when a sample derived from a patient with cancer such as ovarian cancer, endometrial cancer, or thyroid cancer is positive for a biomarker such as CLDN16 protein, it indicates that the cancer patient has a poor prognosis or is likely to have a poor prognosis. Conversely, when a sample derived from a cancer patient is negative for a biomarker such as CLDN16 protein, it indicates that the cancer patient has a good prognosis or is likely to have a good prognosis.

[0102] 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.

[0103] 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%.

[0104] In one embodiment, a biomarker for predicting the prognosis of cancer patients other than the biomarker such as CLDN16 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 CLDN16 protein. Examples of biomarkers for predicting the prognosis of cancer patients other than the biomarker such as CLDN16 protein and antibodies for detecting them are as described above.

[0105] 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 CLDN16 protein. Here, "cellular atypia" refers to a deviation from normal cellular structure, specifically, an increased nucleosomal ratio, cell or nuclei size variation, irregular nuclear shape, increased nuclear chromatin, increased or increased nucleoli, 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, hematoxylin-eosin staining can be used to visualize atypia. "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 boundary with the surrounding tissue. "Metastasis" refers to the discontinuous spread of a malignant tumor from the primary focus to an organ distant from the primary focus.

[0106] 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 by the present invention to be poor, the patient may be subjected to surgery or anticancer drug therapy (e.g., treatment methods including the administration of anticancer drugs such as paclitaxel or carboplatin) 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 testing can be increased to detect cancer recurrence early.

[0107] 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.

[0108] The method of the present invention can be used in combination with other methods (e.g., X-ray imaging; ultrasound (echo) examinations such as transvaginal 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.

[0109] 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.

[0110] (Cancer detection method) In one aspect, the present invention provides a cancer detection method. The cancer detection method of the present invention includes a detection step of detecting a biomarker consisting of CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof (hereinafter referred to as a "biomarker such as CLDN16 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.

[0111] In this embodiment, the "detection step" is a step of measuring the amount of a biomarker for detecting cancer in a sample derived from a subject and determining whether the biomarker is positive or negative based on the measured value (hereinafter referred to as "determination of positive / negative").

[0112] 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.

[0113] As used herein, the term "measurement value of a biomarker for detecting cancer" specifically refers to the measurement value of the amount of a biomarker such as CLDN16 protein in a sample such as a body fluid.

[0114] The following describes (1) the measurement of a biomarker such as CLDN16 protein, and (2) the determination of whether a biomarker such as CLDN16 protein is positive or negative, which constitute the detection step of this embodiment.

[0115] (1) Measurement of biomarkers such as CLDN16 protein The biomarker to be measured by this method may be any of the CLDN16 protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof. Measurement of the amount (expression level) includes measuring the presence or absence of expression, or the magnitude of the expression level or expression concentration.

[0116] When the biomarker to be measured is the CLDN16 protein or a peptide fragment thereof, the measurement method may be any known protein quantification method, and is not particularly limited to, for example, the above-mentioned immunological detection method, aptamer analysis method, and mass spectrometry method.

[0117] Furthermore, when the biomarker to be measured is a CLDN16 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 or the hybridization method using a probe.

[0118] (2) Determining Positivity / Negative of Biomarkers Such as CLDN16 Protein Next, based on the measurement values ​​obtained in (1), it is determined whether the biomarkers such as CLDN16 protein in the sample are positive or negative. The determination of positivity / negative in this method is in accordance with "(2) Determining Positivity / Negative of Biomarkers Such as CLDN16 Protein" described in the above-mentioned prognosis prediction method, and therefore further explanation will be omitted here.

[0119] In the cancer detection method of the present invention, if a sample is positive for a biomarker such as CLDN16 protein based on the detection result obtained in the detection step, it indicates that the subject is highly likely to have cancer. Conversely, if a sample derived from a subject is negative for a biomarker such as CLDN16 protein, it indicates that the subject is low likely to have cancer.

[0120] In one embodiment, a biomarker for detecting cancer other than the biomarker such as CLDN16 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 CLDN16 protein. Examples of the biomarker for detecting cancer other than the biomarker such as CLDN16 protein and the antibody for detecting it are as described above.

[0121] According to the cancer detection method of the present invention, by examining a sample of body fluids (such as ascites or serum) isolated from a subject, it is possible to determine whether or not the subject is susceptible to cancer. Early diagnosis of cancer is possible based on the results of the cancer detection method of the present invention, allowing treatment to be initiated at an early stage. If a high susceptibility to cancer is indicated based on the results of the cancer detection method of the present invention, the patient may be subjected to drug therapy and / or radiation therapy to treat the cancer. Therefore, there is also provided a method for treating cancer, comprising administering at least one of drug therapy and radiation therapy to a cancer patient who has been shown to have a high susceptibility to cancer by the method of the present invention.

[0122] The cancer detection method of the present invention also provides a method for assisting in cancer detection. The cancer detection method of the present invention can be used in combination with other methods (e.g., X-ray imaging; ultrasound (echo) examinations such as transvaginal 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 the method with other methods can improve the accuracy of the cancer detection method of the present invention.

[0123] (Cancer therapeutic agent) In one aspect, the present invention provides a cancer therapeutic agent. The cancer therapeutic agent of the present invention comprises an anti-CLDN16 antibody or a fragment thereof as an active ingredient, and is capable of treating cancers such as ovarian cancer, endometrial cancer, and thyroid cancer in cancer patients. The anti-CLDN16 antibody or a fragment thereof in this aspect conforms to the descriptions of "(1) Anti-CLDN16 antibody" and "(2) Fragment thereof" above, and detailed description here is omitted.

[0124] In this embodiment, the anti-CLDN16 antibody or a fragment thereof preferably has cytotoxic activity, thereby being able to exert an anti-tumor effect.

