WO2020130190A1 - Procédé de fourniture d'informations pour prédire ou diagnostiquer un risque de développer un cancer rénal - Google Patents

Procédé de fourniture d'informations pour prédire ou diagnostiquer un risque de développer un cancer rénal Download PDF

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Publication number
WO2020130190A1
WO2020130190A1 PCT/KR2018/016285 KR2018016285W WO2020130190A1 WO 2020130190 A1 WO2020130190 A1 WO 2020130190A1 KR 2018016285 W KR2018016285 W KR 2018016285W WO 2020130190 A1 WO2020130190 A1 WO 2020130190A1
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kidney cancer
sirt3
sirt1
protein
expression
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PCT/KR2018/016285
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English (en)
Korean (ko)
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화정석
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경상대학교병원
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Priority to PCT/KR2018/016285 priority Critical patent/WO2020130190A1/fr
Publication of WO2020130190A1 publication Critical patent/WO2020130190A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates to a specific protein biomarker that can predict or diagnose the risk of developing kidney cancer.
  • SIRTs are nicotinamide-dependent deacetylases that are well preserved in most species, from bacteria to humans. In mammals, there are a total of seven types of Sirtuin, from SIRT1 to SIRT7.
  • Cancer is a disease with a very heterogeneous cause and progression, and is similar in morphology and pathology, but progresses significantly differently at the molecular level. Recent clinical experience also supports this, with ERBB2 and EGFR targeting drugs successfully used in clinical trials such as Herceptin and Gefitinib being mutated or overexpressed in only about 20 to 30% of all cancer patients. .
  • the present invention relates to a specific protein biomarker capable of predicting or diagnosing the risk of developing kidney cancer, and providing an information providing method of predicting or diagnosing the risk of developing kidney cancer, comprising measuring the expression level of the protein. There is this.
  • the object of the present invention is to provide a screening method for candidates for preventing or treating kidney cancer, comprising measuring the expression level of the protein.
  • the present invention is a composition for diagnosing kidney cancer comprising a nucleotide sequence of a gene encoding the protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence.
  • the purpose is to provide.
  • the object of the present invention is to provide a pharmaceutical composition for preventing or treating kidney cancer, which includes a substance that increases the expression of the protein or promotes activity.
  • a method of providing information for predicting or diagnosing the risk of developing kidney cancer comprising measuring the expression level of SIRT1, SIRT3, or SIRT6 protein in a sample isolated from an individual.
  • a method of screening for a candidate for prevention or treatment of kidney cancer comprising the step of measuring the expression level of SIRT1, SIRT3, or SIRT6 protein before and after treatment by treating the sample separated from the kidney cancer individual.
  • the screening method further comprises the step of selecting the test substance as a candidate for prevention or treatment of kidney cancer.
  • kidney cancer comprising a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence Composition.
  • Kidney cancer diagnostic kit comprising the composition of the above 7.
  • a pharmaceutical composition for preventing or treating kidney cancer comprising a substance that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity.
  • the information providing method of the present invention can quickly and accurately provide information for predicting or diagnosing the risk of developing kidney cancer.
  • the screening method of the present invention can quickly and accurately screen candidates for preventing or treating kidney cancer.
  • the diagnostic composition of the present invention can quickly and accurately diagnose kidney cancer, and the pharmaceutical composition can exert excellent effects in the prevention or treatment of kidney cancer.
  • 3 is a graph of Kaplan-Meier cancer specific survival analysis results, showing that the high SIRT3 expression group has a higher survival rate than the low SIRT3 expression group.
  • the present invention relates to the development of kidney cancer comprising the step of measuring the expression of SIRT1 (GenBank: AAH12499.1), SIRT3 (GenBank: AAD40851.1) or SIRT6 (GenBank: CAG33481.1) protein in a sample isolated from an individual. Provides information provision method for risk prediction or diagnosis.
  • the present invention is based on finding that SIRT1, SIRT3, or SIRT6 is expressed at a specifically low level in a sample obtained from a patient with kidney cancer, and discovering its utility as a novel biomarker or indicator.
  • the sample of the individual and the sample of the control are biological samples, which means all samples obtained from an individual whose expression of SIRT1, SIRT3 or SIRT6 of the present invention can be detected, wherein the biological samples are biopsy, blood, immune cells , May be any one selected from the group consisting of nerve cells and skin tissue, and preferably kidney cancer cells, but is not particularly limited thereto, and may be prepared by treatment using a method commonly used in the technical field of the present invention. .
  • the expression level when the expression level is lower than that of a control object, it may further include a step of predicting a higher risk of developing kidney cancer.
  • a method for measuring the expression level of the indicator factor a method of measuring a concentration in a sample of mRNA, a transcript of a gene encoding an indicator factor, or a concentration in a sample of the indicator factor protein may be selected, but is not limited thereto. It can be carried out by selecting a method commonly used in the technical field of the present invention.
  • RT-PCR reverse transcriptase polymerase reaction
  • Competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • Real-time RT-PCR real-time reverse transcriptase polymerase reaction
  • RPA RNase protection assay
  • the amount of the protein can be confirmed using an antibody that specifically binds to the protein.
  • immunostaining test ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectric Youngdong, tissue immunostaining (immunohistochemistry), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS (fluorescence-activated cell sorting) and protein chip (protein chip), but are not limited thereto. .
  • the expression level of an indicator factor is measured from samples of a suspected kidney cancer sample and a control group (unsuspected patient) according to the above method, the measured results are compared with each other, and then the indicator factor
