WO2019044602A1 - 癌を検出する方法及び検出試薬 - Google Patents
癌を検出する方法及び検出試薬 Download PDFInfo
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- WO2019044602A1 WO2019044602A1 PCT/JP2018/030916 JP2018030916W WO2019044602A1 WO 2019044602 A1 WO2019044602 A1 WO 2019044602A1 JP 2018030916 W JP2018030916 W JP 2018030916W WO 2019044602 A1 WO2019044602 A1 WO 2019044602A1
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- 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
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- 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
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- 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
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- 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
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- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/495—Transforming growth factor [TGF]
Definitions
- the present invention relates to propeptides and their degradation products of growth and differentiation factor 15 (Growth and Differentiation Factor 15, hereinafter also referred to as "GDF 15”) proteins in blood, and a method and reagent for detecting cancer targeted for measurement.
- GDF 15 growth and differentiation factor 15
- Tumor markers for detecting cancer generally include those shown in Table 1.
- Table 1 Tumor markers for detecting cancer.
- the positive rate in the early stage of cancer is low, and there are many problems that can not be detected in benign tumors and false positives in inflammation or cancers with high grade of malignancy. Therefore, it is desirable to develop a method for finding and testing tumor markers that can detect these cancers with high accuracy.
- GDF15 Growth and differentiation factor 15
- macrophage inhibitory cytokine 1 Macrophage Inhibitory Cytokine 1: MIC-1
- nonsteroidal anti-inflammatory drug activating gene 1 Nonsteroidal anti-inflammatory drug-Activated Gene 1: NAG-1
- ProGDF15 is stored in the extracellular matrix via a propeptide, and in the state of forming a dimer from the propeptide by furin-like protease, GDF15 is separated and released into the blood (Non-patent Document 1). It has been reported that pro-GDF15 is fractionated to a full-length molecular weight of about 40,000 and a mature GDF15 fraction to a molecular weight of about 15,000 (Non-patent Document 2).
- GDF15 has been reported to increase the amount of mature body in blood due to various cancers such as pancreatic cancer and colon cancer, and has knowledge of blood volume increase in diseases other than cancer such as heart disease (Patent Document 1) [6], non-patent documents 3-8), and others, application to appetite control and fetal examination during pregnancy has been tried (patent documents 7-8).
- the GDF15 propeptide (hereinafter also referred to as "GDPPP") is a 165-residue polypeptide located on the N-terminal side of pro GDF15. More specifically, the GDF15 propeptide herein has a signal from the initiation methionine to the alanine at residue 29 in the amino acid sequence based on the human GDF15 cDNA (GeneBank Accession No .: NM — 004864) shown in SEQ ID NO: 1 The peptide is followed by at least a sequence from leucine residue 30 to arginine residue 194, or an amino acid sequence having 80% or more identity to the sequence.
- Japan JP 2011-102461 Japanese Patent Application Publication JP-2009-545735 Japanese Unexamined Patent Publication No. 2010-528275 JP JP 2011-523051 gazette Japan JP 2012-515335 gazette Japanese Patent Laid-Open Publication 2015-108077 Japan JP 2011-190262 Japanese Patent Laid-Open No. 2003-532079
- An object of the present invention is to provide a method for detecting cancer easily and with high accuracy, and a reagent that can be used for the method.
- the present inventors conducted intensive studies, and as a result, in pancreatic cancer, colon cancer, lung cancer, breast cancer, esophageal cancer and gastric cancer, GDF15 pro in blood by an immunoassay using an antibody that recognizes GDF15 propeptide.
- GDF15 propeptide is cancer, especially pancreatic
- the present invention has been found to be a marker for detecting cancer, colon cancer, lung cancer, breast cancer, esophagus cancer or gastric cancer, or for differentially detecting non-small cell lung cancer and small cell lung cancer in lung cancer.
- the present invention is as follows.
- a method for detecting cancer which comprises measuring the amount of intact growth and differentiation factor (GDF15) propeptide in a sample (but excluding castration resistant prostate cancer).
- a method of detecting cancer which comprises measuring the amount of GDF15 propeptide fragment in a sample (except for castration resistant prostate cancer).
- a method for detecting cancer which comprises measuring the total amount of intact GDF15 propeptide and GDF15 propeptide fragment in a sample (but excluding castration resistant prostate cancer).
- [4] The method for detecting cancer according to any one of the above [1] to [3], wherein gastric cancer, pancreatic cancer, colon cancer, lung cancer, breast cancer or esophageal cancer is detected, or non-small Method to distinguish and detect cell lung cancer and small cell lung cancer.
- [5] The method according to [2] or [3], wherein the GDF15 propeptide fragment comprises the GDF15 propeptide fragment described in (A) and / or (B) below.
- GDF15 propeptide fragment having the following features: The amino acid sequence from lysine residue 58 to aspartate residue at least the 167th residue of GDF15 amino acid sequence shown in SEQ ID NO: 2 or a sequence having 80% or more identity thereto.
- GDF15 propeptide fragment having the following features:
- the GDF15 amino acid sequence shown in SEQ ID NO: 2 includes an amino acid sequence from glutamic acid at amino acid residue 74 to aspartic acid at least amino acid residue 167, or a sequence having 80% or more identity thereto.
- the measurement is performed using mass spectrometry.
- a reagent for detecting cancer including an antibody that recognizes the GDF15 propeptide (except for castration resistant prostate cancer).
- the present invention provides a method for detecting cancer easily and with high accuracy, and a reagent which can be used for the method.
- the reagent of the present invention detects GDF15 propeptide and GDF15 expression regulation is located downstream of p53, it is speculated that it reflects the therapeutic effect of existing cancer therapeutic agents, particularly taxane anticancer agents. Ru.
- the reagent of the present invention can also be a companion diagnostic in the treatment of cancer.
- FIG. 6 is a box plot of iGDPPP and tGDPPP measurements in healthy individuals, esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer.
- FIG. 6 is a box plot of iGDPPP and tGDPPP measurements in healthy individuals, esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer.