[0125] In this embodiment, the anti-CLDN16 antibody or a fragment thereof can be conjugated to an anti-tumor agent. The antibody can be conjugated to the anti-tumor agent via a spacer having a group reactive with an amino group, a carboxyl group, a hydroxyl group, a thiol group, or the like (e.g., a succinimidyl group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, a hydroxyl group, or the like).

[0126] 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, colofsphamide, estradiol Mustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin ), dynemycin, clodronate, esperamicin, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, adriamycin, epirubicin,Esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine 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, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid acid), triaziquone, roridine A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, oxaliplatin, Examples of such anti-cancer drugs include carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitors, difluoromethylolnitine (DMFO), retinoic acid, and capecitabine.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] The above-mentioned carriers are used to avoid or suppress the degradation of the active ingredient, the anti-CLDN16 antibody or a fragment thereof, by enzymes and the like in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and pharmacological efficacy, and may be used appropriately as needed.

[0131] 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.

[0132] 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.

[0133] The dosage of the antibody or fragment thereof can be selected 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.

[0134] The cancer therapeutic agent or composition for cancer treatment of the present invention may be administered in a single dose or multiple doses. In the case of multiple doses, it can be administered daily or at appropriate time intervals (for example, at intervals of 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 composition for cancer treatment may 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, The dose can be 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 appropriately selected from any amount within the range of, for example, 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg).

[0135] 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.

[0136] Example 1: Identification of genes specifically expressed on the surface of ovarian cancer cells (Objective) To identify new biomarkers for cancer.

[0137] (Methods and Results) Expression data of the CLDN16 gene in various cancer tissues and normal tissues were collected using The Cancer Genome Atlas database (TCGA database) and The Genotype-Tissue Expression database (GTEx database).

[0138] Figure 1 shows the expression levels of the CLDN16 gene in various cancer tissues. Strong expression of the CLDN16 gene was detected in ovarian cancer (OV, Ovarian serous cystadenocarcinoma) and thyroid cancer (THCA, Thyroid carcinoma). Moderate expression of the CLDN16 gene was also detected in uterine corpus endometrial carcinoma (UCEC). In addition to ovarian cancer (OV), uterine cancer (UCEC), and thyroid cancer (THCA), lung cancer (LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma), esophageal cancer (ESCA, Esophageal carcinoma), gastric cancer (STAD, Stomach adenocarcinoma), colorectal cancer (including COAD, Colon adenocarcinoma and READ, Rectum adenocarcinoma), bladder cancer (BLCA, Bladder Urothelial Carcinoma), cervical cancer (CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma), kidney cancer (KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma), liver cancer (LIHC, Liver hepatocellular carcinoma), and pancreatic cancer (PAAD, Pancreatic Increased expression of the CLDN16 gene was also detected in human adenocarcinoma (HNSC) and head and neck cancer (HNSC, Head and Neck squamous cell carcinoma).

[0139] Figure 2 shows the expression levels of the CLDN16 gene in various normal and cancerous tissues of adults. Strong expression of the CLDN16 gene was not detected in various normal tissues, including the ovary, uterus, and thyroid. CLDN16 gene expression in various normal tissues (Normal (GTEx)) was extremely weak compared to cancerous tissues (Cancer (TCGA)).

[0140] These results indicate that CLDN16 gene expression can be a biomarker capable of detecting ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon and rectal cancer), bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, and head and neck cancer, particularly ovarian cancer, uterine cancer, and thyroid cancer, with specificity and high sensitivity.

[0141] Example 2: Preparation of anti-CLDN16 monoclonal antibody (Objective) To develop a monoclonal antibody (anti-CLDN16 monoclonal antibody; hereinafter, sometimes simply referred to as "anti-CLDN16 antibody") that can specifically and sensitively detect CLDN16 protein.

[0142] (Methods and Results) (1) Design of Antigenic Peptide The antigenic peptide was selected from the first extracellular domain (EC1) of CLDN16, a region with low homology to the claudin family (Fig. 3). Specifically, we used a peptide consisting of the amino acid sequence (FDGIRTCDEYDSILA, SEQ ID NO: 11) corresponding to positions 55-69 (initial methionine is at position 1) of the human CLDN16 protein shown in SEQ ID NO: 9, with a cysteine ​​added to the N-terminal side (CFDGIRTCDEYDSILA, SEQ ID NO: 20).

[0143] (2) Preparation of Antigen Peptide Monoclonal antibodies were prepared according to the following procedure based on the method described in Kishiro Y, et al., 1995, Cell Struct Funct, 20(2):151-6.

[0144] 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 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.

[0145] (3) Immunization Anesthetized 8-week-old female rats (Wistar strain) were immunized by injecting 100 μL of the antigen emulsion into both hind legs.

[0146] (4) Preparation of polyethylene glycol (PEG) solution: 5 g of PEG (P2906, Sigma-Aldrich) was dissolved by heating to 70°C. 0.4 mL of dimethyl sulfoxide (D2650, Sigma-Aldrich) was added to 8 mL of Dulbecco's modified Eagle's medium (high glucose; DMEM; D5796, Sigma-Aldrich) and heated to 50°C. This was added to the dissolved PEG and quickly mixed to prepare a PEG solution.

[0147] (5) 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 then filtered through a 100 μm cell strainer (BD Falcon). Approximately 10 7100 μL of mouse multiple myeloma cell line SP2 was added to the culture, 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 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 culture was 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.

[0148] (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.

[0149] 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 incubated 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 incubated 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 incubated 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.

[0150] 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.

[0151] Of the 287 clones, 66 positive clones were obtained by ELISA screening. Furthermore, a positive signal was detected in only one clone by immunohistochemical staining using formalin-fixed / paraffin-embedded specimens (hereinafter referred to as the "anti-CLDN16 monoclonal antibody clone obtained in Example 2" or simply the "anti-CLDN16 antibody clone"). From the above, clones were obtained that simultaneously achieved the advantages of high reactivity in ELISA and specific and highly sensitive detection of CLDN16 protein by immunostaining.