  • the expression level of is measured in a sample of a suspected patient lower than that of a control group, it is possible to predict or diagnose that the risk of developing kidney cancer is high, and the expression level of the indicator factor is similar or higher than a sample of the control group in a sample of the suspected patient.
  • it can provide information that can predict or diagnose a low risk of developing kidney cancer.
  • the present invention provides a method for screening a candidate material for preventing or treating kidney cancer, comprising treating a sample separated from a kidney cancer individual and comparing the expression level of SIRT1, SIRT3, or SIRT6 protein before and after treatment.
  • the test substance is a newly synthesized or known compound, and may include, without limitation, substances expected to exhibit effects in the prevention or treatment of kidney cancer, for example, nucleic acids, nucleotides, proteins, peptides, amino acids, sugars, lipids And it may be at least one selected from the group consisting of compounds, but is not particularly limited thereto.
  • the screening method of the present invention measures the expression level of the indicator factor of the present invention by administering the test substance to the sample, and when the expression of the indicator factor increases compared to before the treatment of the test substance, the test substance is renal cancer.
  • the candidate substance is not screened as a prophylactic or therapeutic agent for renal cancer. can do.
  • the present invention is a kidney cancer comprising a material that specifically binds to a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence.
  • a diagnostic composition comprising a material that specifically binds to a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence.
  • the substance that specifically binds to the protein may be an antibody, and the antibody refers to a specific immunoglobulin directed against an antigenic site, wherein the antibody specifically refers to SIRT1, SIRT3 or SIRT6 protein A binding antibody, and a gene encoding SIRT1, SIRT3 or SIRT6 is cloned into an expression vector to obtain a SIRT1, SIRT3 or SIRT6 protein, and an antibody can be prepared according to a conventional method in the art.
  • the form of the “antibody” includes polyclonal “antibody” or monoclonal “antibody” and includes all immunoglobulin “antibodies”.
  • the antibody does not have the structure of a complete form intact antibody with two light chains and two heavy chains, as well as a complete form with two full length light chains and two heavy chains, but is directed against an antigenic site. Also includes functional fragments of “antibody” molecules that have specific antigen-binding sites (binding domains) and retain antigen-binding functions.
  • kidney cancer is diagnosed by measuring the expression level of SIRT1, SIRT3 or SIRT6 through hybridization using SIRT1, SIRT3 or SIRT6 protein and the antibody. Can.
  • the appropriate antibody selection and hybridization conditions can be appropriately selected according to techniques known in the art.
  • the nucleotide sequence, a sequence complementary to the nucleotide sequence, and a substance specifically binding to the fragment of the nucleotide may be specifically a probe or a primer.
  • the probe means a nucleotide fragment such as RNA or DNA corresponding to a few bases to a few hundred bases, which is capable of specifically binding with nucleotides such as mRNA, and is labeled with a radioactive element, etc. Presence or absence (content) can be checked.
  • the probe may be produced in the form of an oligonucleotide (probe), a single-stranded DNA (probe), a double-stranded DNA (probe), or an RNA-probe, and a gene encoding SIRT1, SIRT3 or SIRT6 Kidney cancer can be diagnosed by performing hybridization using the probe complementary to the mRNA of, and measuring the amount of mRNA expression through the degree of hybridization. The appropriate conditions for selection and hybridization of the probe may be appropriately selected according to techniques known in the art.
  • the primer refers to a short nucleotide sequence that can form a complementary template and base pair with a nucleotide sequence having a short free 3-terminal hydroxyl group and serves as a starting point for template strand copying.
  • the primer is capable of initiating DNA synthesis in the presence of a reagent (i.e., DNA polymerase/polymerase or reverse transcriptase) and four different nucleoside triphosphates for polymerization at an appropriate buffer and temperature, and the SIRT1, Kidney cancer can be diagnosed by measuring the desired expression level of SIRT1, SIRT3 or SIRT6 protein by performing PCR amplification using the mRNA primer of the gene encoding SIRT3 or SIRT6.
  • PCR conditions, and the length of the “primer” set can be appropriately selected according to techniques known in the art.
  • nucleotide sequence of the gene encoding the SIRT1, SIRT3 or SIRT6 the sequence complementary to the nucleotide sequence, or the probe or primer specifically binding to the fragment of the nucleotide is known as the nucleotide sequence of the gene encoding SIRT1, SIRT3 or SIRT6 Therefore, those skilled in the art can design the primer or probe based on the sequence according to conventional methods in the art.
  • the probe or primer can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods, and has a length of 10 to 100 nucleotides (hereinafter referred to as'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 It may be nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 20 to 30 nt, or 20 to 25 nt.
  • the present invention provides a kit for diagnosing kidney cancer comprising the composition.
  • the kit can diagnose kidney cancer by measuring the expression level of SIRT1, SIRT3 or SIRT6 by measuring the expression level of mRNA or SIRT1, SIRT3 or SIRT6 protein of the gene encoding SIRT1, SIRT3 or SIRT6.
  • the kit not only includes a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence, ,
  • the kit may include one or more other components composition, solution, or device suitable for an analytical method for measuring the expression level of SIRT1, SIRT3 or SIRT6 protein used by the kit.
  • kits include test tubes or other suitable containers, reaction buffers, enzymes such as deoxyribonucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each “primer” pair specific for mRNA of the marker gene, DNase, RNase Inhibitors, DEPC-water (dEPC water), sterile water, and the like.
  • dNTPs deoxyribonucleotides
  • Taq-polymerases enzymes such as deoxyribonucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each “primer” pair specific for mRNA of the marker gene, DNase, RNase Inhibitors, DEPC-water (dEPC water), sterile water, and the like.
  • dEPC water DEPC-water
  • sterile water sterile water