- FIG. 6 is a box plot of iGDPPP and tGDPPP measurements in healthy individuals, esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer.
- FIG. 1 is a diagram showing a box plot of CEA measurement values in esophagus cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer. It is the figure which compared iGDPPP, tGDPPP, and CEA measured value in non-small cell lung cancer and small cell lung cancer. It is a figure which shows the result of ROC curve analysis of iGDPPP, tGDPPP, and CEA in non-small cell lung cancer and small cell lung cancer.
- the first aspect of the present invention is a method of detecting cancer (except for castration resistant prostate cancer (hereinafter, also referred to as “CRPC”)).
- Measuring the amount of GDF15 propeptide which is a method based on the characteristic presence of GDF15 propeptide in a biological sample such as cancer blood as compared to a healthy sample.
- cancer except CRPC
- GDF15 propeptide consisting of an amino acid sequence from leucine at residue 30 to arginine at residue 194 of the GDF15 amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “iGDPPP”) And / or GDF15 propeptide fragment, and the GDF15 propeptide fragment includes dNT57-GDPPP (corresponding to residues 58 to 167 of the amino acid sequence of SEQ ID NO: 2), dNT73-GDPP ( The amino acid sequence of SEQ ID NO: 2 (corresponding to residues 74 to 167) and other peptide fragments are included.
- Intact GDF15 propeptide refers to the undegraded GDF15 propeptide.
- the method for measuring the amount of GDF15 propeptide is not particularly limited.
- a method using an antigen-antibody reaction using an antibody that recognizes the GDF15 propeptide, and a method using mass spectrometry can be exemplified.
- the measurement method using an antigen-antibody reaction using an antibody that recognizes the GDF15 propeptide include the following.
- B A method using surface plasmon resonance in which a sample is brought into contact with a chip on which an antibody that recognizes a measurement target is immobilized, and a signal depending on the binding between the antibody and the measurement target is detected.
- (C) A fluorescence polarization immunoassay using an antibody that recognizes a fluorescently labeled measurement target, and the fact that the degree of fluorescence polarization is increased by binding of the antibody and the measurement target.
- (D) A sandwich method in which two types of antibodies with different epitopes (one of which is a labeled antibody) that recognizes an object to be measured are used to form a complex of three of the two antibodies and the object to be measured.
- (E) A method of detecting the polypeptide of the binding protein with a mass spectrometer or the like after concentration of the measurement target in the sample with an antibody that recognizes the measurement target as pretreatment.
- the method (d) is more preferable in processing multi-specimen in that the techniques regarding reagents and devices are well established.
- An antibody that recognizes the GDF15 propeptide recognizes the N-terminal region of the GDF15 propeptide, for example, binds to an antigenic determinant in the region from the 30th leucine residue to the 57th arginine residue of SEQ ID NO: 2
- An antibody can be preferably used to measure the amount of iGDPPP.
- an antibody that recognizes the C-terminal region of the GDF propeptide for example, an antibody that binds to an antigenic determinant within the region from glutamic acid residue 74 to arginine residue 196 in SEQ ID NO: 2, contains iGDPPP and GDPP fragments It can be preferably used for the measurement of the total amount (total GDPP, hereinafter also referred to as “tGDPP”).
- An antibody that recognizes the GDF15 propeptide can be prepared by immunizing an animal using the GDF15 propeptide itself, an oligopeptide consisting of a partial region of the GDF15 propeptide, a polynucleotide encoding an intact or partial region of a pro GDF15 protein, etc. as an immunogen. You can get it.
- the animal used for immunization is not particularly limited as long as it has an antibody-producing ability, and may be a mammal usually used for immunization such as mouse, rat or rabbit, or a bird such as chicken.
- GDF15 propeptide itself or an oligopeptide consisting of a partial region of GDF15 propeptide is used as an immunogen
- the structure may be changed in the process of preparing the protein or the oligopeptide. Therefore, the obtained antibody may not have high specificity or binding ability to the desired antigen, and as a result, the amount of GDF15 propeptide contained in the sample may not be accurately quantified.
- an expression vector containing a polynucleotide encoding an intact or partial region of proGDF15 protein as an immunogen the intact GDF15 propeptide protein as introduced without undergoing structural change in the body of the immunized animal. Alternatively, partial regions are expressed, which is preferable because an antibody having high specificity and avidity (ie, high affinity) can be obtained for the GDF15 propeptide in a sample.
- the antibody that recognizes the GDF15 propeptide may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- hybridoma cells producing an antibody that recognizes the GDF15 propeptide may be appropriately selected from methods in which techniques are established.
- B cells are collected from an animal immunized by the above-described method, and the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and selection of hybridoma cells producing the desired antibody by HAT medium
- hybridoma cells producing a monoclonal antibody that recognizes the GDF15 propeptide can be established.
- Selection of an antibody that recognizes the GDF15 propeptide is carried out using GPI (glycosyl phosphorylidyl inositole) anchored GDF15 pro It may be performed based on the affinity for the peptide or secreted GDF15 propeptide.
- GPI glycosyl phosphorylidyl inositole
- the host is not particularly limited, and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells that are commonly used by those skilled in the art for protein expression, but disulfide bonds or sugar chain addition It is preferable to use, as a host, a mammalian cell capable of expressing a protein having a structure close to that of a naturally occurring GDF15 propeptide by post-translational modification as described above.
- mammalian cells include human fetal kidney-derived cells (HEK) 293T cell line, monkey kidney cell COS7 line, Chinese hamster ovary (CHO) cells or cancer cells isolated from humans, which are conventionally used.
- Purification of the antibody used in the cancer detection method of the present invention may be appropriately selected from methods in which the technology is established.
- the culture supernatant is recovered, and if necessary, after antibody concentration by ammonium sulfate precipitation, protein A, protein G, protein L or the like is immobilized Purification of the antibody is possible by affinity chromatography and / or ion exchange chromatography using a modified carrier.
