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WO2025075127A1 - Biomarqueur et traitement anticancéreux par anticorps - Google Patents

Biomarqueur et traitement anticancéreux par anticorps Download PDF

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WO2025075127A1
WO2025075127A1 PCT/JP2024/035559 JP2024035559W WO2025075127A1 WO 2025075127 A1 WO2025075127 A1 WO 2025075127A1 JP 2024035559 W JP2024035559 W JP 2024035559W WO 2025075127 A1 WO2025075127 A1 WO 2025075127A1
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cancer
cldn16
antibody
amino acid
seq
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英樹 千葉
幸太郎 杉本
信 小林
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Fukushima Medical University PUC
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Fukushima Medical University PUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a cancer biomarker, an anti-claudin 16 (CLDN16) antibody or a fragment thereof, and a cancer therapeutic agent, etc.
  • CLDN16 anti-claudin 16
  • Ovarian cancer In Japan, cancer is the most common cause of death, accounting for approximately 30% of all deaths. Ovarian cancer in particular is one of the gynecological cancers with the poorest prognosis, affecting 11,000 and 240,000 people annually in Japan and worldwide, respectively.
  • Ovarian cancer is difficult to detect early, and no method for early diagnosis has been established. As a result, over 70% of ovarian cancer cases are discovered as advanced cancer, and the five-year survival rate is currently low at approximately 60%.
  • One reason why early diagnosis of ovarian cancer is difficult is that no specific and highly sensitive biomarkers for ovarian cancer, or means for identifying them specifically and with high sensitivity, have been found to date.
  • Ovarian cancer is also known to have the poorest prognosis of gynecological cancers.
  • the current standard treatment for ovarian cancer involves a combination of surgery and chemotherapy, but more than half of treated patients experience recurrence.
  • the 5-year survival rate for recurrent cases is extremely low at approximately 20%, making it a major challenge to improve the prognosis of ovarian cancer patients.
  • no good biomarkers have been found that can be used to determine prognosis.
  • the object of the present invention is to provide a new biomarker that is useful for predicting the prognosis of cancer patients and detecting cancer, a means for specifically recognizing the biomarker, and a new cancer treatment agent.
  • the present inventors have searched for new biomarkers for cancer and found that claudin 16 (CLDN16) protein is highly expressed in ovarian cancer tissue.
  • CLDN16 is expressed in cancer tissues such as ovarian cancer, uterine cancer, and thyroid cancer, but is hardly detected in normal tissues including ovarian tissue, making it an extremely specific biomarker for cancer.
  • the present inventors have further attempted to develop an antibody (anti-CLDN16 antibody) capable of specifically detecting CLDN16 protein. From 66 types of antibody clones obtained by primary screening using ELISA, the present inventors found one type of antibody clone capable of detecting CLDN16 protein by immunohistochemical staining using formalin-fixed and paraffin-embedded specimens.
  • the present invention is based on the above findings and provides the following.
  • 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.
  • CLDN16 claudin 16
  • 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
  • CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:4.
  • the anti-CLDN16 antibody or a fragment thereof comprising a light chain variable region comprising: (5) The anti-CLDN16 antibody or a fragment thereof, The anti-CLDN16 antibody or 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 the prognosis of cancer or for detecting cancer, comprising the anti-CLDN16 antibody or a fragment thereof according to (4) or (5).
  • a method for predicting the prognosis of a cancer patient comprising: The method includes a detection step of detecting the biomarker described in (1) in a sample derived from the cancer patient, and indicates a poor prognosis for the cancer patient if the sample is positive for the biomarker.
  • CDR2 consisting of the amino acid sequence shown in SEQ ID NO:5
  • CDR3 consisting of the amino acid sequence shown in SEQ ID NO:6
  • the cancer therapeutic agent according to (11) or (12) comprising a light chain variable region comprising: (14)
  • the anti-CLDN16 antibody or a fragment thereof comprising: (14)
  • the cancer therapeutic agent according to (13), 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.
  • This specification includes the disclosure of Japanese Patent Application No. 2023-172752, which is the basis of the priority of this application.
  • the present invention provides a new biomarker that is useful for predicting the prognosis of cancer patients and detecting cancer, a means for specifically recognizing the biomarker, and a new cancer treatment agent.
  • Figure 1 shows the expression level of the CLDN16 gene in various human cancer tissues.
  • Figure 1A shows the expression level of the CLDN16 gene in various cancer tissues including ovarian cancer (OV), uterine cancer (UCEC), and thyroid cancer (THCA).
  • Figure 1B shows the expression level of the CLDN16 gene in ovarian cancer (OV) and thyroid cancer (THCA), as well as other cancer tissues (Others).
  • Fig. 2 shows the expression levels of the CLDN16 gene in normal tissues and cancer tissues.
  • Fig. 2A shows the expression levels of the CLDN16 gene in various normal tissues including ovary, uterus, and thyroid.
  • Fig. 2B shows the expression levels of the CLDN16 gene in various tissues including cancer tissues (Cancer (TCGA)) and normal tissues (Normal (GTEx)).
  • FIG. 3 shows the location of the antigen peptide used to generate anti-CLDN16 antibodies.
  • FIG. 3A shows that the antigen peptide is located within the first extracellular domain (EC1).
  • FIG. 3B shows an alignment of the amino acid sequences of EC1 of the human claudin family. The amino acid residues identical to those of the CLDN16 protein are shown in bold. Figure showing the antigen specificity of anti-CLDN16 antibodies.
  • FIG. 1 shows the results of verifying the cross-reactivity of anti-CLDN16 antibody, and shows the results of immunohistochemical staining of HEK293T cells expressing CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN8, CLDN9, CLDN12, CLDN19, and CLDN16, respectively, using anti-CLDN16 antibody.
  • 6 shows the amino acid sequences of the light chain variable region and the heavy chain variable region of an anti-CLDN16 antibody
  • Fig. 6A shows the amino acid sequence of the light chain variable region (SEQ ID NO: 8)
  • Fig. 6B shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 7).
  • Figure 7 shows the proliferation ability of ovarian cancer cells (AMOC2 cells) overexpressing EGFP or CLDN16.
  • 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) verifying that CLDN16 protein is overexpressed in ovarian cancer cells (AMOC2 cells) overexpressing CLDN16.
  • Figure 7B shows the results of evaluating the proliferation ability of the cells.
  • FIG. 1 shows the migration ability of ovarian cancer cells (AMOC2 cells) overexpressing EGFP or CLDN16.
  • FIG. 1 shows the invasive ability of ovarian cancer cells (AMOC2 cells) overexpressing EGFP or CLDN16.
  • This figure shows the results of flow cytometry verifying that anti-CLDN16 antibodies can recognize CLDN16 protein on the surface of live cells.
  • “293T” represents HEK293T cells
  • “293T:EGFP” represents HEK293T cells expressing EGFP
  • “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.
  • the arrowheads indicate the CLDN16 signals on the cell surface, and the arrows indicate the CLDN16 signals within the cells.
  • FIG. 14 shows representative results of immunohistochemical staining of CLDN16 in various normal tissues.
  • FIG. 14 shows representative results of immunohistochemical staining in ovarian cancer tissues (CLDN16-negative cases, CLDN16 weakly positive cases, CLDN16 moderately positive cases, and CLDN16 strongly positive cases).
  • FIG. 14A shows a negative case.
  • FIG. 14B shows a weakly positive case.
  • FIG. 14C shows a moderately positive case.
  • FIG. 14D shows a strongly positive case.
  • 15 shows the results of evaluating staining intensity (SI), positive proportion (PP), and immunoreactivity score (IRS) for 249 ovarian cancer cases.
  • Fig. 15A shows staining intensity (SI)
  • Fig. 15B shows positive proportion (PP)
  • Fig. 15A shows staining intensity (SI)
  • Fig. 15B shows positive proportion (PP)
  • FIG. 1 shows the association between clinicopathological factors and CLDN16 expression (low CLDN16 expression/high CLDN16 expression) in ovarian cancer patients. This is a continuation of Figure 16. Graphs showing Kaplan-Meier curves of relapse-free survival in ovarian cancer patients in the low CLDN16 group (165 cases) and high CLDN16 group (84 cases).
  • Figure 18A shows the overall survival (OS).
  • Figure 18B shows the disease-free survival (DFS).
  • Figure 18C shows the relapse-free survival (RFS).
  • FIG. 1 shows the results of univariate and multivariate analyses of the prognosis (DFS) of ovarian cancer patients based on clinicopathological analysis.
  • FIG. 1 shows the results of immunohistochemical staining of frozen mouse kidney tissue using an anti-OCLN antibody and the anti-CLDN16 antibody of the present invention.