[0152] Example 3: Verification of antigen specificity of anti-CLDN16 monoclonal antibody clones (Objective) The antigen specificity of the anti-CLDN16 monoclonal antibody clones obtained in Example 2 is verified by immunohistochemical staining.

[0153] (Method and Results) A vector expressing human wild-type CLDN16 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, cells were harvested by scraping. The cells were fixed in 10% neutral formalin at 4°C for 16 hours and washed once with 1 mL of PBS. 1 mL of 1% sodium alginate solution was added, and the cell mass was gently suspended using a 1 mL tip with the tip cut off. The cells were then 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-peel coated slides (CRE-12, Matsunami Glass Industry Co., Ltd.), and allowed to dry at room temperature.

[0154] 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 Tris-HCl buffer (TBS). Antigen retrieval was performed in a microwave oven for 10 minutes in 10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA) solution, pH 9.0. After returning to room temperature, the specimens were washed in TBS for 5 minutes. Nonspecific reactions of the primary antibody were prevented using an endogenous avidin-biotin blocking kit (415041, Nichirei Biosciences) and a 0.5% casein (C3400, EMD Millipore) solution diluted with TBS. The former was incubated twice for 10 minutes, and the latter for 10 minutes, all at room temperature. The specimens were then incubated overnight at 4°C with the anti-CLDN16 monoclonal antibody obtained in Example 2 as the primary antibody. After washing three times for 5 minutes with modified Tris-HCl buffer (TBS2T; 11.6 mM Trizma base, 38 mM Trizma hydrochloride, and 300 mM sodium chloride) supplemented with 0.1% Tween 20, the sections were incubated for 15 minutes at room temperature with biotin-labeled tyramide diluted 1:100 in tyramide amplification solution (20% dextran sodium sulfate 5000, 0.2 M Tris-HCl, pH 8.8). After washing three times for 5 minutes with TBS2T, the sections were incubated for 15 minutes at room temperature with horseradish peroxidase-labeled streptavidin (P0397, Agilent Technologies) diluted 5:500 in modified Tris-HCl buffer (TBS2). After washing three times for 5 minutes with TBS2T, the sections were incubated in a DAB substrate kit (425011, Nichirei Biosciences) until a suitable staining image was obtained. For nuclear staining, the specimens were washed with Tissue-Tec® Hematoxylin 3G (Sakura Finetech Japan Co., Ltd.) solution for 5 seconds, followed by 10 minutes of running water. The specimens were then washed with 0.5% hydrochloric acid / 70% ethanol for 5 seconds to remove excess hematoxylin. The specimens were dehydrated with 100% ethanol, cleared with xylene, and prepared into slides using an automatic slide mounting machine (Shiraimatsu Kikai Co., Ltd.).

[0155] Using a similar method, HEK293T cells not transfected with a vector for expressing CLDN16 protein, HEK293T cells transfected with a vector expressing a fusion protein in which an HA tag was fused to the N-terminus or C-terminus of human wild-type CLDN16 protein (hereinafter referred to as "HA-CLDN16" and "CLDN16-HA," respectively), and HEK293T cells transfected with a vector expressing human wild-type CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN8, CLDN9, CLDN12, or CLDN19 protein were prepared, and immunostaining was performed on each cell type.

[0156] The results of immunostaining using anti-CLDN16 monoclonal antibody are shown in Figures 4 and 5. While no CLDN16-positive signal was observed in HEK293T cells that had not been transfected with a vector for CLDN16 protein expression, CLDN16-positive signals were observed in HEK293T cells that had been expressing human wild-type CLDN16 protein, HA-CLDN16 protein, or CLDN16-HA protein (Figure 4). No CLDN16-positive signals were observed in HEK293T cells that had been expressing claudins other than CLDN16, and among the claudin family, only HEK293T cells that had expressed the CLDN16 protein showed CLDN16-positive signals (Figure 5).

[0157] From the above results, it was revealed that the anti-CLDN16 monoclonal antibody obtained in Example 2 is an extremely useful antibody that can detect CLDN16 specifically and with high sensitivity by immunostaining and does not show cross-reactivity with other claudins.

[0158] Example 4: Determination of CDR sequence of anti-CLDN16 monoclonal antibody (Objective) To determine the CDR sequence of the anti-CLDN16 monoclonal antibody clone obtained in Example 2.

[0159] (Methods and Results) The sequences of the heavy and light chain variable regions and CDRs of the anti-CLDN16 monoclonal antibody clone 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, the aqueous phase containing the mRNA was mixed with an equal volume of isopropanol and centrifuged at 15,000 rpm at 4°C for 15 minutes to extract the mRNA. Finally, the mRNA was desalted by adding 1 mL of 70% ethanol and dissolved in 50 μL of water. The mRNA concentration was quantified using a NanoDrop® 2000.

[0160] The 5'-Full RACE Core Set (Takara Bio Inc.) was used to determine the CDRs. All experiments were performed using the manufacturer's instructions and accompanying reagents. The resulting DNA fragments were cloned into the pGEM®-T Easy Vector (Promega) and then sequenced. DNA sequencing was performed by Macrogen Japan Co., Ltd., and CDRs were identified according to the Kabat antibody numbering system.

[0161] The sequences of the heavy and light chain variable regions and CDRs of the anti-CLDN16 monoclonal antibody clone were determined, and the results are shown below (FIG. 6).