- the labeled antibody used in the antigen-antibody reaction by the sandwich method described above may be prepared by labeling the antibody purified by the method described above with an enzyme such as peroxidase or alkaline phosphatase, etc. You can do it using the enzyme
- proteins such as albumin, immunoglobulin and transferrin, which are abundant in blood, are removed with Agilent Human 14 as a pretreatment step, and then further fractionated by ion exchange, gel filtration, reverse phase HPLC, etc. It is preferable to do.
- Measurement is by tandem mass spectrometry (MS / MS), liquid chromatography, tandem mass spectrometry (LC / MS / MS), matrix assisted laser desorption ionization time of flight mass spectrometry (matrix assisted laser desorption ionizat-ion time-of-flight) Mass spectrometry, MALDI-TOF / MS), surface enhanced laser ionization mass spectrometry (SELDI-MS) and the like can be used.
- pancreatic cancer, colon cancer, lung cancer, breast cancer, gastric cancer or esophageal cancer is detected when the amount of GDF15 propeptide obtained by measurement exceeds the standard value (Cutoff value) calculated from the control. It is preferable to determine that the In addition, as a method for differentially detecting non-small cell lung cancer and small cell lung cancer according to the present invention, it is determined that small cell lung cancer is detected when the reference value (Cutoff value) calculated from non small cell lung cancer is exceeded. It is preferable to do.
- the amount of GDF15 propeptide used for determination may be either a measured value or a converted concentration value.
- a conversion concentration value says the value converted from a measured value based on the analytical curve prepared by using GDF15 propeptide as a standard sample.
- the determination of the concentration of the standard sample may be a value converted from the measured value based on a standard curve of the standard peptide using mass spectrometry.
- the reference value measures normal subjects and pancreatic cancer, colon cancer, lung cancer, breast cancer, esophageal cancer or gastric cancer, or non-small cell lung cancer and small cell lung cancer, respectively, and receiver operating characteristics (ROC) It can set suitably to the measured value which shows optimal sensitivity and specificity by curve analysis.
- a second aspect of the present invention detects pancreatic cancer, colon cancer, lung cancer, breast cancer, esophageal cancer, or gastric cancer, which comprises an antibody that recognizes GDF15 propeptide.
- it is a reagent for differentially detecting non-small cell lung cancer and small cell lung cancer.
- the antibody is usually an antibody that binds to an antigenic determinant within the region from the 30th leucine residue to the 196th arginine residue of proGDF15 represented by SEQ ID NO: 2.
- the GDF15 propeptide to be detected in this embodiment includes an intact GDF15 propeptide and / or a GDF15 propeptide fragment, and the GDF15 propeptide fragment includes dNT57-GDPPP, dNT73-GDPPP, and other peptide fragments.
- the reagent of the present invention When the reagent of the present invention is used in the above-mentioned sandwich method, it is necessary to include two types of antibodies with different epitopes as the above-mentioned antibodies.
- the detection reagent of the present invention may further include a detection reagent for a tumor marker of cancer, including an antibody that recognizes a tumor marker of cancer.
- a detection reagent for a tumor marker of cancer including an antibody that recognizes a tumor marker of cancer.
- tumor markers for cancer include those shown in Table 1.
- the antibody contained in the reagent of the present invention may be the antibody itself, may be labeled, or may be immobilized on a solid phase.
- the present invention is not limited to this.
- the reagent of the present invention can be prepared by the methods shown in (I) to (III) below.
- (I) First, among two types of antibodies (hereinafter referred to as “antibody 1” and “antibody 2") that recognize the GDF 15 propeptide used in the sandwich method (hereinafter referred to as “antibody 1” and “antibody 2”), B / F (Bound / Free) separable carrier.
- the bonding method may be physical bonding utilizing hydrophobic bonding, or chemical bonding using a linker reagent or the like capable of crosslinking between two substances.
- the carrier surface is blocked with bovine serum albumin, skimmed milk, a commercially available blocking agent for immunoassay, etc. to avoid nonspecific binding, and used as a primary reagent.
- the other antibody 2 is labeled, and a solution containing the obtained labeled antibody is prepared as a secondary reagent.
- substances to be labeled for antibody 2 include enzymes such as peroxidase, alkaline phosphatase, fluorescent substances, chemiluminescent substances, substances detectable with a detection device such as radioisotope, or substances in which a partner to be specifically bound to avidin such as biotin exists Etc. is preferred.
- a buffer capable of satisfactorily performing an antigen-antibody reaction such as a phosphate buffer, a Tris-HCl buffer and the like is preferable.
- the reagent of the present invention thus prepared may be lyophilized as required.
- antibody 1 is bound to a carrier and blocking treatment is carried out in the same manner as described in (I) to (II) above to prepare an antibody-immobilized carrier.
- the buffer may be further added to prepare a reagent.
- the following methods (IV) to (VI) may be used.
- the primary reagent prepared in (II) and the sample are brought into contact at a constant temperature for a certain period of time.
- the reaction conditions may be in the range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
- Unreacted material is removed by B / F separation, and then it is contacted with the secondary reagent prepared in (III) for a certain period of time under a constant temperature to form a sandwich complex.
- the reaction conditions may be in the range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
- the amount of reagent components such as antibodies contained in the detection reagent may be appropriately set according to various conditions such as the amount of sample, the type of sample, the type of reagent, and the detection method. Specifically, for example, when 50 ⁇ L of 2.5-fold diluted serum or plasma is used as a sample and the amount of GDF 15 propeptide is measured by a sandwich method as described later, 50 ⁇ L of the sample is reacted with the antibody
- the amount of antibody bound to the carrier per system may be 100 ng to 1000 ⁇ g, and the amount of labeled antibody may be 2 ng to 20 ⁇ g.
- the cancer detection reagent of the present invention can also be used for detection by a manual method, and can also be used for detection using an automatic immunodiagnostic apparatus.
- detection using an automatic immunodiagnostic apparatus can be performed without being affected by endogenous measurement-disturbing agents contained in the sample or a competitor enzyme, and GDF15 propeptide in the sample and cancer tumors can be detected in a short time. It is preferable because the concentration of the marker can be quantified.
