WO2017150314A1 - 去勢抵抗性前立腺癌を検出する方法及び検出試薬 - Google Patents
去勢抵抗性前立腺癌を検出する方法及び検出試薬 Download PDFInfo
- Publication number
- WO2017150314A1 WO2017150314A1 PCT/JP2017/006705 JP2017006705W WO2017150314A1 WO 2017150314 A1 WO2017150314 A1 WO 2017150314A1 JP 2017006705 W JP2017006705 W JP 2017006705W WO 2017150314 A1 WO2017150314 A1 WO 2017150314A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gdf15
- antibody
- prostate cancer
- propeptide
- gdf15 propeptide
- Prior art date
Links
Images
Classifications
-
- 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
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- 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
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/525—Tumour necrosis factor [TNF]
-
- 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
-
- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
Definitions
- the present invention relates to a propeptide of a growth and differentiation factor 15 (Growth and Differentiation Factor 15, hereinafter also referred to as “GDF15”) protein in the blood and a degradation product thereof, and a detection method and detection of castration resistant prostate cancer using them as a measurement target It relates to a reagent.
- GDF15 Growth and Differentiation Factor 15
- Growth differentiation factor 15 is macrophage inhibitory cytokine 1 (MacroagephInhibitoryhCytokine 1: MIC-1) and non-steroidal anti-inflammatory drug activation gene 1 (Nosteroidoanti-inflammatory drug-Activated Gene 1: NAG-1). It is the same protein and belongs to the TGF- ⁇ family. GDF15 is expressed as preproGDF15 containing a secretion signal and a propeptide, and then the secretion signal is cleaved and secreted out of the cell as proGDF15.
- ProGDF15 is stored in the extracellular matrix via the propeptide, and GDF15 is cleaved and released into the blood in a state where a dimer is formed from the propeptide by a furin-like protease (Non-patent Document 1). It has been reported that pro-GDF15 is fractionated with a molecular weight of around 40,000, and a mature GDF15 is fractionated with a molecular weight of around 15,000 (Non-patent Document 2).
- GDF15 increases the amount of mature body in the blood in various diseases such as prostate cancer (Prostate cancer: PCa), ovarian cancer and heart disease (Patent Documents 1 to 6, Non-Patent Documents 3 to 7).
- prostate cancer has an increased amount of mature GDF15 in blood compared to prostate hypertrophy (BPH), and combined with PSA measurement results in improved diagnostic performance, It is known that there is a correlation between metastasis and the amount of mature GDF15 in the blood and it is useful for prognosis determination (Patent Documents 9 to 10, Non-Patent Documents 8 to 10).
- Non-patent Document 2 Comprehensive proteome analysis data (PeptideAtras, Protein Name: ENSP000002809, Build: Human Plasma Non-Glyco 2015-09) suggests the presence of GDF15 propeptide in blood, but there is a degradation product of GDF15 propeptide To do was not known to date. In addition, it was not clear whether the disease was detected using the protein as a measurement target or the effect.
- CRPC patients have been treated with anticancer drugs, but in recent years, three new therapeutic drugs targeting CRPC (two hormone therapeutic drugs and one anticancer drug) have been covered by insurance, and the order of use of the therapeutic drugs And differentiation of the timing to shift to anticancer drug treatment is regarded as important.
- PSA prostate specific antigen
- Castration resistance is relatively fast in patients with high Gleason scores and advanced stages, but a clear diagnosis by PSA is not possible, and castration resistance is several months to 10 years depending on the person. Since the acquisition period is wide and the pathological condition such as bone metastasis is often deteriorated, there is an urgent need for a simple and more accurate method for identifying castration resistance by blood diagnosis.
- Patent Publication 2011-102461 Patent Publication 2009-545735 Patent Publication 2010-528275 Patent Publication 2011-523051 Patent Publication 2012-515335 Patent Publication 2015-108077 Patent Publication 2011-190262 Patent Publication 2003-532079 Patent Publication 2013-174614 Patent Publication 2011-509403
- An object of the present invention is to provide a method for detecting castration resistant prostate cancer (CRPC) simply and with high accuracy, and a reagent that can be used in the method.
- CRPC castration resistant prostate cancer
- GDF15 propeptide and its degradation product are secreted in the culture supernatant of cancer cell lines, and GDF15 is also present in the blood of cancer patients.
- the presence of the propeptide and its degradation product was confirmed.
- proteomic analysis was performed on culture supernatants of prostate cancer (PCa) and castration resistant prostate (CRPC) cell lines, not only GDF15 mature but also GDF15 propeptide and its degradation products are present in the culture supernatant. I found out.