  • the results of evaluating the expression of CLDN16 protein in human ovarian cancer cell lines are shown in Fig. 21A.
  • N.C.” indicates HEK293T cells as a negative control
  • P.C.” indicates HEK293T cells expressing CLDN16 (positive control).
  • Vehicle shows a negative control that does not contain an antibody or antibody-drug conjugate
  • IgG shows the result of administering rat IgG to cells
  • CLDN16-Ab-MMAE shows the result of administering anti-CLDN16 antibody bound to the antitumor agent MMAE to cells
  • CLDN16-Ab shows the result of administering anti-CLDN16 antibody not bound to the antitumor agent MMAE to cells. 1 shows that the CLDN16 antibody-drug conjugate does not induce nephrotoxicity.
  • MMAE normal rat IgG
  • CLDN16-Ab normal rat IgG
  • CLDN16-Ab normal rat IgG
  • CLDN16-Ab-MMAE CLDN16 antibody-drug conjugate
  • the present invention provides a biomarker for cancer.
  • the biomarker of the present invention comprises a claudin 16 (CLDN16) protein or a peptide fragment thereof, or a transcription product of the CLDN16 gene or a nucleic acid fragment thereof.
  • CLDN16 claudin 16
  • a transcription product of the CLDN16 gene or a nucleic acid fragment thereof which may be expressed in cancer, as a biomarker, it becomes possible to determine the malignancy of cancer, predict the prognosis of cancer patients, and further detect cancer, such as for early diagnosis.
  • cancer is not limited, but examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma.
  • Specific types of cancer include, for example, 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, pediatric tumors such as neuroblastoma, leukemia, and lymphoma.
  • the cancer is preferably 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, for example, ovarian cancer, uterine cancer, or thyroid cancer.
  • colorectal cancer includes colon cancer and rectal cancer.
  • ovarian cancer refers to cancer that occurs 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. In this specification, the cell of origin of ovarian cancer is not particularly limited.
  • stage of ovarian cancer can be classified into stage I (tumor is localized to the ovary), stage II (tumor is present in one or both ovaries and shows spread to the pelvis), stage III (tumor is present in one or both ovaries and shows peritoneal dissemination or lymph node metastasis), and stage IV (excluding peritoneal dissemination and showing distant metastasis).
  • Uterine cancer refers to uterine cancer that occurs in the uterine body, and is also called endometrial cancer because it originates from the endometrium. Uterine cancer is classified into histological types such as endometrioid cancer, serous cancer, and clear cell carcinoma depending on the state of the cancer tissue.
  • stage of uterine cancer can be classified into stage I (cancer is limited to the uterine body), stage II (cancer has invaded the cervical stroma but has not spread beyond the uterus), stage III (cancer has spread outside the uterus but has not spread beyond the small pelvic cavity or has spread to regional lymph nodes), and stage IV (cancer has spread beyond the small pelvic cavity or has clearly invaded the bladder/intestinal mucosa and/or there is distant metastasis).
  • thyroid cancer refers to cancer that occurs in the thyroid gland.
  • Thyroid cancer can be classified according to histological type into papillary cancer, follicular cancer, poorly differentiated cancer, medullary cancer, undifferentiated cancer, malignant lymphoma, etc.
  • Thyroid cancer can be classified into stages 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 exceeding 4 cm), stage III, and stage IV.
  • prognosis refers to the reduction in tumor burden, 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, 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 (hereinafter sometimes referred to as "ovarian cancer, uterine cancer, thyroid cancer, etc.” in this specification).
  • cancer treatment e.g., surgery, chemotherapy (drug therapy), or radiation therapy, etc.
  • cancer treatment e.g., surgery, chemotherapy (drug therapy), or radiation therapy, etc.
  • cancer treatment e.g., surgery, chemotherapy (drug therapy), or
  • Prognosis prediction may be a prediction of recurrence risk (e.g., recurrence-free survival), metastasis risk, survival time, survival rate after a certain period of time from surgery (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, or 20 years or later), relapse-free survival (RFS), or disease-specific survival (DFS).
  • prognosis prediction includes prediction of recurrence risk (e.g., relapse-free survival) or prediction of metastasis risk.
  • 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.
  • a "cancer patient” is, for example, a mammal, preferably a primate, and more preferably a human.
  • "Cancer patients” such as “ovarian cancer patients,” “uterine cancer patients,” and “thyroid cancer patients” are also, for example, mammals, preferably primates, and more preferably humans.
  • 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.
  • malignancy By determining the malignancy of cancer such as ovarian cancer, uterine cancer, or thyroid cancer, it becomes possible to predict the prognosis and select poor prognosis cases with high infiltration, metastasis, and recurrence potential. In this specification, the determination of malignancy also includes the prediction of prognosis.
  • 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).
  • 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 as "CLDN16 protein, etc.” in this specification).
  • CLDN16 protein a transcription product of the CLDN16 gene or a nucleic acid fragment thereof
  • the human CLDN16 protein derived from the human CLDN16 gene and the transcription product (mRNA) of the human CLDN16 gene can be biomarkers in the present invention.
  • Claudin 16 is a transmembrane protein whose N-terminus is present intracellularly and whose C-terminus is present intracellularly. CLDN16 forms tight junctions, and in normal kidney tissue, it is known to control the reabsorption of magnesium ions as an intercellular pore or ion concentration sensor.
  • the biological species from which CLDN16 is derived is not limited, but an example of such a protein is the human CLDN16 protein consisting of the amino acid sequence shown in SEQ ID NO:9.
  • amino acid identity refers to the percentage (%) of matching amino acid residues out of 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, as necessary. 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.
  • multiple 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.
  • 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 base sequence shown in SEQ ID NO:10.
  • CLDN16 genes also include 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.
  • CLDN16 genes include nucleotide sequences in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 10, or CLDN16 genes 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.
  • genes include genes consisting of a nucleotide sequence that hybridizes under highly stringent conditions with a nucleic acid fragment consisting of a portion of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10, and encoding a protein having activity functionally equivalent to that of the CLDN16 protein.
  • 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 as necessary to maximize the degree of base identity between the two.
  • the base sequence information of the CLDN16 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, based on the known base sequence information of the CLDN16 gene shown in SEQ ID NO: 10, genes with high base identity can be searched and obtained from the database.
  • CLDN16 gene transcription product means CLDN16 mRNA.
  • the mRNA may be either pre-mRNA or mature mRNA. Normally, pre-mRNA is immediately spliced in the nucleus to become mature mRNA, so the transcription product of the CLDN16 gene that is essentially the biomarker of the present invention is CLDN16 mature mRNA.
  • 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.
  • the peptide may be composed 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.
  • a peptide fragment containing an antigen epitope recognized by the anti-CLDN16 antibody of the present invention described below for example, a peptide fragment containing or included in the amino acid sequence shown in SEQ ID NO: 11 or 20, is preferred.
  • nucleic acid fragment refers to a nucleic acid fragment consisting of a part of the base sequence constituting CLDN16 mRNA, which can be identified as a fragment of CLDN16 mRNA from the base sequence constituting the fragment.
  • nucleic acid 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 bases of the full-length base sequence of CLDN16 mRNA.
  • the term "subject” refers to a human individual who provides a sample and is subjected to testing. In principle, it is an individual, but in this specification, it may also include tissues and cells of human origin. Furthermore, an individual may be not only a healthy individual, but also a patient with some kind of disease (e.g., malignant tumor), or an individual who may be affected by a disease (e.g., malignant tumor).
  • a disease e.g., malignant tumor
  • 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.
  • healthy human cells are also included in the broad definition of healthy individuals. Therefore, healthy individuals are not only referred to at the individual level, but also when they are in a healthy state at the cellular level, such as normal parts of tissue taken from a cancer patient.
  • amino acid substitution refers to substitution within a conservative amino acid group that has similar properties such as charge, side chain, polarity, and aromaticity among the 20 types of amino acids that make up natural proteins. Examples include substitutions within the uncharged polar amino acid group 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 cause changes in the properties of peptides.
  • hybridizing under high stringency 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, 100 ⁇ g/mL denatured fragmented salmon sperm DNA at 65-68°C, followed by washing in a washing solution of 2xSSC, 0.1% SDS starting at room temperature, lowering the salt concentration in the washing solution to 0.1xSSC, and raising the temperature to 68°C until no background signal is detected.
  • high stringency hybridization conditions please refer to Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • the biomarker for predicting the prognosis of cancer patients of the present invention can predict the prognosis of cancer 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 poor prognosis cases with high potential for invasion, metastasis, and/or recurrence from among cancers.