- the method for detecting the cancer of the present invention and the sample (test sample) targeted by the detection reagent of the present invention include whole blood, blood cells, serum, blood components such as plasma, extract of cells or tissues, urine, cerebrospinal cord Liquid etc. It is preferable to use a body fluid such as a blood component or urine as a sample, because cancer can be detected simply and noninvasively, and the blood component is used as a sample in consideration of ease of sample collection and versatility to other examination items. Is particularly preferred.
- the dilution ratio of the sample may be appropriately selected according to the type and condition of the sample to be used from no dilution to 100-fold dilution. For example, in the case of serum or plasma, 50 ⁇ L of a 2.5-fold diluted sample is used Good.
- Example 2 Epitope analysis of various monoclonal antibodies The antigen recognition site of each antibody prepared in Example 1 was expressed with intact GDF15 propeptide (iGDPPP) and cells expressing variants of N-terminal deleted GDF15 propeptide fragment (dNT-GDPPP) The culture supernatant was identified.
- iGDPPP intact GDF15 propeptide
- dNT-GDPPP N-terminal deleted GDF15 propeptide fragment
- a plasmid capable of expressing secreted iGDPPP and 4 kinds of dNT-GDPP was prepared by further inserting an oligonucleotide encoding the No. -240300 gazette).
- the structures of the various recombinant GDPPs produced are shown in FIG. 1 and a specific preparation method is shown below.
- iGDPPP is from residues 30 to 196
- dNT37-GDPPP is from residues 38 to 196
- dNT59-GDPP is 60.
- Residues to residues 196 and dNT77-GDP P correspond to residues 78 to 196 and dNT94-GDP P corresponds to residues 95 and 196, respectively.
- Example 3 Preparation of GDF15 Propeptide Measurement Reagent Using the anti-GPDP monoclonal antibody produced in Example 1, two different GDPP measurement reagents were prepared by changing the combination of antibodies.
- One is a combination of an antibody that recognizes the N-terminal region of GDPP (TS-GDPPP02) and an antibody that recognizes the C-terminal region (TS-GDPPP04) to detect intact GDPP (iGDPP).
- the other is a combination of antibodies that recognize the C-terminal region (TS-GDPPP04 and TS-GDPPP08), which detect both iGDPPP and dNT-GDPPP.
- the value detected by the latter is taken as the total GDPP (tGDPP).
- the specific preparation method is described below.
- a GDF15 propeptide measurement reagent was prepared by adding 50 ⁇ L of a solution (Tris buffer solution containing 3% BSA, pH 8.0) and performing lyophilization. The prepared GDF15 propeptide measurement reagent was sealed with nitrogen under nitrogen, and stored at 4 ° C. until measurement.
- Example 4 Preparation of GDF15 propeptide standard product
- the secretory iGDPPP culture supernatant prepared in Example 3 contains a degradation product, so the Strep-II tag (N-terminal side of recombinant iGDPPP ( Only full-length GDF15 propeptide was purified using a commercially available purification kit (manufactured by IBA) using IBA company). Secreted iGDPPP after purification was evaluated by Western blotting. Various purified samples were developed by SDS-PAGE according to a conventional method, and transferred to a PVDF membrane (Bio-Rad).
- a blocking one solution Nacalai Tesque, Inc.
- an alkaline phosphatase labeled anti-BNC antibody was added at 1 ⁇ g / sheet, and allowed to react overnight at 4 ° C.
- the obtained chemiluminescence was detected by LAS 4000 image analyzer (manufactured by GE Healthcare) using ECL Select reagent (manufactured by GE Healthcare).
- Example 5 Performance Evaluation of GDF15 Propeptide Measurement Reagent
- the recombinant GDPP supernatant prepared in Example 4 as a sample containing GDF15 propeptide and the culture supernatant of prostate cancer cell line LnCap were each diluted 10-fold with FBS.
- a total of three pseudo-analyte samples of only FBS were prepared as samples not containing GDF15 propeptide.
- Performance evaluation of the two GDF15 propeptide measurement reagents prepared in Example 4 was carried out using a fully automatic enzyme immunoassay apparatus AIA-2000 (manufactured by Tosoh Corp .: manufacturing and sales notification number 13B3X90002000009).
- the measurement by the fully automatic enzyme immunoassay device AIA-2000 is (1) Automatically dispense 20 ⁇ L of the diluted sample and 80 ⁇ L of a diluted solution containing a surfactant into a container containing the GDF 15 propeptide measurement reagent prepared in Example 3, (2) Perform antigen-antibody reaction for 10 minutes under constant temperature of 37 ° C. (3) Wash 8 times with a buffer solution containing a surfactant, (4) Add 4-methylumbelliferyl phosphate, The concentration of 4-methyl umbelliferone produced by alkaline phosphatase per unit time was determined as the measurement value (n mol / (L ⁇ s)).
- Example 6 Measurement of clinical sample The breakdown of the serum sample panel (total of 123 cases) used in this example is shown in Table 4. Healthy human serum samples were purchased from Bioreclamation IVT, and various cancer serum samples were purchased from PROMEDDX, and it is clearly stated in the product inserts of each company that they were collected according to a protocol approved by the ethics committee.
- iGDPPP and tGDPPP measurement reagents prepared in Example 3. Box plots of various measurement values are shown in Fig. 4.
- iGPDP minimum value of tGDPP measurement, 25th percentile, median, 75% percentile, maximum value and measurement range in 95% confidence interval for each sample group It is shown in Table 5.
- iGdPP and tGdPP measurements were clearly higher in all cancer types compared to healthy groups, and especially in pancreatic cancer and colon cancer, which are major cancer types in the digestive system, tended to be higher .
- Fig. 5 shows the results of receiver operating characteristic (ROC) curve analysis between healthy human groups and various cancer sample groups in iGDPPP and tGDPP, and P in the AUC (Area Under the Curve) and the significant difference test.