[0162] Amino acid sequence of heavy chain variable region: DRQMGLLLCLVTFPRCVLSQVQLEESGPGLVQPSQTLSLTCTVSGFSLTSNGVSWVRQPPGKGLEWIAAISSAGNTYYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYFCNTLYYGYNRGYFDYWGQGVMVTVSSAETTAPSVYP (SEQ ID NO: 7) Amino acid sequence of light chain variable region: MTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVSNRFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPLTFGSGTKLEIKRADAAPTVSIFPPSMEHEMVSWTVLLIRTPKTARTA (SEQ ID NO: 8) Amino acid sequence of heavy chain CDR1: GFSLTSN (SEQ ID NO: 1) Amino acid sequence of heavy chain CDR2: SSAGN (SEQ ID NO: 2) Heavy chain CDR3 amino acid sequence: LYYGYNRGYFDY (SEQ ID NO: 3) Light chain CDR1 amino acid sequence: RSSQSLVHNNGNTYLS (SEQ ID NO: 4) Light chain CDR2 amino acid sequence: KVSNRFS (SEQ ID NO: 5) Light chain CDR3 amino acid sequence: GQGTQYPLT (SEQ ID NO: 6)

[0163] Example 5: Functional analysis of CLDN16 protein (Objective) An ovarian cancer cell line, AMOC2, that overexpresses CLDN16 protein is prepared, and the effects on its proliferation, migration, and invasive abilities are examined.

[0164] (Methods) (1) Generation of ovarian cancer cell lines overexpressing CLDN16 protein. The ovarian cancer cell line AMOC2 was used as the cell line for transduction. AMOC2 was provided by Dr. Tsuyoshi Saito of Sapporo Medical University. AMOC2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin, and amphotericin B (Fujifilm Wako Pure Chemical Industries). Hereafter, this medium will be referred to as DMEM (10% FBS + 1% PS) medium. A vector expressing human wild-type CLDN16 protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or a vector expressing EGFP as a negative control, was packaged into a lentivirus and transduced using a polybrene solution (12996-81, Nacalai Tesque) at a final concentration of 10 μg / mL to generate CLDN16-expressing ovarian cancer cell line #1, CLDN16-expressing ovarian cancer cell line #2, and an EGFP-expressing ovarian cancer cell line.

[0165] Next, Western blot analysis was performed to verify the overexpression of CLDN16 protein in CLDN16-expressing ovarian cancer cell lines #1 and #2. Proteins were extracted using a lysate prepared by adding the protease inhibitor Complete mini EDTA-free (Roche Diagnostics) to CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich) at a concentration of 1 tablet per 10 mL. SDS-PAGE, transfer, and antibody reaction were performed sequentially according to standard protocols. Chemiluminescence was generated using EzWestLumi Plus ECL (Atto) and images were captured using a CCD imager, Image Quant LAS4000 (GE Healthcare). The primary antibody was a 500-fold diluted anti-CLDN16 antibody (provided by Dr. Mikio Furuse of the National Institute for Physiological Sciences) and incubated overnight at 4°C. The secondary antibody was a 2,000-fold diluted ECL™ Rabbit IgG, HRP-linked whole antibody (Sigma-Aldrich) and incubated for 1 hour. The diluent used was Can Get Signal® Immunoreaction Enhancer Solution (Toyobo). The results of the Western blot are shown in Figure 7A. CLDN16 protein overexpression was confirmed in CLDN16-expressing ovarian cancer cell line #1 and CLDN16-expressing ovarian cancer cell line #2.

[0166] (2) Evaluation of Proliferation Potential. Cell proliferation potential was evaluated using the following method. Each cell line obtained using the method described in (1) above was cultured in a 10-cm dish, and formalin-fixed, paraffin-embedded specimens were prepared using the same method as in Example 3 above. The specimens were deparaffinized in xylene for 10 minutes and then dexylened in 100% ethanol. Endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide / methanol for 10 minutes, followed by washing with phosphate-buffered saline (PBS) for 5 minutes. The specimens were placed in 10 mM citric acid solution, pH 6.0, and subjected to antigen retrieval in a microwave oven for 10 minutes. After returning to room temperature, the specimens were washed with PBS for 5 minutes and then treated with a 5% solution of skim milk (Morinaga Milk Industry) diluted with PBS to prevent nonspecific reactions of the primary antibody. The specimens were then incubated overnight at 4°C with Ki-67 antibody (M7240, Agilent Technologies) as the primary antibody. After washing three times with PBS for 5 minutes, the sections were incubated with Histofine Simple Stain Rat MAX-PO (MULTI) (product number 424151, Nichirei Biosciences) as a secondary antibody for 60 minutes at room temperature. After washing three times with PBS for 5 minutes, the sections were incubated with a DAB substrate kit (product number 425011, Nichirei Biosciences) until appropriate staining was achieved. For nuclear staining, the sections were shaken in Tissue-Tec® Hematoxylin 3G (Sakura Finetech Japan Co., Ltd.) solution for 5 seconds and then washed in running water for 10 minutes. Next, the sections were shaken in 0.5% hydrochloric acid / 70% ethanol for 5 seconds to remove excess hematoxylin. The sections were dehydrated with 100% ethanol, cleared with xylene, and then prepared into slides using an automatic slide mounter (Shiraimatsu Kikai Co., Ltd.). The number of positive cells was calculated using ImageJ, and statistical analysis was performed.

[0167] (3) Evaluation of migration activity Cell migration activity assay was performed as follows. First, 500 μL of DMEM (10% FBS + 1% PS) medium was placed in each well of a 24-well plate, and a 24-well culture insert (#353-097, Corning) was placed inside. Then, 1 × 10 cultured cells were placed inside the 24-well culture insert. 5The cells were placed in a 24-well culture insert and cultured for 24 hours in a culture incubator (37°C, 5% CO2, 95% air). To measure the number of migrating cells, the 24-well culture insert was first washed with PBS and then fixed with 4% paraformaldehyde solution at room temperature for 10 minutes. After washing again with PBS, the cells were stained with 0.2% crystal violet solution at room temperature for 10 minutes. Finally, the cells inside the 24-well culture insert were removed with a cotton swab, and the number of cells outside was calculated using ImageJ, and this was used as the number of migrating cells.