  • the cancer prognosis predicting biomarker 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 prognosis predicting biomarker for cancer patients.
  • the cancer detection biomarker of the present invention can detect any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer, with high accuracy. For example, early diagnosis or early detection of ovarian cancer, uterine cancer, or thyroid cancer becomes possible.
  • the present invention also 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 detecting any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer.
  • 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, uterine cancer, or thyroid cancer, and an anti-CLDN16 antibody or a fragment thereof for detecting any cancer, such as ovarian cancer, uterine 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 a CLDN16 protein or a peptide fragment thereof that can be expressed in highly malignant cancers, such as ovarian cancer, uterine cancer, or thyroid cancer.
  • the anti-CLDN16 antibody or a fragment thereof of the present invention can detect cancer by detecting a CLDN16 protein or a peptide fragment thereof that can be expressed in cancers, such as ovarian cancer, uterine cancer, or thyroid cancer.
  • Anti-CLDN16 Antibody refers to an antibody that exhibits immunoreactivity against the CLDN16 protein or a peptide fragment thereof.
  • 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.
  • the anti-CLDN16 antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody, so long as it recognizes the CLDN16 protein or a peptide fragment thereof and exhibits immune responsiveness.
  • a monoclonal antibody with a stable antibody titer is preferable.
  • polyclonal antibody refers to a group of multiple different immunoglobulins that can specifically bind to and recognize an antigen.
  • 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 a recombinant antibody or synthetic antibody that contains at least one pair of a light chain variable region ( VL region) and a heavy chain variable region ( VH region) contained in an immunoglobulin.
  • FR framework region
  • CDR complementarity determining region
  • the location of the epitope of the CLDN16 protein or a peptide fragment thereof recognized by the anti-CLDN16 antibody of the present invention is not particularly limited.
  • An example of an epitope recognized by the anti-CLDN16 antibody of the present invention is a peptide sequence contained in the amino acid sequence shown in SEQ ID NO: 11 or 20.
  • 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 described below.
  • 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.
  • 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
  • CDR3 consists of the amino acid sequence shown in SEQ ID NO:3.
  • 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
  • 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.
  • Amino acid sequence of the heavy chain variable region DRQMGLLLCLVTFPRCVLSQVQLEESGPGLVQPSQTLSLTCTVSGFSLTSNGVSWVRQPPGKGLEWIAAISSAGNTYYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYFCNTLYYGYNRGYFDYWGQGVMVTVSSAETTAPSVYP (SEQ ID NO: 7) Amino acid sequence of the light chain variable region: MTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVSNRFSGISDRFSGSGSGTDFT LKISRVEPDDLGVYYCGQGTQYPLTFGSGTKLEIKRADAAPTVSIFPPSMEHEMVSWTVLLIRTPKTARTA (SEQ ID NO: 8) Heavy chain CDR1 amino acid sequence: GFSLTSN (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: SSAGN (SEQ ID NO:2) Heavy chain C
  • nucleic acid 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 base sequence shown in SEQ ID NO: 18.
  • 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 base sequence shown in SEQ ID NO: 19.
  • nucleic acid encoding the CDR1, CDR2, and CDR3 of the heavy chain variable region in the above antibody clone is a nucleic acid consisting of the base sequences shown in SEQ ID NO: 12, 13, and 14, respectively.
  • nucleic acid encoding the CDR1, CDR2, and CDR3 of the light chain variable region in the above antibody clone is a nucleic acid consisting of the base sequences shown in SEQ ID NO: 15, 16, and 17, respectively.
  • Recombinant antibody refers to a chimeric antibody or a humanized antibody.
  • a “chimeric antibody” is an antibody produced 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.
  • C region constant region
  • an antibody in which the C region of a rat monoclonal antibody is replaced with the C region of a human antibody corresponds to this.
  • a specific example is an antibody in which the heavy chain variable region of a human antibody against an arbitrary antigen is replaced with the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 in the anti-CLDN16 antibody clone obtained in Example 2 above, and the light chain variable region of the human antibody is replaced with the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. This can reduce the immune response against the antibody in the human body.
  • a "humanized antibody” is a mosaic antibody in which the CDR of a human antibody is replaced with the CDR of an antibody derived from a mammal other than human.
  • 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 order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus.
  • FRs are relatively conserved regions that constitute the framework of the variable region, and the CDRs directly contribute to the antigen-binding specificity of the antibody.
  • a humanized antibody can be constructed as a human antibody that inherits the antigen-binding specificity of a rat antibody clone, for example, by replacing a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a rat-derived antibody clone with a set of CDR1, CDR2, and CDR3 in the light chain or heavy chain of a human antibody against any antigen.
  • Specific examples include antibodies in which CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3 derived from the heavy chain of the rat-derived anti-CLDN16 antibody clone obtained in Example 2 above are replaced with heavy chain CDR1, CDR2, and CDR3 of a human antibody, respectively, and CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6 derived from the light chain of the above-mentioned antibody clone are replaced with light chain CDR1, CDR2, and CDR3 of a human antibody, respectively.
  • Such humanized antibodies are derived from human antibodies except for the CDRs, and therefore can reduce the immune response against the antibody in the human body more than chimeric antibodies.
  • synthetic antibody refers to an antibody synthesized chemically or by using recombinant DNA techniques.
  • an antibody newly synthesized by recombinant DNA techniques can be mentioned.
  • scFv single chain fragment of variable region
  • diabody diabody
  • triabody triabody
  • tetrabody etc.
  • a pair of variable regions (light chain variable region VL and heavy chain variable region VH ) that form a functional antigen-binding site are located on separate polypeptide chains called light chain and heavy chain.
  • An scFv is a synthetic antibody with a molecular weight of about 35 kDa or less that has a structure in which VL and VH are linked by a flexible linker of sufficient length in an immunoglobulin molecule and are included in one polypeptide chain.
  • a pair of variable regions can self-assemble with each other to form one functional antigen-binding site.
  • An scFv can be obtained by incorporating a recombinant DNA encoding it into a vector using a known technique and expressing it.
  • a diabody is a molecule having a structure based on the dimeric structure of scFv (Holliger et al., 1993, Proc. Natl. Acad. Sci.
  • a diabody is a bivalent antibody fragment.
  • Triabodies and tetrabodies are trivalent and tetravalent antibodies, respectively, based on the scFv structure and having the trimer and tetramer structures, similar to diabodies.
  • the diabody, triabody, and tetrabody may be a multispecific antibody.
  • the term "multispecific antibody” refers to a multivalent antibody, i.e., an antibody having multiple antigen-binding sites in one molecule, each of which binds to a different epitope.
  • a diabody may be a bispecific antibody in which each antigen-binding site binds to a different epitope.
  • the anti-CLDN16 antibody of the present invention may be 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 epitope.
  • the anti-CLDN16 antibody of the present invention can also be modified.
  • Modification here includes functional modifications, such as glycosylation, that are necessary for antigen-specific binding activity, and labeling modifications that are necessary for antibody detection.
  • Glycosylation modifications on anti-CLDN16 antibodies are performed to adjust the affinity of the anti-CLDN16 antibodies for the target CLDN16 protein or a peptide fragment thereof. Specifically, for example, modifications can be made to eliminate glycosylation at a site by introducing a substitution into an amino acid residue that constitutes glycosylation in the FR of the anti-CLDN16 antibody to remove the glycosylation site.
  • labels for anti-CLDN16 antibodies include labels with 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., 3H , 14C , 35S ), or biotin or (strept)avidin.
  • fluorescent dyes e.g., rhodamine, Texas Red, Cy3, Cy5
  • fluorescent proteins e.g., PE, APC, GFP
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase
  • radioisotopes e.g., 3H , 14C , 35S
  • biotin or (strept)avidin e.g., 3H , 14C , 35S
  • the anti-CLDN16 antibody of the present invention preferably has a dissociation constant with the CLDN16 protein of 10 ⁇ 7 M or less, and preferably has a high affinity of, for example, 10 ⁇ 8 M or less, more preferably 10 ⁇ 9 M or less, and particularly preferably 10 ⁇ 10 M or less.
  • the dissociation constant can be measured using a technique known in the art. For example, it may be measured using a Biacore system (GE Healthcare) with a kinetic evaluation kit software.
  • fragment thereof refers to an antibody fragment that consists of a part of an anti-CLDN16 antibody and exhibits immune reactivity to the CLDN16 protein or a fragment thereof, similar to the anti-CLDN16 antibody, such as Fab, F(ab') 2 , and Fab'.