- GDF15 propeptide in blood shows a significant increase in prostate cancer specimens after acquiring castration resistance by immunoassay using an antibody that recognizes GDF15 propeptide in prostate tumors and prostatic hypertrophy.
- GDF15 propeptide can be a detection marker for castration resistant prostate cancer, and the present invention has been completed.
- the present invention is as follows. [1] A method for detecting castration resistant prostate cancer, comprising measuring the amount of intact GDF15 propeptide in a specimen. [2] A method for detecting castration resistant prostate cancer, comprising measuring the amount of GDF15 propeptide fragment in a specimen. [3] A method for detecting castration resistant prostate cancer, comprising measuring the total amount of intact GDF15 propeptide and GDF15 propeptide fragment in a specimen. [4] The method according to [2] or [3], wherein the GDF15 propeptide fragment includes the GDF15 propeptide fragment described in (A) and / or (B) below.
- a GDF15 propeptide fragment having the following characteristics (a) and (b): (A) The amino acid sequence from the lysine at the 58th residue to the aspartic acid at the 167th residue of the GDF15 amino acid sequence shown in SEQ ID NO: 2 or a sequence having 80% or more identity thereto. (B) It is fractionated to a molecular weight of about 17,000 by reducing SDS-PAGE. (B) A GDF15 propeptide fragment having the following characteristics (c) and (d): (C) The amino acid sequence from the 74th glutamic acid to at least the 167th aspartic acid of the GDF15 amino acid sequence shown in SEQ ID NO: 2 or a sequence having 80% or more identity thereto.
- the present invention provides a novel peptide fragment that can be a diagnostic marker for castration resistant prostate cancer (CRPC).
- CRPC castration resistant prostate cancer
- the present invention also provides a method for detecting CRPC simply and with high accuracy, and a reagent that can be used in the method.
- the reagent of the present invention detects GDF15 propeptide and GDF15 expression control is located downstream of p53, it is speculated that it reflects the therapeutic effect of existing prostate cancer therapeutic agents, particularly taxane anticancer agents. Is done. Therefore, the reagent of the present invention can be a companion diagnostic agent in the treatment of prostate cancer.
- the shaded portion indicates the peptide detected by proteome analysis.
- the horizontal axis represents absorbance.
- the horizontal bar indicates the median value of each disease group.
- the horizontal bar indicates the median value of each disease group.
- the horizontal bar indicates the median value of each disease group.
- the solid line indicates the plasma value, and the broken line indicates the serum value.
- a thin solid line indicates PSA
- a broken line indicates mGDF15
- a thick solid line indicates iGDPP.
- the first aspect of the present invention is a GDF15 propeptide fragment which is a degradation product of the GDF15 propeptide. From the expression / secretion mode of GDF15, it has been considered that GDF15 propeptide is localized in the extracellular matrix even after GDF15 mature body secretion. When the present inventors performed proteome analysis of the culture supernatant of PCa and CRPC cell lines, it was found that not only mature GDF15 but also GDF15 propeptide and its degradation product were present in the culture supernatant. Furthermore, the present inventors have found that GDF15 propeptide and its degradation products are secreted into the blood.
- GDF15 propeptide As the expression level increases, GDF15 propeptide is also secreted into the blood, and GDF15 propeptide undergoes some characteristic processing in the process, and it is speculated that GDF15 propeptide fragment exists as a degradation product thereof. The Until now, the existence of degradation products (fragments) of GDF15 propeptide has not been known.
- GDF15 propeptide (hereinafter also referred to as “GDPP”) is a 165-residue polypeptide located on the N-terminal side of proGDF15. More specifically, the GDF15 propeptide in the present specification is a signal from the starting methionine to the 29th alanine in the amino acid sequence based on the human GDF15 cDNA shown in SEQ ID NO: 1 (GenBank Accession No .: NM — 004864). It includes at least a sequence from leucine at the 30th residue to arginine at the 194th residue following the peptide, or an amino acid sequence having 80% or more identity with the above sequence.
- the GDF15 propeptide fragment is considered to have a plurality of forms, but includes the following two types confirmed by Examples described later.
- the first is a CDF-side fragment of the GDF15 propeptide in which processing has occurred on the N-terminal side of the 58th lysine of SEQ ID NO: 2 in GDPP, and the 57th residue has been deleted (hereinafter referred to as “dNT57”).
- dNT57 -Also referred to as “GDPP”). More specifically, it includes a sequence from the 58th lysine of SEQ ID NO: 2 to at least 167th aspartic acid, or an amino acid sequence having 80% or more identity with the above sequence.