- ROC receiver operating characteristic
- iGDPPP and tGDPPP were statistically significantly different from those of healthy people in all cancer types, and were found to have excellent cancer detection performance.
- AUC is 0.9 or more, and it has been shown to be extremely useful for differentiating healthy from cancer.
- Example 7 Performance Comparison with CA19-9
- the digestive cancer detection performance of CA19-9 which is a representative marker of digestive organ cancer
- iGDPPP and tGDPPP was compared.
- the results of analysis of healthy, pancreatic cancer and colon cancer serum samples measured in Example 6 with CA19-9 (Tosoh Corporation: Production and sales notification number 20400 AMZ00913000) measurement reagent are shown in FIG. 6.
- ROC analysis of healthy and pancreatic cancer or colon cancer The AUCs determined from and the P values in the significant difference test are shown in Table 7.
- CA19-9 tended to be higher in the pancreatic cancer group or the colon cancer group compared to the healthy group, and ROC analysis showed that it is an excellent digestive system tumor marker.
- iGDPPP or tGDPPP was shown to be superior to CA19-9 in pancreatic cancer and colon cancer detection performance.
- Example 8 Calculation of Sensitivity and Specificity of iGDPPP, tGDPP, and CA19-9
- the sensitivity and specificity of various markers are calculated using the ROC analysis results of iGDPPP, tGDPP, and CA19-9 shown in Examples 6 and 7. did.
- the sensitivity and specificity derived from the maximum value of Youden Index (sensitivity-(100-specificity)) and the cutoff value are shown in Table 8.
- Both iGDPPP and tGDPPP were found to exhibit excellent cancer detection performance with lung cancer, pancreatic cancer and colon cancer with sensitivity and specificity of 85% or more.
- iGDPPP and tGDPPP were shown to be superior to CA19-9 in the detection performance of pancreatic cancer and colon cancer.
- it was shown that iGDPPP and tGDPPP could be detected as positive in 3 of 3 CA19-9 negative pancreatic cancer samples and in 6 of 8 CA19-9 negative colorectal cancer samples.
- Example 9 Measurement of clinical samples (esophageal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer) The breakdown of the serum sample panel (total 120 cases) used in this example is shown in Table 9. Healthy human serum samples were purchased from Bioreclamation IVT, and various cancer serum samples were purchased from PROMEDDX or Bioreclamation IVT, and it is clearly stated in the product inserts of each company that they were collected according to a protocol approved by the ethics committee.
- iGDPPP and tGDPPP measurement reagents prepared in Example 3. Box plots of various measurements (Box Plot) are shown in Figure 7.
- the iGDPPP and tGDPP measurements were clearly higher in all cancer types, esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer, compared to the healthy group.
- Table 11 shows AUCs calculated from ROC curve analysis between esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer groups and P values in a significant difference test between healthy human group and iGDPPP and tGDPPP.
- the iGDPPP and tGDPPP had an AUC of 0.9 or more in any of the cancer types, and a statistically significant difference was observed, which revealed that they have excellent cancer detection performance.
- Example 10 Calculation of sensitivity and specificity of iGDPPP, tGDPPP in esophagus cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer Sensitivity of various markers using ROC curve analysis results of iGDPPP and tGDPPP shown in Example 9 And the specificity was calculated.
- the sensitivity and specificity derived from the maximum value of Youden Index (sensitivity-(100-specificity)) and the cutoff value are shown in Table 12.
- iGdPP and tGdPP were found to have excellent cancer detection performance with sensitivity and specificity of 85% or higher (except for iGdPP for gastric cancer) in esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer groups It became.
- Example 11 Comparison of positive rate of iGDPPP, tGDPPP and CEA in esophageal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer Esophageal cancer, gastric cancer, non-esophageal cancer with iGDPPP and tGDPP which are representative markers of general cancer
- the positive rates in small cell lung cancer and small cell lung cancer were compared.
- the esophagus cancer, stomach cancer, non-small cell lung cancer and small cell lung cancer specimens described in Example 9 were analyzed using a CEA measurement reagent (manufactured by Tosoh Corporation: manufacturing and sales notification number 20100 EZZ00112000).
- the CEA has a cutoff value of 5 ng / mL or more, and iGDPPP and tGDPPP have calculated positive rates with the cutoff values described in Example 10.
- a box plot of CEA measurements is shown in FIG. 8 and a list of positive rates is shown in Table 13.
- iGDPPP and tGDPPP were superior in esophageal cancer, gastric cancer, non-small cell lung cancer and small cell lung cancer groups, and their positive rate was about twice as high as CEA. It became clear to have cancer detection performance.
- Example 12 Comparison of histotyping ability of iGDPPP, tGDPPP and CEA in lung cancer
- the histotypes of lung cancer are mainly divided into non-small cells (about 85%) and small cells (about 15%). Different types of treatment have different treatment policies, so differentiation of tissue types is considered important. Therefore, the histotyping performance of non-small cell lung cancer and small cell lung cancer was compared between iGDPPP, tGDPPP and CEA.
- the comparison analysis results of CEA, iGDPPP and tGDPPP non-small cell lung cancer or small cell lung cancer sample groups are shown in FIG. 9 and the ROC curve analysis results are shown in FIG.
- ROC curve analysis also showed that CEA has an AUC of 0.5 and does not have differentiation performance, while iGDPPP and tGDPPP have an AUC of about 0.7, indicating that it has good differentiation performance.
- a method for detecting GDF 15 propeptide capable of detecting lung cancer, pancreatic cancer, colon cancer, breast cancer, esophagus cancer or gastric cancer, or differentially detecting non-small cell lung cancer and small cell lung cancer And reagents are provided.
- various cancers that are difficult to distinguish with conventional markers can be detected easily and accurately with blood diagnosis and the like.
- the detection of cancer can be simplified, and the selection of therapeutic methods and the determination of therapeutic effects can be made, which is very useful in industry.