[0168] (4) Evaluation of Invasion Ability The invasion ability assay was performed as follows. First, 500 μL of DMEM (10% FBS + 1% PS) medium was placed in each well of a 24-well plate, and a BioCoat® Matrigel Invasion Chamber (#354480, Corning) was placed inside. 1 × 10 cultured cells were then placed inside the BioCoat® Matrigel Invasion Chamber. 5 The cells were placed in a culture incubator (37°C, 5% CO2, 95% air) and cultured for 24 hours. To measure the number of invasive cells, the BioCoat® Matrigel invasion chamber was first washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde solution at room temperature for 10 minutes. After washing again with PBS, the cells were stained with 0.2% crystal violet solution at room temperature for 10 minutes. Finally, the cells inside the BioCoat® Matrigel invasion chamber were removed with a cotton swab, and the number of cells outside was calculated using ImageJ, which was used as the number of invasive cells.

[0169] (Results) The proliferation, migration, and invasion abilities of the CLDN16-expressing ovarian cancer cell lines #1 and #2 and the EGFP-expressing ovarian cancer cell line were evaluated. The results are shown in Figure 7B, Figure 8, and Figure 9, respectively. Both the CLDN16-expressing ovarian cancer cell lines #1 and #2 showed significantly enhanced proliferation, migration, and invasion abilities compared with the EGFP-expressing ovarian cancer cell line. These results demonstrated that CLDN16 can enhance the malignancy of ovarian cancer cells.

[0170] Example 6: Verification of antibody binding ability to CLDN16 protein on the surface of living cells (Objective) The binding specificity of the anti-CLDN16 monoclonal antibody obtained in Example 2 to CLDN16 protein on the surface of living cells is verified by flow cytometry.

[0171] (Method and Results) Using the same method as in Example 3, HEK293T cells without a vector introduced therein, HEK293T cells introduced with a vector expressing human wild-type CLDN16 protein, and HEK293T cells introduced with a vector expressing EGFP protein were prepared.

[0172] Flow cytometry was performed as follows: anti-CLDN16 antibody or normal rat IgG antibody (147-09521, Fujifilm Wako Pure Chemical Industries, Ltd.) was added as the primary antibody to the cell suspension at a final concentration of 100 μg / mL. Subsequently, antibody-bound cells were stained with Alexa647-labeled anti-rat IgG antibody (563-78551, Fujifilm Wako Pure Chemical Industries, Ltd.), and fluorescence was analyzed by flow cytometry (On-chip Flow, On-chip Biotechnologies).

[0173] The results are shown in Figure 10. Antibody binding was not detected in HEK293T cells without a vector or in HEK293T cells transfected with a vector expressing EGFP protein, whereas binding of the anti-CLDN16 monoclonal antibody was detected in HEK293T cells transfected with a vector expressing human wild-type CLDN16 protein. These results demonstrate that the anti-CLDN16 monoclonal antibody can specifically recognize the CLDN16 protein on the surface of live cells.

[0174] Example 7: Verification of uptake of anti-CLDN16 antibody into cancer cells (Objective) It was verified that the anti-CLDN16 monoclonal antibody obtained in Example 2 can be taken up into cancer cells.

[0175] (Methods and Results) A CLDN16-expressing ovarian cancer cell line overexpressing wild-type CLDN16 protein and an EGFP-expressing ovarian cancer cell line were prepared by a method similar to that described in Example 5(1).

[0176] Anti-CLDN16 monoclonal antibody or rat IgG antibody (17316, Immuno-Biological Laboratories, Inc.) was added to the cell culture medium at a final concentration of 10 μg / mL. The cells were cultured for 3 hours in a culture incubator (37°C, 5% CO2, 95% air), washed three times with PBS, and then fixed for 10 minutes with 4% paraformaldehyde solution. After three washes with PBS, the cells were permeabilized with 0.1% Triton X-100 / PBS solution for 10 minutes and blocked with 5% skim milk for 30 minutes. Subsequently, Cy3-labeled anti-rat IgG antibody (564-71901, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to stain the antibody-bound cells, allowing detection of antibody uptake within the cancer cells.

[0177] The results are shown in Figure 11. While no uptake of the anti-CLDN16 antibody was detected in the EGFP-expressing ovarian cancer cell line, intracellular uptake of the anti-CLDN16 antibody was detected in the CLDN16-expressing ovarian cancer cell line (Figure 11, arrow). The arrowhead indicates the CLDN16 signal on the cell surface. On the other hand, the rat IgG antibody was not internalized into the CLDN16-expressing ovarian cancer cell line. These results demonstrate that the anti-CLDN16 monoclonal antibody can be internalized into ovarian cancer cells based on the CLDN16 protein. These results demonstrate that the anti-CLDN16 antibody of the present invention enables the delivery of drugs such as antitumor agents to ovarian cancer cells and can specifically kill cancer cells.

[0178] Example 8: Verification of the cytotoxic effect of anti-CLDN16 antibody on cancer cells (Objective) It was verified that the anti-CLDN16 monoclonal antibody obtained in Example 2 exhibits a cytotoxic effect on cancer cells.

[0179] (Methods and Results) An ovarian cancer cell line overexpressing wild-type CLDN16 protein was prepared by the same method as in Example 5(1) (hereinafter referred to as "CLDN16-expressing ovarian cancer cells").

[0180] CLDN16-expressing ovarian cancer cells were seeded in 96-well plates and cultured in DMEM (10% FBS + 1% PS) medium. 1.5 μg of cytotoxic toxin MMAF (Monomethyl Auristatin F)-conjugated anti-rat IgG antibody (AR101-AF, Moradec; hereafter referred to as "ADC-rat IgG") and 3 μg of rat anti-CLDN16 monoclonal antibody were added to the culture medium. After 24 hours of culture, a cell viability assay was performed by adding MTS reagent (CellTiter96 Aqueous One Solution, G3580, Promega) to each well. The MTS reagent is reduced by viable cells and converted to a colored formazan product, allowing the number of viable cells in the culture medium to be assessed by measuring the absorbance at 490 nm.