  • Fab is an antibody fragment produced when an IgG molecule is cleaved by papain on the N-terminal side of the disulfide bond in the hinge region, and is composed of C H1 and V H adjacent to V H of the three domains (C H1 , C H2 , C H3 ) that make up the H-chain constant region (heavy chain constant region : hereafter abbreviated as CH ) , and a full-length L chain.
  • F(ab') 2 is a dimer of Fab' produced by cleaving an IgG molecule with pepsin at the C-terminal side of the disulfide bond in the hinge region.
  • Fab' has a slightly longer H chain than Fab due to the hinge region, but has a structure substantially similar to that of Fab.
  • Fab' can be obtained by reducing F(ab') 2 under mild conditions to cleave the disulfide bond in the hinge region. All of these antibody fragments contain an antigen-binding site and therefore have the ability to specifically bind to an antigen epitope.
  • the anti-CLDN16 antibody of the present invention can be obtained by a conventional method in the art. If the amino acid sequence of the 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.
  • an antigen 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 antigen peptide").
  • an example of an antigen peptide that can be used as an immunogen for the anti-CLDN16 antibody of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 11 or 20.
  • the CLDN16 antigen peptide can be prepared, for example, using chemical synthesis or DNA recombination technology.
  • the present invention provides a kit for predicting cancer prognosis and a kit for detecting cancer.
  • the kit for predicting cancer prognosis or the kit for detecting cancer 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 kit for predicting cancer prognosis or the kit for detecting cancer of the present invention comprises, as a selected component, an antibody (hereinafter referred to as “another antibody for predicting prognosis”) against a biomarker for predicting cancer prognosis other than the CLDN16 protein or a peptide fragment thereof (hereinafter referred to as “another biomarker for predicting prognosis”).
  • 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 anti-CLDN16 antibody contained in the cancer prognosis prediction kit or cancer detection kit of the present invention may be a single type or multiple types.
  • the cancer prognosis prediction kit or cancer detection kit of the present invention may further include one or more types of other prognosis prediction antibodies or other detection antibodies, or immunoreactive fragments thereof, as selected constituents.
  • the other prognosis prediction antibodies or other detection antibodies may be any antibodies that can improve the accuracy of prognosis prediction or detection of cancer patients, such as ovarian cancer, uterine cancer, or thyroid cancer, when used in combination with the above-mentioned anti-CLDN16 antibody, and an antibody against any cancer prognosis prediction biomarker or cancer detection biomarker can be used.
  • a biomarker can be selected from known cancer biomarkers.
  • the cancer prognosis prediction kit or cancer detection kit of the present invention may contain, in addition to the essential components described above, 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.
  • the present invention provides a method for predicting the prognosis of cancer.
  • the method for predicting the prognosis of cancer of the present invention includes a detection step of detecting a biomarker consisting of a 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 determination result of whether the biomarker is positive/negative.
  • the detection step can be performed in vitro.
  • the "detection step” refers to 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").
  • detection includes measurement, qualitative, quantitative, and semi-quantitative.
  • a "sample” is taken from a subject or a group of subjects, or a healthy subject or a group of healthy subjects, and is subjected to the prognosis prediction method for cancer patients of this embodiment, and is, for example, a tissue or cell.
  • the "tissue” and “cell” to be subjected to the prognosis prediction method of the present invention are, for example, tissues and cells of a subject suffering from any cancer, such as ovarian cancer, uterine cancer, or thyroid cancer, and corresponding tissues and cells in a healthy subject.
  • the sample to be subjected to the prognosis prediction method of the present invention is, but is not limited to, a specimen taken by biopsy or resected by surgery from a subject suffering from cancer.
  • a part of the cancer e.g., tissue or cell
  • the invasive front taken by biopsy or resected by surgery.
  • the amount of sample required in the prognosis prediction method of the present invention is not particularly limited. For tissue or cells, at least 10 ⁇ g, preferably at least 0.1 mg, is desirable, and the same applies to biopsy material.
  • the sample can be prepared and processed as necessary so as to enable detection of a biomarker for predicting the prognosis of a cancer patient. For example, when detecting a biomarker by immunohistochemical staining, a paraffin-embedded section may be prepared from a patient-derived sample. Also, for example, when detecting a biomarker by Western blotting or RT-PCR, a protein extract or an mRNA extract may be prepared from a patient-derived sample.
  • Such preparation may include homogenization or cell lysis, removal of impurities by centrifugation or filtration, addition of a protease inhibitor, etc., as appropriate. Details of these processes are described in detail in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, and may be used as a reference.
  • the "measurement value of a biomarker for predicting cancer prognosis” specifically refers to the measurement value of the amount of a biomarker such as CLDN16 protein in a sample.
  • a "measurement value” refers to a measurement value obtained by measuring a biomarker.
  • the measurement value may be an absolute value expressed in units such as ng (nanogram) or ⁇ g (microgram) as the amount of protein in a sample, or may be a relative value expressed in terms of absorbance or fluorescence intensity of a labeling molecule relative to a control value, or may be a score calculated from the spatial distribution of the biomarker in the sample (e.g., the staining pattern) using a certain formula.
  • the control value may be a measurement 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.
  • the following describes the steps of the detection process of this embodiment: (1) measuring a biomarker such as CLDN16 protein, and (2) determining whether a biomarker such as CLDN16 protein is positive or negative.
  • 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 includes measuring the presence or absence of expression, or the magnitude of the expression amount or expression concentration.
  • the term "measurement” includes detection, qualitative, quantitative, and semi-quantitative.
  • the measurement method may be any known protein quantification method, and includes, but is not limited to, immunological detection, aptamer analysis, and mass spectrometry.
  • an "immunological detection method” is a method for quantifying a target molecule using an antibody or a binding fragment thereof that specifically binds to the target molecule.
  • Known immunological detection methods include, for example, enzyme immunoassay (including ELISA and EIA), fluorescent immunoassay, radioimmunoassay (RIA), luminescence immunoassay, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), immunoturbidimetry, latex agglutination immunoassay, latex turbidimetry, erythrocyte agglutination reaction, particle agglutination reaction, gold colloid method, capillary electrophoresis, Western blot, and immunohistochemistry (immunostaining), and any of these detection methods may be used in this method. Although not limited thereto, immunohistochemistry is preferred.
  • the method for quantifying the CLDN16 protein or its peptide fragments is not limited. For example, it may be quantified as a score calculated using a certain formula based on the observation of the staining pattern of tissue sections.
  • the antibody used in the immunological detection in this step may be either a monoclonal antibody or a polyclonal antibody, 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, for example, 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.
  • the antibody used in the immunological detection in this step is the anti-CLDN16 antibody or a fragment thereof described above.
  • the "aptamer analysis method” is a method for quantifying a target molecule, a biomarker protein for predicting prognosis, using an aptamer that binds strongly and specifically to a target substance due to its three-dimensional structure.
  • aptamers can be broadly divided into nucleic acid aptamers and peptide aptamers, but either type of aptamer is acceptable.
  • Nucleic acid aptamer refers to an aptamer composed of nucleic acid.
  • 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.
  • examples of the aptamer include an anti-CLDN16 RNA aptamer or an anti-CLDN16 DNA aptamer.
  • Nucleic acid aptamers can be prepared using methods known in the art, such as the SELEX (systematic evolution of ligands by exponential enrichment) method, with the CLDN16 protein or a portion thereof as the target molecule.
  • SELEX systematic evolution of ligands by exponential enrichment
  • the SELEX method is a well-known method, and a specific method may be carried out, for example, according to Pan et al. (Proc. Natl. Acad. Sci. U.S.A., 1995, 92: 11509-11513).
  • 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.
  • examples include anti-CLDN16 peptide aptamers.
  • Peptide aptamers may be produced based on known production methods in the field. For example, see Whaley, S. R., et al., Nature, 2000, 405, 665-668. Usually, they can be produced using phage display or cell surface display methods.
  • the above-mentioned antibody or aptamer may be labeled as necessary. Labeling may be performed using a labeling substance known in the art. In the case of an antibody or peptide aptamer, it may be labeled with, for example, a fluorescent dye (fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5), a fluorescent protein (e.g., PE, APC, GFP), an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), a radioisotope (e.g., 3H , 14C , 35S ), or biotin or (strept)avidin.
  • a fluorescent dye fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5
  • a fluorescent protein e.g., PE, APC, GFP
  • an enzyme e.g., horseradish peroxidase, alkaline phosphat
  • examples of the labeling substance include radioisotopes (e.g., 32 P, 3 H, 14 C), DIG, biotin, fluorescent dyes (e.g., FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA), and luminescent substances (e.g., acridinium ester).