  • the kit provides immunological detection of a material that specifically binds to a nucleotide sequence of a gene encoding a SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence.
  • a suitable buffer solution a secondary antibody labeled with a coloring enzyme or a fluorescent substance, a coloring substrate.
  • the substrate may be a nitrocellulose membrane, a 96 well plate synthesized from polyvinyl resin, a 96 well plate synthesized from polystyrene resin, glass slide glass, and the like, and the chrominase may be peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, FITC, RITC, etc. can be used for the fluorescent material, and the color development substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like can be used.
  • ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
  • OPD o- Phenylenediamine
  • TMB tetramethyl benzidine
  • the kit may be a microarray for diagnosing kidney cancer capable of measuring the mRNA expression level of a gene encoding SIRT1, SIRT3 or SIRT6 protein or a protein thereof.
  • the microarray can be easily prepared by a person skilled in the art according to a method known in the art using the index factor, and according to one embodiment, to the mRNA or fragment thereof of the gene encoding the SIRT1, SIRT3 or SIRT6 protein.
  • the cDNA of the corresponding sequence may be a microarray attached to the substrate as a #probe.
  • the present invention provides a composition for preventing or treating kidney cancer comprising a substance that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity.
  • Treatment means an approach to obtain beneficial or desirable clinical results.
  • beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease range, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state Improvement or temporary relief and alleviation (partially or entirely), detectable or not.
  • treatment may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. The treatments include treatments that are required for disorders that have already occurred as well as preventive disorders.
  • Prevention means any action that suppresses or delays the onset of a related disease. It will be apparent to those skilled in the art that the compositions herein can prevent related diseases when administered prior to initial symptoms, or onset.
  • Substances that increase the expression of SIRT1, SIRT3 or SIRT6 protein or promote activity are low molecular weight drugs, gene drugs, protein drugs, extracts, nucleic acids, oligonucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, pressure Tamers, chemicals, enzymes, amino acids, sugars, lipids, compounds (natural compounds and/or synthetic compounds), and at least one selected from the group consisting of components, but may increase SIRT1, SIRT3 or SIRT6 protein expression Or, if the substance has an effect of promoting activity, it is not particularly limited.
  • composition may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • a pharmaceutical carrier that is acceptable in a composition formulated as a liquid solution, as a sterile and biocompatible material, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • diluents such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.
  • composition of the present invention is applicable to any formulation containing an agent that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity as an active ingredient, and can be prepared as an oral or parenteral formulation.
  • the pharmaceutical formulations of the present invention are oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal or parenteral; intramuscular and subcutaneous. And forms suitable for administration by inhalation (including intravenous) or administration by inhalation or insufflation.
  • the composition of the present invention is administered in a pharmaceutically effective amount.
  • the effective dose level depends on the type of patient's disease, severity, drug activity, sensitivity to the drug, duration of administration, rate of administration and release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. Can be determined.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.
  • the dosage of the composition of the present invention can vary widely depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity, and an appropriate dosage is, for example,
  • the amount of drugs accumulated in the body and/or the expression of the SIRT1, SIRT3 or SIRT6 protein to be used may be increased or may vary depending on the specific efficacy level of the substance promoting the activity.
  • the EC50 measured as effective in the in vivo animal model and in vitro may be 0.01 ⁇ g to 1 g per 1 kg of body weight, and in a unit period of daily, weekly, monthly or yearly, It may be administered once or several times per unit period, or may be continuously administered for a long period of time using an infusion pump. The number of repeated doses is determined taking into account the time the drug stays in the body and the concentration of the drug in the body. The composition may be administered for relapse even after being treated according to the course of disease treatment.
  • composition of the present invention may further contain a compound that maintains/increases the solubility and/or absorption of one or more active ingredients exhibiting the same or similar function in relation to the treatment of kidney cancer.
  • chemotherapeutic agents, anti-inflammatory agents, anti-viral agents and/or immunomodulators may be further included.
  • compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • the excised cancer samples were fixed overnight in 20% neutral formalin buffer. Samples were thoroughly tested, excised, and inserted into paraffin blocks. Portions representing tumor tissue and adjacent normal tissues (tissues at least 10 mm from the tumor) were selected from each specimen by microscopic examination of H&E (Hematoxylin and Eosin) stained sections. One 2.0 mm tumor tissue core and one 2.0 mm normal tissue core were arrayed per case.
  • H&E Hematoxylin and Eosin
  • SIRT1 clone H-95; Santa Cruz Biotechnology
  • SIRT2 clone H-300; Santa Cruz Biotechnology
  • SIRT3 clone C73E3; Cell Signaling Technology
  • SIRT4-7 ab90485, ab78982, ab118487, and ab78977; Abcam, respectively.
  • the expression levels of SIRT 1 to 7 are the intensity of staining (0, no staining; 1, weak staining; 2, medium staining; and 3, strong staining) and the ratio of staining positive cells (0, ⁇ 30%; 1,30- 49%; 2,50-69%; and 3, ⁇ 70%) were scored semi-quantitatively.
  • the sum index was obtained by combining the staining intensity and the ratio of cells. Scoring was performed repeatedly from 2 pathologists who did not provide clinical information. The grade of expression was evaluated by calculating the average value of the measured scores, and a final average score of 4 or higher was considered to indicate high expression in the sample. On the other hand, tumors were considered to have a low expression level.