[0181] The results of the cell viability assay are shown in Figure 12. No cytotoxic effect was observed in the untreated group in which cells were not administered any antibody, or in the control group in which cells were administered only with ADC-rat IgG. On the other hand, a significant cytotoxic effect was induced in the group in which CLDN16-expressing ovarian cancer cell lines were treated with a combination of anti-CLDN16 monoclonal antibody and ADC-rat IgG. These results demonstrate that the anti-CLDN16 antibody of the present invention can specifically kill CLDN16-expressing ovarian cancer cells.

[0182] Experimental Example 9: Immunohistochemistry in normal tissues (Objective) Using the anti-CLDN16 antibody obtained in Example 2, the expression of CLDN16 protein in human normal tissues was evaluated.

[0183] (Methods and Results) (1) Tissue specimen collection Tissue specimens were collected and CLDN16 expression was evaluated from 15 patients who underwent pathological autopsy at Fukushima Medical University Hospital between 2013 and 2014. The collection of tissue materials was approved by the Fukushima Medical University Ethics Committee (approval number 2019-0311) and was carried out in compliance with ethical guidelines for clinical research.

[0184] (2) Immunohistochemical staining and evaluation of CLDN16 expression level All collected human normal tissues were fixed in 10% formalin, embedded in paraffin, and subjected to hematoxylin eosin (HE) staining and immunohistochemical staining. Immunohistochemical staining was performed in the same manner as in Example 3.

[0185] An example of CLDN16 staining results in human normal tissues is shown in Figure 13. It was revealed that CLDN16 is expressed in some normal tissues, such as the kidney, but not in other normal tissues (brain, heart, liver, lung, ovary, and thyroid).

[0186] Example 10: Immunohistochemistry of ovarian cancer tissue, and disease-free survival analysis and clinicopathological analysis based thereon (Objective) To detect CLDN16 protein in ovarian cancer tissue using the anti-CLDN16 antibody obtained in Example 2, and to clarify the relationship between CLDN16 expression and prognosis.

[0187] (Methods and Results) (1) Tissue Specimen Collection. Tissue specimens were collected from 173 patients diagnosed with ovarian cancer who underwent surgery at Fukushima Medical University Hospital between 2003 and 2015, and from 76 patients diagnosed with ovarian cancer who underwent surgery at Iwaki City Medical Center between 2010 and 2015. CLDN16 expression was evaluated in a total of 249 cases. Only patients with known survival information for 3 to 5 years after diagnosis were included; deaths unrelated to the underlying disease were excluded. Tissue specimen collection was approved by the Fukushima Medical University Ethics Committee (approval number 2019-0311) and was conducted in compliance with ethical guidelines for clinical research.

[0188] (2) Immunohistochemical staining and evaluation of CLDN16 expression level All collected ovarian cancer tissues were fixed in 10% formalin, embedded in paraffin, and subjected to hematoxylin eosin (HE) staining and immunohistochemical staining. Immunohistochemical staining was performed in the same manner as in Example 3.

[0189] 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 Positive Proportion (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 score was calculated by multiplying PP and SI. Furthermore, since the majority of cases had PP=3 or PP=4, evaluation was based on the SI score, with SI scores of 0 / 1 / 2 classified as the low CLDN16 group and SI score of 3 classified as the high CLDN16 group.

[0190] An example of the CLDN16 staining results in ovarian cancer tissues (negative, weakly positive, moderately positive, and strongly positive) is shown in Figure 14, and the analysis results of SI, PP, and IRS are shown in Figure 15. It was revealed that CLDN16 is expressed in the majority of ovarian cancer cases, and that moderate or higher expression was observed in more than 80% of ovarian cancer cases.

[0191] (3) Clinicopathological analysis Clinicopathological analysis was performed on 249 ovarian cancer patients. CLDN16 was evaluated based on the SI score as in (2) above, and analysis was performed using StatFlex Ver. 7.0.11 (Artec, Osaka).

[0192] First, the results of analyzing the association between each clinicopathological factor and CLDN16 expression (low or high CLDN16 expression) are shown in Figures 16 and 17. In Figures 16 and 17, high CLDN16 expression showed a significant correlation with stage (FIGO stage), histological type, pT classification (pT), and peritoneal dissemination, but did not show a significant correlation with cancer antigen 125 (CA-125), a conventionally known representative ovarian cancer marker.

[0193] (4) Analysis of Recurrence-Free Survival Rates. Next, the overall survival (OS), disease-free survival (DFS), and relapse-free survival (RFS) rates were calculated for each year after surgery for the low CLDN16 group (84 cases) and the high CLDN16 group (165 cases) 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.

[0194] The results of analyzing the recurrence-free survival rate are shown in Figure 18. These results indicate that ovarian cancer patients in the low CLDN16 group tended to have a good prognosis after ovarian cancer resection surgery, whereas ovarian cancer patients in the high CLDN16 group tended to have a poor prognosis after ovarian cancer resection surgery.

[0195] Next, univariate and multivariate analyses for disease-free survival (DFS) were performed using a logistic regression model. The results are shown in Figure 19. This suggests that high expression of CLDN16 may be associated with disease-free survival.

[0196] Example 11: Verification of reactivity of anti-CLDN16 antibody with mouse CLDN16 (Objective) The anti-CLDN16 antibody obtained in Example 2 was produced using a peptide fragment derived from human CLDN16 protein as an antigen. In this Example, we verify that this anti-CLDN16 antibody also reacts with mouse CLDN16. In mouse kidneys, the occludin (OCLN) protein is known to be expressed in tight junctions. Therefore, we performed double staining of mouse kidney tissue using the anti-CLDN16 antibody of the present invention and an anti-OCLN antibody to demonstrate that the anti-CLDN16 antibody of the present invention also reacts with mouse CLDN16.