  • radioisotopes e.g., 32 P, 3 H, 14 C
  • DIG diotin
  • biotin e.g., fluorescent dyes (e.g., FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA)
  • luminescent substances e.g., acridinium ester
  • 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 mass spectrometry (ICP-MS).
  • LC-MS high performance liquid chromatography mass spectrometry
  • LC-MS/MS high performance liquid chromatography tandem mass spectrometry
  • GC-MS gas chromatography mass spectrometry
  • GC-MS/MS gas chromatography tandem mass spectrometry
  • CE-MS capillary electrophoresis mass spectrometry
  • ICP-MS inductively coupled plasma mass spectrometry
  • the measurement method may be a 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.
  • Nucleic acid amplification method 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.
  • nucleic acid amplification methods include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction).
  • 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 the target nucleic acid to be detected is used as a probe, and the target nucleic acid or its fragment is detected and quantified by utilizing base pairing between the nucleic acid and the probe.
  • detection means such as the Northern hybridization method (Northern blot hybridization method), the in situ hybridization method, and the microarray method.
  • Nucleic acid strands such as primers and probes can be appropriately designed based on known biomarker sequence information using methods known to those skilled in the art, and can be obtained using known production methods such as chemical synthesis.
  • the method for determining positivity/negative based on a measured value is not limited.
  • 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.
  • 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, it is determined to be negative.
  • the cutoff value is 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 the receiver operating characteristic curve (ROC curve). There are no particular limitations on the method for setting the cutoff value.
  • 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 measurement value 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.
  • 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 from healthy subjects not suffering from any cancer such as ovarian cancer, uterine cancer, or thyroid cancer, or the measurement values of samples from a group of healthy subjects, and a positive determination may be made when the measurement value of the subject is higher than the cutoff value.
  • the measurements obtained from the control group can also 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 measurements obtained from the control subjects is used as the cutoff value, and values above this value are considered positive and values below this value are considered negative, then if the subject's measurement is at or above the 95th percentile, it can be determined to be positive.
  • the measurements of control healthy subjects do not necessarily need to be taken 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, it is possible to reuse measurements of control healthy subjects that have been previously measured.
  • the number of stained tumor cells relative to the total number of tumor cells exceeds a certain percentage (e.g., 10%, 15%, or 20%), it may be determined to be positive, and if the number of stained tumor cells relative to the total number of tumor cells is equal to or less than that certain percentage, it may be determined to be negative.
  • a certain percentage e.g. 10%, 15%, or 20%
  • the prognosis of a cancer patient is predicted or indicated based on the detection results of a biomarker such as CLDN16 protein obtained in the above detection step.
  • a biomarker such as CLDN16 protein
  • a sample derived from a cancer patient such as ovarian cancer, uterine cancer, or thyroid cancer
  • a biomarker such as CLDN16 protein
  • 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 highly likely to have a good prognosis.
  • poor prognosis refers to poor clinical outcome (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).
  • 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.
  • survival rate refers to cumulative survival rate.
  • good prognosis refers to a good clinical outcome.
  • the recurrence-free survival rate or survival rate 5 years after cancer resection surgery may be 50% or more, 55% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%.
  • a biomarker for predicting the prognosis of a cancer patient 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 result and the detection result of the biomarker such as CLDN16 protein.
  • the biomarker for predicting the prognosis of a cancer patient other than the biomarker such as CLDN16 protein and the antibodies for detecting it are as described above.
  • cellular atypia refers to a deviation from a normal cell structure, specifically an increase in the nucleoplasmic ratio, uneven size of cells or nuclei, irregular nuclear shape, an increase in nuclear chromatin, an increase or increase in nucleoli, an increase in nuclear division figures, and/or the appearance of abnormal nuclear division figures.
  • structural atypia refers to a deviation from a normal tissue structure, that is, irregularity of tissue structure.
  • the method of detecting cellular atypia or structural atypia is not limited. For example, it can be visualized using hematoxylin-eosin staining.
  • Invasion refers to the continuous progression of a malignant tumor while destroying the surrounding normal tissues and organs, and can be determined by the unclear boundary with the surrounding tissue.
  • Metalastasis is the discontinuous spread of a malignant tumor from the primary site to an organ distant from the primary site.
  • the prognosis prediction method of the present invention by examining a biological sample extracted 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, the risk of recurrence or metastasis can be determined, and based on the results, a treatment plan (e.g., type of anticancer drug, dosage, administration interval, etc.) can be determined, or the interval for testing for cancer recurrence and metastasis can be determined.
  • the patient may be subjected to surgery or anticancer drug therapy (for example, a treatment method including administration of an anticancer drug such as paclitaxel or carboplatin) to prevent cancer recurrence, improve the prognosis, or improve the survival rate. Therefore, a method for preventing cancer recurrence, improving the prognosis, or improving the survival rate, which includes performing at least one of the above on a cancer patient shown to have a poor prognosis by the method of the present invention, is also provided. In addition, if a cancer patient's prognosis is predicted to be poor, the frequency of testing can be increased to detect cancer recurrence early.
  • anticancer drug therapy for example, a treatment method including administration of an anticancer drug such as paclitaxel or carboplatin
  • the prognosis prediction method of the present invention also provides a method for assisting in predicting the prognosis of cancer. It also provides a method for determining the malignancy of a cancer in a subject, and a method for assisting in determining the malignancy of a cancer in a subject.
  • the method of the present invention can be used in combination with other methods (e.g., X-rays; 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., classification by stage, tumor size, the presence or absence of lymph node metastasis, histological grade, etc.).
  • other methods e.g., X-rays; 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
  • methods for treating cancer and methods for improving the prognosis of cancer patients are provided, which include a step of predicting the prognosis of cancer using the method of the present invention.
  • the present invention provides a method for detecting cancer.
  • the method for detecting cancer of the present invention includes a detection step of detecting a biomarker consisting of a 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 possibility that the subject is affected by cancer is indicated based on the determination result of whether the biomarker is positive/negative.
  • the detection step can be performed in vitro.
  • the method for detecting cancer of the present invention enables early diagnosis of cancer.
  • the "detection process” refers to a process 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”).
  • a “sample” is something collected from a subject or a group of subjects, or a healthy subject or a group of healthy subjects, and is subjected to the cancer detection method of this embodiment, and is, for example, a body fluid.
  • body fluids include blood (including serum, plasma, and interstitial fluid), lymph, extracts of each tissue or cell, ascites, pleural effusion, saliva, and urine.
  • Blood may be serum or plasma prepared from blood.
  • the "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.
  • the following describes the steps of the detection process of this embodiment: (1) measuring a biomarker such as CLDN16 protein, and (2) determining whether a biomarker such as CLDN16 protein is positive or negative.
  • 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 amount) includes measuring the presence or absence of expression, or the magnitude of the expression amount or expression concentration, etc.
  • the measurement method may be any known protein quantification method, and includes, but is not limited to, the above-mentioned immunological detection method, aptamer analysis method, and mass spectrometry.
  • 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.
  • a sample is positive for a biomarker such as CLDN16 protein obtained in the above detection step, it is indicated 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 is indicated that the subject is low likely to have cancer.
  • 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 result and the detection result of the biomarker such as CLDN16 protein.
  • examples of the biomarker for detecting cancer other than the biomarker such as CLDN16 protein and the antibodies for detecting them are as described above.
  • the cancer detection method of the present invention by examining a sample of body fluids (ascites, serum, etc.) isolated from a subject, it is possible to determine whether the subject is likely to have cancer. Early diagnosis of cancer is possible based on the results of the cancer detection method of the present invention, making it possible to start treatment at an early stage. If a high probability of cancer is shown 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.
  • a method of treating cancer which includes performing at least one of drug therapy and radiation therapy on a cancer patient shown by the method of the present invention to have a high probability of cancer.
  • the cancer detection method of the present invention can be used in combination with other methods (e.g., X-ray photography; ultrasound (echo) examinations such as transvaginal ultrasound; endoscopy; mammography; palpation; internal 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 tissue diagnosis; tumor marker tests; and/or genetic diagnosis) or other factors (e.g., classification by stage, tumor size, the presence or absence of lymph node metastasis, histological grade, etc.). Combining with other methods can improve the accuracy of the cancer detection method of the present invention.
  • other methods e.g., X-ray photography; ultrasound (echo) examinations such as transvaginal ultrasound; endoscopy; mammography; palpation; internal examination; rectal examination; CT examination; MRI examinations such as pelvic MRI examinations, imaging examinations such as PET-CT examinations; blood tests; path
  • the present invention provides a cancer therapeutic agent.
  • the cancer therapeutic agent of the present invention contains an anti-CLDN16 antibody or a fragment thereof as an active ingredient, and can treat cancers such as ovarian cancer, uterine cancer, and thyroid cancer in cancer patients.