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Abstract

La présente invention concerne un biomarqueur de protéine spécifique permettant de prédire ou diagnostiquer le risque de développer un cancer rénal, et concerne un procédé de fourniture d'informations, un procédé de criblage, une composition pour le diagnostic et une composition pharmaceutique l'utilisant.
PCT/KR2018/016285 2018-12-20 2018-12-20 Procédé de fourniture d'informations pour prédire ou diagnostiquer un risque de développer un cancer rénal WO2020130190A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120047334A (ko) * 2009-07-24 2012-05-11 지드익 바이오텍 에이아이이. 자궁내막암 마커
KR20170082372A (ko) * 2016-01-06 2017-07-14 한국화학연구원 Ｓｉｒｔ６의 활성 조절용 조성물
KR20170084376A (ko) * 2016-01-07 2017-07-20 경북대학교 산학협력단 암 치료 표적 Ｓｉｒｔ２ 및 그를 사용하여 암 치료제를 스크리닝하는 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120047334A (ko) * 2009-07-24 2012-05-11 지드익 바이오텍 에이아이이. 자궁내막암 마커
KR20170082372A (ko) * 2016-01-06 2017-07-14 한국화학연구원 Ｓｉｒｔ６의 활성 조절용 조성물
KR20170084376A (ko) * 2016-01-07 2017-07-20 경북대학교 산학협력단 암 치료 표적 Ｓｉｒｔ２ 및 그를 사용하여 암 치료제를 스크리닝하는 방법

Non-Patent Citations (2)

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Title
GUAN, Y.: "SIRT1 and Kidney Function", KIDNEY DISEASES, vol. 1, 2015, pages 258 - 265, XP055721109 *
LIU, H.: "SIRT3 Overexpression Inhibits Growth of Kidney Tumor Cells and Enhances Mitochondrial Biogenesis", JOURNAL OF PROTEOME RESEARCH, 10 August 2018 (2018-08-10), XP055721110 *

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