[0197] (Methods and Results) Frozen sections were prepared from mouse kidneys and subjected to fluorescent immunostaining using anti-OCLN antibody (Thermo Fisher Scientific, Cat#71-1500) and anti-CLDN16 antibody obtained in Example 2. The specimens were first fixed with 4% paraformaldehyde solution for 10 minutes at room temperature and then washed with PBS. Subsequently, the specimens were incubated with 0.1% TritonX-100 / PBS solution for 10 minutes at room temperature to permeabilize the cell membrane. After washing with PBS, the specimens were incubated with 0.5% Casein / PBS solution for 10 minutes to prevent nonspecific reactions with the primary antibody. The specimens were incubated overnight at 4°C with the anti-CLDN16 antibody and anti-OCLN antibody obtained in Example 2. After washing three times for 5 minutes with PBS, the specimens were incubated with Alexa488-labeled anti-rat IgG antibody and Cy3-labeled anti-rabbit IgG antibody, both diluted 200-fold, at room temperature for 1 hour. After washing with PBS three times for 5 minutes, the sections were mounted using Fluoro-Gel II (Electron Microscopy Science) and observed under a fluorescence microscope.

[0198] The staining results are shown in Figure 20. Staining with the anti-OCLN antibody detected OCLN protein in the tight junction regions of mouse kidney epithelium. The staining pattern with the anti-CLDN16 antibody was consistent with that of the anti-OCLN antibody. These results demonstrated that the anti-CLDN16 antibody of the present invention also reacts with mouse CLDN16 in fixed kidney tissue.

[0199] Example 12: Evaluation of CLDN16 protein expression in human ovarian cancer cell lines (Objective) Using an anti-CLDN16 antibody (provided by Dr. Mikio Furuse of the National Institute for Physiological Sciences), the presence or absence of CLDN16 protein expression in representative human ovarian cancer cell lines was evaluated.

[0200] (Methods and Results) The ovarian cancer cell lines used were AMOC2 (provided by Dr. Tsuyoshi Saito of Sapporo Medical University, as described above), SKOV3 (provided by Professor Takaya Fujimori of Fukushima Medical University), OVCAR3 (RIKEN BRC, RCB2135), and OVSAHO (JCRB Cell Bank, JCRB1046). Western blotting was performed on these ovarian cancer cell lines using the same method as described in Example 5(1). In the Western blotting, HEK293T cells expressing CLDN16 were used as a positive control (PC).

[0201] The results of Western blotting are shown in Figure 21A. CLDN16 expression was strong in OVCAR3 cells and moderate in OVSAHO cells. On the other hand, CLDN16 expression was extremely weak in SKOV3 cells and almost undetectable in AMOC2 cells.

[0202] Example 13: Preparation of a human ovarian cancer cell line in which the CLDN16 gene has been knocked out (Objective) The CLDN16 gene is knocked out in the above-mentioned human ovarian cancer cell line.

[0203] (Methods and Results) To knockout the CLDN16 gene in the SKOV3 and OVSAHO cells described above, two DNA oligos, gRNA1 (SEQ ID NOs: 21 and 22) and gRNA2 (SEQ ID NOs: 23 and 24), were incubated at 98°C for 10 minutes and then allowed to anneal at room temperature for 30 minutes. The oligos were cloned into the PX459 plasmid (Add gene, pSpCas9(BB)-2A-Puro (PX459) V2.0), and the plasmid was transfected into the cells using Lipofectamine 3000 reagent (Thermo Fisher). Cells lacking the plasmid were removed using puromycin, and CLDN16 gene knockout cells were established. Western blotting was performed on the resulting cell lines using an anti-CLDN16 antibody, using the same method as described in Example 5(1).

[0204] FIG. 21B shows that no CLDN16 expression was detected in SKOV3 cells and OVSAHO cells in which the CLDN16 gene was knocked out.

[0205] Example 14: Verification of cytotoxic effect of CLDN16 antibody-drug conjugate against ovarian cancer cell lines (Objective) A CLDN16 antibody-drug conjugate is prepared by conjugating the anti-CLDN16 antibody obtained in Example 2 with an antitumor agent. The cytotoxic effect of this antibody-drug conjugate is evaluated by administering it to ovarian cancer cell lines.

[0206] (Methods and Results) (1) Preparation of CLDN16 Antibody-Drug Conjugate To conjugate the antitumor agent monomethyl auristatin F (MMAE) to the anti-CLDN16 antibody obtained in Example 2, the antibody solvent was replaced with Dulbecco's phosphate-buffered saline (Fujifilm Wako Pure Chemical Industries, Ltd., 048-29805) by dialysis using Slide-A-Lyzer™ Dialysis Cassettes, 10K MWCO (ThermoFisher, 66830). Dithiothreitol (DTT) was then added to a final concentration of 1 mM, and the mixture was shaken at 150 rpm for 30 minutes at room temperature. The DTT was then removed by redialysis using Slide-A-Lyzer™ Dialysis Cassettes. The antibody was then mixed with 0.4 mg / mL dithiobis(2-nitrobenzoic acid) at a ratio of 1:4 and allowed to stand at room temperature for 10 minutes. The absorbance at 421 nm was measured, and the thiol group content per antibody molecule was calculated to be 5.18. Next, the linker payload MC-Val-Cit-PAB-MMAE (MedChemiExpress, HY-15575) was added to a final concentration of 68.4 μM, four times the molar concentration of SH groups, and the mixture was shaken at 150 rpm at room temperature for 60 minutes. The mixture was then redialyzed using Slide-A-Lyzer™ Dialysis Cassettes to remove unreacted linker payload.