  • the anti-CLDN16 antibody or a fragment thereof in this aspect is similar to the above-mentioned descriptions of "(1) Anti-CLDN16 antibody” and "(2) Fragment thereof", and detailed description here is omitted.
  • the anti-CLDN16 antibody or a fragment thereof preferably has cytotoxic activity, thereby exerting an antitumor effect.
  • the anti-CLDN16 antibody or a fragment thereof can be conjugated to an antitumor agent.
  • the antibody can be conjugated to the antitumor agent via a spacer having a group reactive with an amino group, a carboxyl group, a hydroxyl group, a thiol group, etc. (e.g., a succinimidyl group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, a hydroxyl group, etc.).
  • Antitumor agents include known antitumor agents and their pharma- ceutically acceptable salts or derivatives.
  • known antitumor agents include paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, bryostatin, and callystatin.
  • the present invention also provides a composition for treating cancer that contains, in addition to the above-mentioned cancer therapeutic agent, other cancer therapeutic agents and a pharma- ceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to additives commonly used in the field of formulation technology. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, dissolution aids, and other additives.
  • the solvent may be, for example, water or any other pharma- ceutically acceptable aqueous solution, or a pharma-ceutically acceptable organic solvent.
  • aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer.
  • adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.
  • the above-mentioned carriers are used to avoid or inhibit the decomposition of the active ingredient, the anti-CLDN16 antibody or a fragment thereof, by enzymes in the body, to facilitate formulation and administration, and to maintain the dosage form and efficacy, and may be used appropriately as needed.
  • the subjects to which the cancer therapeutic agent or composition for cancer treatment of the present invention is administered are mammals, including, for example, primates, pet animals, livestock, and sports animals, and humans are particularly preferred.
  • the preferred administration form of the cancer therapeutic agent or composition for cancer treatment of the present invention may be administered orally or parenterally.
  • parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, and administration by blood transfusion.
  • the administration form can be appropriately selected depending on the age, weight, sex, symptoms, etc. of the patient.
  • the dosage of the antibody or fragment thereof can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration, or from the range of 0.001 mg/body to 100,000 mg/body per patient, but is not necessarily limited to these values.
  • 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 may be administered every day or at appropriate time intervals (e.g., 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 of 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, It 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 (for example, 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10
  • Example 1 Identification of genes specifically expressed on the surface of ovarian cancer cells (the purpose) Identify new biomarkers for cancer.
  • Figure 1 shows the expression level 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).
  • OV Ovarian serous cystadenocarcinoma
  • THCA thyroid carcinoma
  • UCEC uterine corpus endometrial carcinoma
  • ovarian cancer OV
  • uterine cancer UCEC
  • thyroid cancer THCA
  • lung cancer Lung adenocarcinoma
  • LUSC Lung squamous cell carcinoma
  • esophageal cancer ESA
  • gastric cancer STAD, Stomach adenocarcinoma
  • colorectal cancer including COAD, Colon adenocarcinoma and READ, Rectum adenocarcinoma
  • bladder cancer BLCA, Bladder Urothelial Carcinoma
  • cervical cancer CEC, Cervical squamous cell carcinoma
  • Increased expression of the CLDN16 gene was also detected in human cerebrospinal fluid (CEF) carcinoma and endocervical adenocarcinoma, renal cancer (KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma), liver cancer (LIHC, Liver hepatocellular carcinoma),
  • Figure 2 shows the expression level of the CLDN16 gene in various adult normal and cancer tissues. Strong expression of the CLDN16 gene was not detected in various normal tissues, including the ovary, uterus, and thyroid. Expression of the CLDN16 gene in various normal tissues (Normal (GTEx)) was extremely weak compared to cancer tissues (Cancer (TCGA)).
  • expression of the CLDN16 gene can be a biomarker that can specifically and sensitively detect 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, especially ovarian cancer, uterine cancer, and thyroid cancer.
  • Example 2 Preparation of anti-CLDN16 monoclonal antibody (the purpose)
  • Anti-CLDN16 monoclonal antibody hereinafter, sometimes simply referred to as "anti-CLDN16 antibody”
  • anti-CLDN16 antibody a monoclonal antibody that can detect CLDN16 protein specifically and with high sensitivity.
  • a 10 mg/mL KLH solution was prepared by dissolving 2 mg of ImjectTM Maleimide Activated mcKLH (Thermo Fisher Scientific) in 200 ⁇ L of ultrapure water.
  • the antigen peptide was dissolved in ultrapure water to prepare a 5 mg/mL antigen peptide solution.
  • 200 ⁇ L of each of the KLH solution and antigen peptide solution were mixed and left to stand at room temperature for 2 hours.
  • the mixture was transferred to a boiled dialysis membrane and dialyzed using PBS as the external solution. The resulting solution was used as the antigen solution.
  • PEG polyethylene glycol
  • Cell fusion and cell culture Iliac lymph nodes were removed 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 ⁇ m cell strainer (BD Falcon). Approximately 10 7 mouse multiple myeloma cell line SP2 was added to the dish, mixed well with a pipette, centrifuged at 1,200 rpm/min for 5 minutes, and the supernatant was aspirated. PEG solution at 37°C was slowly dripped over about 1 minute, left for 2 minutes, and then 9 mL of DMEM medium was slowly dripped over 5 minutes.
  • BD Falcon 100 ⁇ m cell strainer
  • Hybridoma medium (78% GIT medium [Fujifilm Wako Industries, Ltd.], 2% HAT Supplement [Thermo Fisher Scientific], 10% BM Condimmed H1 Hybridoma Cloning Supplement [Roche], 10% fetal bovine serum) was added to 40 mL, and 100 ⁇ L of the medium was seeded into four 96-well culture dishes and then cultured in a CO2 incubator at 37°C.
  • the peptide solution containing the antigen peptide solution was adjusted to 3 ⁇ g/mL, and 50 ⁇ L was added to each well of a 96-well ELISA plate and left to stand overnight at 4 ° C. Then, the antigen peptide solution was removed from each well, and the plate was washed once with 200 ⁇ L of 0.1% Tween 20-added Tris-HCl buffer (TBS-T), and then 200 ⁇ L of blocking solution (1% bovine serum albumin/TBS) was added and left to stand at 37 ° C for 1 hour.
  • TBS-T 0.1% Tween 20-added Tris-HCl buffer
  • blocking solution 1% bovine serum albumin/TBS
  • the blocking solution was removed from each well, and the plate was washed once with 200 ⁇ L of TBS-T, and then 50 ⁇ L of culture supernatant was added and reacted at 37 ° C for 1 hour. After removing the culture supernatant from each well, the plate was washed three times with 200 ⁇ L of TBS-T. Next, 50 ⁇ L of ECLTM Rat IgG, HRP-linked whole antibody (Cytiva) diluted 2,000-fold with the blocking solution was added to each well as a secondary antibody, and the reaction was allowed to proceed for 1 hour at 37° C.
  • the secondary antibody solution was removed from each well, and the wells were washed three times with TBS-T, after which coloring was carried out using TMB Substrate Set (BioLegend) according to the method recommended by the manufacturer, and the absorbance at a wavelength of 490 nm (OD 490 ) was measured.
  • TMB Substrate Set BioLegend
  • 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.
  • Example 3 Verification of antigen specificity of anti-CLDN16 monoclonal antibody clones (the purpose) The antigen specificity of the anti-CLDN16 monoclonal antibody clone obtained in Example 2 will be verified by immunohistochemical staining.
  • the specimens were deparaffinized in xylene for 10 min, then dexylened in 100% ethanol. Endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide/methanol for 10 min, followed by washing in Tris-HCl buffer (TBS) for 5 min. Antigen retrieval was performed in a microwave oven for 10 min in 10 mM Tris, 1 mM ethylenediaminetetraacetic acid solution, pH 9.0.
  • the specimens were washed in TBS for 5 min and nonspecific reactions of the primary antibody were prevented using an endogenous avidin-biotin blocking kit (415041, Nichirei Biosciences) and a casein (C3400, EMD Millipore) solution diluted to 0.5% in TBS.
  • the former was reacted twice for 10 min, and the latter was reacted for 10 min, all at room temperature.
  • the specimens were then incubated overnight at 4°C using the anti-CLDN16 monoclonal antibody obtained in Example 2 as the primary antibody.
  • TBS2T modified Tris-HCl buffer
  • 11.6 mM Trisma base 11.6 mM Trisma base
  • 38 mM Trisma hydrochloride 38 mM Trisma hydrochloride
  • 300 mM sodium chloride 0.1% Tween 20
  • biotin-labeled tyramide diluted 100-fold in tyramide amplification solution (20% dextran sodium sulfate 5000, 0.2 M Tris-HCl pH 8.8).