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Abstract
Description
[1]検体において、インタクト増殖分化因子(GDF15)プロペプチド量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
[2]検体において、GDF15プロペプチド断片量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
[3]検体において、インタクトGDF15プロペプチド量とGDF15プロペプチド断片量との合計量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
[4]上述の[1]~[3]の何れか一項に記載の癌を検出する方法において、胃癌、膵臓癌、大腸癌、肺癌、乳癌、若しくは食道癌を検出する、又は、非小細胞肺癌と小細胞肺癌とを鑑別して検出する方法。
[5]前記GDF15プロペプチド断片が、以下の(A)及び/又は(B)に記載のGDF15プロペプチド断片を含む、[2]又は[3]に記載の方法。
配列番号2に示すGDF15アミノ酸配列の58残基目のリジンから少なくとも167残基目のアスパラギン酸までのアミノ酸配列、又はこれと80%以上の同一性を有する配列を含む。
配列番号2に示すGDF15アミノ酸配列の74残基目のグルタミン酸から少なくとも167残基目のアスパラギン酸までのアミノ酸配列、又はこれと80%以上の同一性を有する配列を含む。
[6]GDF15プロペプチドを認識する抗体を用いた抗原抗体反応を用いて前記測定を行う、[1]~[5]の何れか一項に記載の方法。
[7]質量分析法を用いて前記測定を行う、[1]~[5]の何れか一項に記載の方法。
[8]GDF15プロペプチドを認識する抗体を含む、癌を検出するための試薬(但し、去勢抵抗性前立腺癌を除く)。
本発明の第一の態様は、癌を検出する方法(但し、去勢抵抗性前立腺癌(以下、「CRPC)とも記す)を除く)であり、検体においてGDF15プロペプチド量を測定することを含む。これは、健常な検体と比べて、癌の血液等の生体試料中に特徴的にGDF15プロペプチドが存在することに基づく方法である。この方法により、後述する実施例が示すように、従来知られた腫瘍マーカー(CA19-9、CEA)を測定した場合に比べて、癌(但し、CRPCを除く)、例えば膵臓癌、大腸癌、肺癌、乳癌、食道癌、若しくは胃癌を検出する、又は、非小細胞肺癌と小細胞肺がんとを鑑別して検出する際に、高い感度と特異度で検出することができる。
(a)標識した測定対象及び測定対象を認識する抗体を用い、標識した測定対象及び検体に含まれる測定対象が、前記抗体に競合的に結合することを利用した競合法。
(b)測定対象を認識する抗体を固定化したチップに検体を接触させ、当該抗体と測定対象との結合に依存したシグナルを検出する表面プラズモン共鳴を用いた方法。
(c)蛍光標識した測定対象を認識する抗体を用い、当該抗体と測定対象とが結合することで蛍光偏光度が上昇することを利用した蛍光偏光免疫測定法。
(d)エピトープの異なる2種類の、測定対象を認識する抗体(うち1つは標識した抗体)を用い、当該2つの抗体と測定対象との3者の複合体を形成させるサンドイッチ法。
(e)前処理として測定対象を認識する抗体により検体中の測定対象を濃縮後、その結合タンパクのポリペプチドを質量分析装置等により検出する方法。
本発明の第二の態様は、GDF15プロペプチドを認識する抗体を含む、膵臓癌、大腸癌、肺癌、乳癌、食道癌、若しくは胃癌を検出する、又は、非小細胞肺癌と小細胞肺癌とを鑑別して検出するための試薬である。前記抗体は、通常は、配列番号2で表されるプロGDF15の30残基目のロイシンから196残基目のアルギニンまでの領域内の抗原決定基に結合する抗体である。
(I)まず、サンドイッチ法で用いる、GDF15プロペプチドを認識する、エピトープの異なる2種類の抗体(以下、「抗体1」及び「抗体2」とする)のうち、抗体1をイムノプレートや磁性粒子等のB/F(Bound/Free)分離可能な担体に結合させる。結合方法は、疎水結合を利用した物理的結合であってもよいし、2物質間を架橋可能なリンカー試薬などを用いた化学的結合であってもよい。
(II)担体に前記抗体1を結合させた後、非特異的結合を避けるため、担体表面を牛血清アルブミン、スキムミルク、市販のイムノアッセイ用ブロッキング剤などでブロッキング処理を行ない1次試薬とする。
(III)他方の抗体2を標識し、得られた標識抗体を含む溶液を2次試薬として準備する。抗体2に標識する物質としては、ペルオキシダーゼ、アルカリ性ホスファターゼといった酵素、蛍光物質、化学発光物質、ラジオアイソトープなどの検出装置で検出可能な物質、又はビオチンに対するアビジンなど特異的に結合する相手が存在する物質等が好ましい。また、2次試薬の溶液としては、抗原抗体反応が良好に行える緩衝液、例えばリン酸緩衝液、Tris-HCl緩衝液等が好ましい。
(IV)(II)で作製した1次試薬と検体とを一定時間、一定温度のもと接触させる。反応条件は、温度4℃から40℃の範囲で、5分から180分間反応させればよい。
(V)未反応物質をB/F分離により除去し、続いて(III)で作製した2次試薬と一定時間、一定温度のもと接触させ、サンドイッチ複合体を形成させる。反応条件は、温度4℃から40℃の範囲で、5分から180分間反応させればよい。
(VI)未反応物質をB/F分離により除去し、標識抗体の標識物質を定量し、既知濃度のGDF15プロペプチド溶液を標準とし作成した検量線により、検体中のヒトGDF15プロペプチド濃度を定量する。
既知の方法(DNA免疫:特開2013-061321号公報)により、GDF15プロペプチドを認識するモノクローナル抗体を5種類作製した。
実施例1で作製した各抗体の抗原認識部位を、インタクトGDF15プロペプチド(iGDPP)及びN末端欠損型GDF15プロペプチド断片(dNT-GDPP)のバリアント発現細胞培養上清により同定した。