[0207] (2) Verification of Cytotoxicity The ovarian cancer cell line AMOC2 overexpressing the CLDN16 protein prepared in Example 5 (hereinafter referred to as "AMOC2:CLDN16 cells") and the ovarian cancer cell line AMOC2 transfected with the EGFP gene (hereinafter referred to as "AMOC2:EGFP cells") were adjusted to 1,000 cells / 100 μL and seeded at 5,000 cells per well in a 96-well cell culture plate. The ovarian cancer cell line OVSAHO (hereinafter referred to as "OVSAHO:CLDN16 cells") in which the CLDN16 gene was disrupted prepared in Example 13 were adjusted to 1,000 cells / 100 μL and seeded at 5,000 cells per well. KO cells) and SKOV3 (hereinafter referred to as "SKOV3:CLDN16 KOCLDN16-independent OVSAHO cells (hereinafter referred to as "control OVSAHO cells") and SKOV3 cells with undisrupted CLDN16 genes (hereinafter referred to as "control OVSAHO cells") were adjusted to 5,000 cells / 100 μL and seeded in the same manner. After 24 hours, normal rat IgG (Fujifilm Wako Pure Chemical Industries, Ltd., 147-09521) and CLDN16 antibody were added at 200 nM, and CLDN16-ADC was added at 25 nM, 50 nM, or 100 nM. After 48 hours, the medium containing no antibody-drug conjugates was replaced. After another 24 hours, the absorbance at 490 nm was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS; Promega, G3581) to determine the relative number of viable cells.

[0208] The results of the MTS assay for AMOC2 cells are shown in Figure 22B. Figure 22B shows that the viability of AMOC2:CLDN16 cells was reduced in a dose-dependent manner by the antibody-drug conjugate, whereas the viability of AMOC2:EGFP cells was not affected. These results indicate that the cytotoxic effect of the antibody-drug conjugate on AMOC2 cells was dependent on the expression of CLDN16.

[0209] The results of the MTS assay for OVSAHO cells are shown in Figure 22C. Figure 22C shows that the antibody-drug conjugate dose-dependently reduced the viability of control OVSAHO cells, while the OVSAHO:CLDN16 KO The results show that cell viability was not affected. These results indicate that the cytotoxic effect of antibody-drug conjugates on OVSAHO cells is dependent on the expression of endogenous CLDN16.

[0210] The results of the MTS assay for SKOV3 cells, which express very little endogenous CLDN16, are shown in Figure 22C. Figure 22C shows that the antibody-drug conjugate dose-dependently reduced the viability of control SKOV3 cells, while the SKOV3:CLDN16 KOThe results show that cell viability was not affected. These results indicate that the cytotoxic effect of antibody-drug conjugates on SKOV3 cells is dependent on the expression of endogenous CLDN16.

[0211] Example 15: Verification of nephrotoxicity of CLDN16 antibody-drug conjugate in vivo (Objective) CLDN16 is known to be strongly expressed in the ascending limb of the loop of Henle in normal kidneys. Therefore, the CLDN16 antibody-drug conjugate prepared in Example 14 was administered to mice to verify the presence or absence of nephrotoxicity in vivo.

[0212] (Methods and Results) Human severe combined immunodeficiency model mice, CB-17 / Icr-scid / scidJcl (SCID; CLEA Japan), were injected four times weekly into the tail vein with MMAE, normal rat IgG, CLDN16 antibody, and the CLDN16 antibody-drug conjugate (CLDN16-ADC) prepared in Example 14. The antibody dose was 0.1 mg per mouse, equivalent to 5 mg / kg, and the volume per injection was adjusted to 100 μL. MMAE was administered at 1.87 μg per injection, equivalent to the molar amount of CLDN16-ADC. After 4 weeks, the mice were euthanized and their kidneys were removed. The kidneys were fixed in 10% neutral formalin and then subjected to histological examination using HE specimens. The results are shown in Figure 23. In mice administered CLDN16-ADC, no significant changes were observed in HE images of renal tissue, as in the groups administered MMAE, normal rat IgG, or CLDN16 antibody. This indicates that CLDN16-ADC does not induce nephrotoxicity. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A cancer biomarker consisting of claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof.

2. The biomarker of claim 1, wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer.

3. A biomarker according to claim 1 or 2 for predicting the prognosis of a cancer patient having said cancer or for detecting said cancer.

4. An anti-CLDN16 antibody or a fragment thereof, comprising a heavy chain variable region comprising 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, and a light chain variable region comprising 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.

5. The anti-CLDN16 antibody or a fragment thereof according to claim 4, comprising: a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

8.

6. A kit for predicting the prognosis of cancer or for detecting cancer, comprising the anti-CLDN16 antibody or a fragment thereof according to claim 4 or 5.

7. The kit of claim 6, wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer.

8. A method for predicting the prognosis of a cancer patient, comprising: a detection step of detecting a biomarker described in claim 1 in a sample derived from the cancer patient, the method indicating a poor prognosis for the cancer patient if the sample is positive for the biomarker.

9. The method of claim 8, wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer.

10. The method of claim 8 or 9, wherein the biomarker is detected using an anti-CLDN16 antibody or a fragment thereof described in claim 4.

11. A cancer therapeutic agent comprising an anti-CLDN16 antibody or a fragment thereof as an active ingredient.

12. The cancer therapeutic agent according to claim 11, wherein the cancer is ovarian cancer, uterine cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, kidney cancer, liver cancer, pancreatic cancer, or head and neck cancer.

13. The cancer therapeutic agent described in claim 11 or 12, wherein the anti-CLDN16 antibody or a fragment thereof comprises a heavy chain variable region comprising 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, and a light chain variable region comprising 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.

14. The cancer therapeutic agent according to claim 13, wherein the anti-CLDN16 antibody or a fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

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