  • TBS2T horseradish peroxidase-labeled streptavidin
  • P0397 horseradish peroxidase-labeled streptavidin
  • TBS2T modified Tris-HCl buffer
  • the specimens were reacted in a DAB substrate kit (425011, Nichirei Biosciences) until a suitable staining image was obtained.
  • the specimens were shaken and washed for 5 seconds in Tissue-Tec (registered trademark) Hematoxylin 3G (Sakura Finetech Japan Co., Ltd.) solution, and then washed for 10 minutes with running water.
  • the specimens were then shaken and washed for 5 seconds in a 0.5% hydrochloric acid/70% ethanol solution to remove excess hematoxylin.
  • the specimens were dehydrated using 100% ethanol, cleared with xylene, and prepared into slides using an automatic slide mounting machine (Shiraimatsu Kikai Co., Ltd.).
  • HEK293T cells were prepared that did not incorporate a vector for expressing CLDN16 protein, HEK293T cells that incorporated 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 that incorporated a vector expressing human wild-type CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN8, CLDN9, CLDN12, or CLDN19 protein, and immunostaining was performed on each cell.
  • Example 2 The above results demonstrate 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 immunohistochemistry and does not show cross-reactivity with other claudins.
  • Example 4 Determination of CDR sequence of anti-CLDN16 monoclonal antibody (the purpose) The CDR sequence of the anti-CLDN16 monoclonal antibody clone obtained in Example 2 is determined.
  • the sequences of the heavy and light chain variable regions and each CDR of the anti-CLDN16 monoclonal antibody clone were determined. Specifically, the hybridoma was first mixed with 1 mL of TRIzol (registered trademark) reagent (Thermo Fisher Scientific) and 200 ⁇ L of chloroform, and centrifuged at 4° C. and 15,000 rpm for 15 minutes. Next, the aqueous phase containing the mRNA was mixed with an equal amount of isopropanol, and centrifuged at 4° C. and 15,000 rpm for 15 minutes to extract the mRNA. Finally, 1 mL of 70% ethanol was added to desalt the mRNA, which was dissolved in 50 ⁇ L of water, and the concentration of the mRNA was quantified using NanoDrop (registered trademark) 2000.
  • the 5'-Full RACE Core Set (Takara Bio Inc.) was used to determine the CDRs. All experiments were performed using the instruction manual and accompanying reagents, and the resulting DNA fragments were cloned into the pGEM (registered trademark)-T Easy Vector (Promega) before DNA sequencing. DNA sequencing was performed by Macrogen Japan Inc., and CDRs were identified according to the Kabat antibody numbering system.
  • Amino acid sequence of the heavy chain variable region DRQMGLLLCLVTFPRCVLSQVQLEESGPGLVQPSQTLSLTCTVSGFSLTSNGVSWVRQPPGKGLEWIAAISSAGNTYYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYFCNTLYYGYNRGYFDYWGQGVMVTVSSAETTAPSVYP (SEQ ID NO: 7) Amino acid sequence of the light chain variable region: MTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVSNRFSGISDRFSGSGSGTDFT LKISRVEPDDLGVYYCGQGTQYPLTFGSGTKLEIKRADAAPTVSIFPPSMEHEMVSWTVLLIRTPKTARTA (SEQ ID NO: 8) Heavy chain CDR1 amino acid sequence: GFSLTSN (SEQ ID NO: 1) Heavy chain CDR2 amino acid sequence: SSAGN (SEQ ID NO:2) Heavy chain C
  • Example 5 Functional analysis of CLDN16 protein (the purpose)
  • AMOC2 ovarian cancer cell line
  • AMOC2 was used as the cell line for transduction.
  • AMOC2 was provided by Dr. Tsuyoshi Saito of Sapporo Medical University.
  • the culture medium for AMOC2 cells was Dulbecco's modified Eagle's medium (DMEM, Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum and 1% mixture of penicillin, streptomycin, and amphotericin B (Fujifilm Wako Pure Chemical Industries).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS + 1% PS amphotericin B
  • a polybrene solution 12996-81, Nacalai Tesque
  • the cell proliferation ability was evaluated by the following method. Each cell line obtained by the method described in (1) above was cultured on a 10 cm dish, and a formalin-fixed paraffin-embedded specimen was prepared by the same method as in Example 3 above. The specimen was deparaffinized in xylene for 10 minutes, and then dexylened in 100% ethanol. The specimen was treated with 0.3% hydrogen peroxide/methanol for 10 minutes to inactivate endogenous peroxidase, and then washed with phosphate-buffered saline (PBS) for 5 minutes. The specimen was placed in a 10 mM citric acid solution, pH 6.0, and antigen retrieval was performed for 10 minutes using a microwave oven.
  • PBS phosphate-buffered saline
  • the specimen was washed with PBS for 5 minutes, and a non-specific reaction of the primary antibody was prevented using a solution of skim milk (Morinaga Milk Industry) diluted to 5% with PBS.
  • the specimen was then reacted overnight at 4°C with Ki-67 antibody (M7240, Agilent Technologies) as the primary antibody.
  • Ki-67 antibody M7240, Agilent Technologies
  • the specimens were washed three times for 5 minutes with PBS, and reacted with Histofine Simple Stain Rat MAX-PO (MULTI) (424151, Nichirei Biosciences) as a secondary antibody for 60 minutes at room temperature.
  • the specimens were reacted in a DAB substrate kit (425011, Nichirei Biosciences) until an appropriate stained image was obtained.
  • a DAB substrate kit (425011, Nichirei Biosciences)
  • the specimens were shaken and washed for 5 seconds in a Tissue-Tec (registered trademark) Hematoxylin 3G (Sakura Finetech Japan Co., Ltd.) solution, and washed with running water for 10 minutes.
  • the specimens were shaken and washed for 5 seconds in a 0.5% hydrochloric acid/70% ethanol solution to remove excess hematoxylin.
  • the specimens were dehydrated using 100% ethanol, cleared with xylene, and prepared into slides using an automatic slide mounting machine (Shiraimatsu Kikai Co., Ltd.). The number of positive cells was calculated using ImageJ, and statistical analysis was performed.
  • the cell migration ability assay was performed by the following method. First, 500 ⁇ L 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 in it. Furthermore, 1 ⁇ 10 5 cultured cells were placed inside the 24-well culture insert, and cultured for 24 hours in a culture incubator (37 °C, 5% CO 2 , 95% Air). To measure the number of migrating cells, the 24-well culture insert was first washed with PBS and 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, which was used as the number of migrating cells.
  • the invasive ability assay was performed by the following method. First, 500 ⁇ L of DMEM (10% FBS + 1% PS) medium was placed in each well of a 24-well plate, and a BioCoat (registered trademark) Matrigel Invasion Chamber (#354480, Corning) was placed. Furthermore, 1 ⁇ 10 5 cultured cells were placed inside the BioCoat (registered trademark) Matrigel Invasion Chamber, and cultured for 24 hours in a culture incubator (37 ° C, 5% CO 2, 95% Air).
  • the BioCoat (registered trademark) Matrigel Invasion Chamber was washed with phosphate buffered saline (PBS) and 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 for 10 minutes at room temperature. Finally, the cells inside the BioCoat (registered trademark) Matrigel invasion chamber were removed with a cotton swab, and the number of cells outside was calculated using ImageJ, which was taken as the number of invaded cells.
  • PBS phosphate buffered saline
  • Example 6 Verification of antibody binding ability to CLDN16 protein on the surface of living cells (the purpose) The binding specificity of the anti-CLDN16 monoclonal antibody obtained in Example 2 to the CLDN16 protein on the surface of living cells will be verified by flow cytometry.
  • HEK293T cells without a vector HEK293T cells with a vector expressing human wild-type CLDN16 protein, and HEK293T cells with a vector expressing EGFP protein were prepared.
  • Flow cytometry was performed as follows. Anti-CLDN16 antibody or normal rat IgG antibody (147-09521, Fujifilm Wako Pure Chemical) was added as the primary antibody to the cell suspension to a final concentration of 100 ⁇ g/mL. The antibody-bound cells were then stained with Alexa647-labeled anti-rat IgG antibody (563-78551, Fujifilm Wako Pure Chemical), and the fluorescence was analyzed by flow cytometry (On-chip Flow, On-chip Biotechnologies).
  • Example 7 Verification of uptake of anti-CLDN16 antibody into cancer cells (the purpose) We will verify whether the anti-CLDN16 monoclonal antibody obtained in Example 2 can be taken up into cancer cells.