(3)プラスミドpFLAG1に挿入したポリヌクレオチドにより発現される各種分泌型GDPPにおいて、N末端側にFLAGタグ、C末端側にBNCタグが付加されていることを確認するために、一過性発現細胞である293T細胞株を用い、下記の方法で検証した。
(4)常法に従い、(2)で構築した各種分泌型GDPP発現プラスミドを293T細胞株へ導入して各種分泌型GDPPを一過性発現させ、培養72時間後の培養液を遠心分離し、上清を各種分泌型GDPP溶液として回収した。
(5-1)ウサギ抗FLAGポリクローナル抗体(ROCKLAND社製)を100ng/ウェルになるようカーボネート緩衝液(pH9.8)で希釈し、MaxiSorp96穴プレート(NUNC社製)に固相化した。
(5-2)4℃にて一晩反応後、TBS(Tris-Buffered Saline)により3回洗浄し、3%ウシ血清アルブミン(BSA;Bovine Serum Albumin)を含むTBS溶液を250μL/ウェルにて各ウェルに添加し、室温で2時間放置した。
(5-3)TBSにより3回洗浄を行ない、各種分泌型GDPP溶液、及び、陰性対照として発現プラスミドを導入していない293T細胞株の培養上清を、50μL/ウェルにて添加し、室温で1時間放置した。
(5-5)TBS-Tにより3回洗浄を行なった後、1%BSA/TBS-Tで10000倍希釈した西洋ワサビペルオキシダーゼ(HRP)標識抗マウスイムノグロブリンG-Fc抗体(SIGMA社製)溶液を50μL/ウェルにて添加し、室温で1時間放置した。
(5-6)TBS-Tにより4回洗浄を行ない、TMB Microwell Peroxidase Substrate(KPL社製)を添加後、1mol/Lリン酸溶液で反応停止し、吸光測定プレートリーダーにて450nmの吸光値を測定した。
(5-7)ELISA解析の結果を図2に、各抗体の抗原認識部位を表3に示す。
実施例1で作製した抗GDPPモノクローナル抗体を用い、抗体の組み合わせを変えて2種類のGDPP測定試薬を作製した。1つはGDPPのN末端領域を認識する抗体(TS-GDPP02)とC末端領域を認識する抗体(TS-GDPP04)の組み合わせで、インタクトGDPP(iGDPP)を検出する。もう一方は、C末端領域を認識する抗体どうしの組み合わせ(TS-GDPP04とTS-GDPP08)で、iGDPPとdNT-GDPPの両方を検出する。後者で検出される値を、総GDPP(tGDPP)とする。以下に、具体的な調製方法を記載する。
(2)抗GDF15プロペプチドモノクローナル抗体(TS-GDPP04)をアルカリフォスファターゼ標識キット(同仁化学社製)にて、抗GDF15プロペプチド標識抗体を調製した。
(3)磁力透過性の容器(容量1.2mL)に、(1)で調製した12個の抗体固定化担体を入れた後、(2)で調製した標識抗体を0.5μg/mL含む緩衝液(3%BSAを含むトリス緩衝液、pH8.0)50μLを添加し、凍結乾燥を実施することで、GDF15プロペプチド測定試薬を作製した。なお、作製したGDF15プロペプチド測定試薬は窒素充填下にて密閉封印シールを施し、測定まで4℃で保管した。
実施例3で調製した分泌型iGDPP培養上清中には分解物が混在しているため、組換えiGDPPのN末端側にあるStrep-IIタグ(IBA社製)を利用し、市販の精製キット(IBA社製)で全長GDF15プロペプチドのみを精製した。精製後の分泌型iGDPPを、ウェスタンブロッティング法により評価した。各種精製サンプルを常法に従いSDS-PAGEで展開し、PVDF膜(バイオラッド社製)に転写した。ブロッキングワン溶液(ナカライテスク社製)にてブロッキング後、当該ブロッキング溶液に添加しアルカリフォスファターゼ標識抗BNC抗体を1μg/シートにて添加し、4℃で一晩反応させた。TBS-Tで洗浄後、ECL Select試薬(GEヘルスケア社製)を用い、得られた化学発光をLAS 4000画像解析装置(GEヘルスケア社製)により検出した。
GDF15プロペプチドを含むサンプルとして実施例4で作製した組換えGDPP上清、及び前立腺癌細胞株LnCapの培養上清をそれぞれFBSで10倍希釈し、GDF15プロペプチドを含まないサンプルとしてFBSのみの計3種の擬似検体サンプルをそれぞれ調製した。全自動エンザイムイムノアッセイ装置AIA-2000(東ソー社製:製造販売届出番号13B3X90002000009)を用いて実施例4で作製した2種のGDF15プロペプチド測定試薬の性能評価を実施した。全自動エンザイムイムノアッセイ装置AIA-2000による測定は、
(1)希釈サンプル20μLと界面活性剤を含む希釈液80μLを、実施例3で作製したGDF15プロペプチド測定試薬を収容した容器に自動で分注し、
(2)37℃恒温下で10分間の抗原抗体反応を行ない、
(3)界面活性剤を含む緩衝液にて8回の洗浄を行ない、
(4)4-メチルウンベリフェリルリン酸塩を添加する、
ことで行い、単位時間当たりのアルカリフォスファターゼによる4-メチルウンベリフェロン生成濃度をもって測定値(nmol/(L・s))とした。AIA測定の結果、FBSを除くいずれの擬似検体サンプルも5点測定の変動係数が3%以下を示し、実施例3で作製したGDF15プロペプチド測定試薬にて得られる結果が信頼し得る結果であることが証明された。
本実施例で使用した血清検体パネル(総計123症例)の内訳を表4に示す。健常人血清検体はBioreclamationIVT社より、各種癌血清検体はPROMEDDX社から購入し、各社の製品添付書類に倫理委員会承認済のプロトコルで収集されたことが明記されている。
iGDPPおよびtGDPP測定値は、健常群と比較していずれの癌種群においても明瞭に高値を示し、特に消化器系の主要な癌種である膵癌および大腸癌で測定値が高い傾向が認められた。