  • CLDN16-expressing ovarian cancer cell line that overexpresses 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).
  • 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 ⁇ g/mL.
  • the cells were cultured for 3 hours in a culture incubator (37°C, 5% CO 2 , 95% air), washed three times with PBS, and then fixed for 10 minutes with 4% paraformaldehyde solution. After washing three times with PBS, the cell membrane was permeabilized for 10 minutes with 0.1% TritonX-100/PBS solution, and blocked for 30 minutes with 5% skim milk.
  • Cy3-labeled anti-rat IgG antibody (564-71901, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to stain the antibody-bound cells, and the antibody taken up into the cancer cells was detected.
  • Example 8 Verification of anti-CLDN16 antibody-induced cancer cell cytotoxicity (the purpose) It will be verified that the anti-CLDN16 monoclonal antibody obtained in Example 2 exhibits a cytotoxic effect against cancer cells.
  • CLDN16-expressing ovarian cancer cells An ovarian cancer cell line overexpressing wild-type CLDN16 protein was prepared by a method similar to that described in Example 5(1) (hereinafter referred to as "CLDN16-expressing ovarian cancer cells").
  • CLDN16-expressing ovarian cancer cells were seeded in 96-well plates and cultured in DMEM (10% FBS + 1% PS) medium. 1.5 ⁇ g of anti-rat IgG antibody conjugated with the cytotoxic toxin MMAF (Monomethyl Auristatin F) (AR101-AF, Moradec; hereafter referred to as "ADC-rat IgG") and 3 ⁇ g of rat anti-CLDN16 monoclonal antibody were added to the 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 converted to a colored formazan product when reduced by viable cells, and the number of viable cells in the medium can be evaluated by measuring the absorbance at 490 nm.
  • MTS reagent CellTiter96 Aqueous One Solution, G3580, Promega
  • CLDN16 staining results in human normal tissues is shown in Figure 13. It was revealed that CLDN16 is expressed in some normal kidney tissues, but not in other normal tissues (brain, heart, liver, lung, ovary, and thyroid).
  • Example 10 Immunohistochemistry in ovarian cancer tissue, and disease-free survival rate analysis and clinicopathological analysis based thereon (the purpose) Using the anti-CLDN16 antibody obtained in Example 2, CLDN16 protein will be detected in ovarian cancer tissues, and the relationship between CLDN16 expression and prognosis will be clarified.
  • Tissue specimens were collected from 173 patients who underwent surgery for ovarian cancer diagnosed at Fukushima Medical University Hospital between 2003 and 2015, and from 76 patients who underwent surgery for ovarian cancer diagnosed at Iwaki City Medical Center between 2010 and 2015, and CLDN16 expression was evaluated in a total of 249 cases. The subjects were limited to patients whose survival information for 3 to 5 years after diagnosis was available, and deaths unrelated to the underlying disease were excluded. The collection of tissue materials was approved by the Fukushima Medical University Ethics Committee (approval number 2019-0311) and was conducted in compliance with the ethical guidelines for clinical research.
  • Frozen sections were prepared from mouse kidneys, and fluorescent immunostaining was performed using anti-OCLN antibody (Thermo Fisher Scientific, Cat#71-1500) and anti-CLDN16 antibody obtained in Example 2.
  • anti-OCLN antibody Thermo Fisher Scientific, Cat#71-1500
  • anti-CLDN16 antibody obtained in Example 2.
  • the specimen was fixed with 4% paraformaldehyde solution at room temperature for 10 minutes, and then washed with PBS. Then, the specimen was incubated with 0.1% TritonX-100/PBS solution at room temperature for 10 minutes to permeabilize the cell membrane. The specimen was washed with PBS and incubated with 0.5% Casein/PBS solution for 10 minutes to prevent nonspecific reaction with the primary antibody.
  • the anti-CLDN16 antibody and anti-OCLN antibody obtained in Example 2 were reacted overnight at 4°C, washed three times for 5 minutes with PBS, and then reacted with Alexa488-labeled anti-rat IgG antibody and Cy3-labeled anti-rabbit IgG antibody diluted 200 times at room temperature for 1 hour. After washing three times for 5 minutes with PBS, the specimen was mounted using Fluoro-Gel II (Electron microscopy science) and observed under a fluorescent microscope.
  • Example 12 Evaluation of CLDN16 protein expression in human ovarian cancer cell lines (the purpose) Using anti-CLDN16 antibody (provided by Dr. Mikio Furuse of the National Institute for Physiological Sciences), the presence or absence of CLDN16 protein expression will be evaluated in representative human ovarian cancer cell lines.
  • Figure 21B shows that CLDN16 expression was not detected in SKOV3 cells and OVSAHO cells in which the CLDN16 gene was knocked out.
  • AMOC2:CLDN16 cells ovarian cancer cell line AMOC2 in which the CLDN16 protein was overexpressed, prepared in Example 5
  • AMOC2:EGFP cells ovarian cancer cell line AMOC2 in which the EGFP gene was introduced
  • OVSAHO ovarian cancer cell lines
  • SKOV3 SKOV3:CLDN16 KO cells
  • control OVSAHO cells OVSAHO cells and control OVSAHO cells
  • FIG. 22B shows that the viability of AMOC2:CLDN16 cells was decreased in a dose-dependent manner by the antibody-drug conjugate, whereas the viability of AMOC2:EGFP cells was not affected. This result indicates that AMOC2 cells administered the antibody-drug conjugate exhibited a cytotoxic effect that was dependent on the expression of CLDN16.
  • Figure 22C shows that the viability of control OVSAHO cells was decreased in a dose-dependent manner by the antibody-drug conjugate, whereas the viability of OVSAHO:CLDN16 KO cells was not affected. This result indicates that the cytotoxic effect of the antibody-drug conjugate on OVSAHO cells is dependent on the expression of endogenous CLDN16.
  • Figure 22C shows that the antibody-drug conjugate reduced the viability of control SKOV3 cells in a dose-dependent manner, whereas the viability of SKOV3:CLDN16 KO cells was unaffected. This result indicates that the antibody-drug conjugate administered to SKOV3 cells exerts a cytotoxic effect that is dependent on the expression of endogenous CLDN16.
  • Example 15 Verification of nephrotoxicity of CLDN16 antibody-drug conjugate in vivo (the purpose) It is known that CLDN16 is 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 examine whether it has nephrotoxicity in vivo.
  • MMAE normal rat IgG, CLDN16 antibody, and the CLDN16 antibody-drug conjugate (hereinafter referred to as "CLDN16-ADC") prepared in Example 14 were injected into the tail vein of a human severe combined immunodeficiency model mouse CB-17/Icr-scid/scidJcl (SCID; Japan CLEA) four times once a week.
  • the antibody dose was 0.1 mg/mouse, which corresponds to 5 mg/kg, and the administration volume was adjusted to 100 ⁇ L per injection.
  • MMAE was administered at 1.87 ⁇ g per injection so that the molar amount was equal to that of CLDN16-ADC.
  • mice After 4 weeks, the mice were euthanized and the kidneys were removed. The kidneys were fixed with 10% neutral formalin, and HE specimens were prepared and examined histologically. The results are shown in FIG. 23.
  • CLDN16-ADC In the mice administered with CLDN16-ADC, no significant changes were observed in the HE images of the kidney tissue, as in the groups administered with MMAE, normal rat IgG, and CLDN16 antibody. This demonstrated that CLDN16-ADC does not induce nephrotoxicity. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

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Abstract

La présente invention concerne : un nouveau biomarqueur utile pour la prédiction du pronostic d'un patient cancéreux ou pour la détection d'un cancer ; un moyen capable de reconnaître spécifiquement le biomarqueur ; et un nouvel agent thérapeutique anticancéreux.　L'invention concerne donc un biomarqueur de cancer, le biomarqueur comprenant une protéine claudine 16 (CLDN16) ou un fragment peptidique de celle-ci, ou un produit de transcription du gène CLDN16 ou un fragment d'acide nucléique de celui-ci. L'invention concerne également un anticorps anti-CLDN16 ou un fragment de celui-ci, qui comprend : une région variable de chaîne lourde qui comprend une CDR1 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 1, une CDR2 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 2, et une CDR3 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 3 ; et une région variable de chaîne légère qui comprend une CDR1 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 4, une CDR2 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 5, et une CDR3 comprenant la séquence d'acides aminés représentée par SEQ ID NO : 6.
PCT/JP2024/035559 2023-10-04 2024-10-04 Biomarqueur et traitement anticancéreux par anticorps Pending WO2025075127A1 (fr)

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