消化器癌の代表的なマーカーであるCA19-9とiGDPPおよびtGDPPの消化器癌検出性能を比較した。CA19-9(東ソー社製:製造販売届出番号20400AMZ00913000)測定試薬により、実施例6で測定した健常、膵癌および大腸癌血清検体を解析した結果を図6に、健常と膵癌または大腸癌のROC解析から求めたAUCおよび有意差検定におけるP値を表7に示す。CA19-9は健常群と比較して膵癌群または大腸癌群で高値傾向を示し、ROC解析からも優れた消化器系腫瘍マーカーであることが示された。一方、iGDPPまたはtGDPPはCA19-9に比べて膵癌および大腸癌検出性能が優れていることが示された。
実施例6および7に示すiGDPP、tGDPPおよびCA19-9のROC解析結果を用いて、各種マーカーの感度および特異度を算出した。Youden Index(感度-(100-特異度))の最大値から導き出された感度および特異度、並びにカットオフ値を表8に示す。iGDPP並びにtGDPPは肺癌、膵癌および大腸癌で感度および特異度がいずれも85%以上となり、優れた癌検出性能を有することが明らかとなった。また、iGDPP並びにtGDPPはCA19-9に比べて膵癌並びに大腸癌の検出性能が優れていることが示された。さらに、iGDPP並びにtGDPPはCA19-9陰性膵癌検体3例中3例、CA19-9陰性大腸癌検体8例中6例を陽性と検出できることが示された。
本実施例で使用した血清検体パネル(総計120症例)の内訳を表9に示す。健常人血清検体はBioreclamationIVT社より、各種癌血清検体はPROMEDDX社またはBioreclamationIVT社から購入し、各社の製品添付書類に倫理委員会承認済のプロトコルで収集されたことが明記されている。
iGDPPおよびtGDPP測定値は、健常群と比較して食道癌、胃癌、非小細胞肺癌、小細胞肺癌のいずれの癌種群においても明瞭に高値を示した。
実施例9に示すiGDPPおよびtGDPPのROC曲線解析結果を用いて、各種マーカーの感度および特異度を算出した。Youden Index(感度-(100-特異度))の最大値から導き出された感度および特異度、並びにカットオフ値を表12に示す。iGDPP並びにtGDPPは食道癌、胃癌、非小細胞肺癌、小細胞肺癌群で感度および特異度がいずれも85%以上となり(胃癌のiGDPPによる感度を除く)、優れた癌検出性能を有することが明らかとなった。
癌全般の代表的なマーカーであるCEAとiGDPPおよびtGDPPの食道癌、胃癌、非小細胞肺癌、小細胞肺癌における陽性率を比較した。CEA測定試薬(東ソー社製:製造販売届出番号20100EZZ00112000)により、実施例9記載の食道癌、胃癌、非小細胞肺癌、小細胞肺癌検体を解析した。CEAはカットオフ値5ng/mL以上、iGDPPおよびtGDPPは実施例10記載のカットオフ値で陽性率を算出した。CEA測定値のボックスプロットを図8に、陽性率一覧を表13に示す。CEAは様々な癌種で上昇傾向が認められたものの、iGDPP並びにtGDPPは食道癌、胃癌、非小細胞肺癌、小細胞肺癌群でいずれもCEAに比べて陽性率が2倍程度高く、優れた癌検出性能を有することが明らかとなった。
肺癌の組織型は主に非小細胞(約85%)と小細胞(約15%)に大別されるが、組織型毎に治療方針が異なるため、組織型の鑑別が重要とされている。そこで、iGDPP、tGDPPおよびCEAの非小細胞肺癌と小細胞肺癌の組織型鑑別性能を比較した。CEA、iGDPPおよびtGDPPの非小細胞肺癌又は小細胞肺癌検体群の比較解析結果を図9に、ROC曲線解析結果を図10に示す。CEAは非小細胞肺癌で高値傾向を示したものの、有意差は認められなかった(マン=ホイットニーのU検定、p=0.9747)。一方、iGDPPおよびtGDPPは小細胞肺癌で高値傾向を示し、iGDPPは有意差が認められた(マン=ホイットニーのU検定、p=0.0452)。ROC曲線解析においても、CEAはAUCが0.5であり鑑別性能を有しないことが示された一方、iGDPPおよびtGDPPはAUCが0.7程度であり、良好な鑑別性能を有することが示された。
Claims (8)
- 検体において、インタクト増殖分化因子(GDF15)プロペプチド量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
- 検体において、GDF15プロペプチド断片量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
- 検体において、インタクトGDF15プロペプチド量とGDF15プロペプチド断片量との合計量を測定することを含む、癌を検出する方法(但し、去勢抵抗性前立腺癌を除く)。
- 請求項1~3の何れか一項に記載の癌を検出する方法において、胃癌、膵臓癌、大腸癌、肺癌、乳癌、若しくは食道癌を検出する、又は、非小細胞肺癌と小細胞肺癌とを鑑別して検出する方法。
- 前記GDF15プロペプチド断片が、以下の(A)及び/又は(B)に記載のGDF15プロペプチド断片を含む、請求項2又は3に記載の方法。
(A)以下の特徴を有する、GDF15プロペプチド断片。
配列番号2に示すGDF15アミノ酸配列の58残基目のリジンから少なくとも167残基目のアスパラギン酸までのアミノ酸配列、又はこれと80%以上の同一性を有する配列を含む。
(B)以下の特徴を有する、GDF15プロペプチド断片。
配列番号2に示すGDF15アミノ酸配列の74残基目のグルタミン酸から少なくとも167残基目のアスパラギン酸までのアミノ酸配列、又はこれと80%以上の同一性を有する配列を含む。 - GDF15プロペプチドを認識する抗体を用いた抗原抗体反応を用いて前記測定を行う、請求項1~5の何れか一項に記載の方法。
- 質量分析法を用いて前記測定を行う、請求項1~5の何れか一項に記載の方法。
- GDF15プロペプチドを認識する抗体を含む、癌を検出するための試薬(但し、去勢抵抗性前立腺癌を除く)。
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