- the second is a C-terminal fragment of GDF15 propeptide in which processing occurred on the N-terminal side of the 74th glutamic acid of SEQ ID NO: 2 in GDPP and deletion of the 73rd residue was made (hereinafter referred to as “dNT73”).
- dNT73 glutamic acid of SEQ ID NO: 2 in GDPP and deletion of the 73rd residue was made
- GDPP -Also referred to as “GDPP”). More specifically, it includes a sequence from glutamic acid at residue 74 to aspartic acid at least residue 167 in SEQ ID NO: 2, or an amino acid sequence having 80% or more identity with the above sequence.
- the identity is preferably 90% or more, more preferably 95% or more.
- the polypeptide of the present invention may be a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the above sequence.
- the term “several” means preferably 2 to 20, more preferably 2 to 10, and still more preferably 2 to 5. Further, by reducing SDS-PAGE, GDF15 propeptide is detected at a molecular weight of about 20,000, dNT57-GDPP is detected at a molecular weight of 17,000, and dNT73-GDPP is detected at a molecular weight of about 15,000.
- a molecular weight marker preferably Precision Plus Protein Prestained Standard (BIO -RAD's) molecular weights of 20,000, 17,000 and 15,000 are detected at almost the same positions.
- the second aspect of the present invention is a method for detecting castration resistant prostate cancer (CRPC), which comprises measuring the amount of GDF15 propeptide in a specimen. Including. This is a method based on the characteristic presence of the GDF15 propeptide in a biological sample such as blood of CRPC compared to PCa before acquiring castration resistance. By this method, as shown in the examples described later, castration-resistant prostate cancer can be detected with higher sensitivity and specificity than when a conventionally known tumor marker (PSA) or GDF15 mature body alone is measured. Can do.
- PSA tumor marker
- the GDF15 propeptide to be measured in this embodiment includes an intact GDF15 propeptide consisting of the amino acid sequence from the 30th leucine to the 194th arginine of the GDF15 amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “iGDPP”). And / or GDF15 propeptide fragments, which include dNT57-GDPP, dNT73-GDPP, and other peptide fragments. Intact GDF15 propeptide refers to an 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 specimen is brought into contact with a chip on which an antibody for recognizing 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 utilizing the fact that the degree of fluorescence polarization is increased by binding the antibody to the measurement target.
- (D) A sandwich method in which two types of antibodies with different epitopes that recognize the measurement target (one of which is a labeled antibody) are used to form a ternary complex between the two antibodies and the measurement target.
- (E) A method of detecting a binding protein polypeptide with a mass spectrometer or the like after concentrating a measurement target in a specimen with an antibody that recognizes the measurement target as a pretreatment.
- the methods (d) and (e) are simple and highly versatile, the method (d) is more preferable for processing a large number of specimens in terms of sufficiently established techniques relating to reagents and devices.
- the 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 leucine at the 30th residue to the arginine at the 57th residue of SEQ ID NO: 2.
- the antibody can be preferably used for measurement of the amount of iGDPP.
- An antibody that recognizes the C-terminal region of the GDF propeptide for example, an antibody that binds to an antigenic determinant in the region from glutamic acid at residue 74 to arginine at residue 194 of SEQ ID NO: 2, 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 obtained by immunizing an animal using the GDF15 propeptide itself, an oligopeptide consisting of a partial region of the GDF15 propeptide, a polynucleotide encoding the intact or partial region of the proGDF15 protein as an immunogen.
- the animal used for immunization is not particularly limited as long as it has an antibody-producing ability, and may be a mammal such as a mouse, a rat, or a rabbit, which is usually used for immunization, or a bird such as a chicken.
- the structure of the protein or the oligopeptide may change during the preparation process. For this reason, the obtained antibody may not have high specificity or binding power 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 the intact or partial region of pro-GDF15 protein is used as an immunogen, the intact of GDF15 pro-peptide protein as introduced without undergoing structural changes in the immunized animal body.
- an antibody having high specificity and binding force that is, high affinity
- the antibody that recognizes the GDF15 propeptide may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- Establishment of a hybridoma cell that produces an antibody that recognizes the GDF15 propeptide may be appropriately selected from methods for which techniques have been established. As an example, selection of a hybridoma cell that collects a B cell from an animal immunized by the above-described method, fuses the B cell and myeloma cell electrically or in the presence of polyethylene glycol, and produces a desired antibody in a HAT medium. And the selected hybridoma cells are monocloned by the limiting dilution method, so that hybridoma cells producing a monoclonal antibody that recognizes the GDF15 propeptide can be established.
- Selection of an antibody that recognizes the GDF15 propeptide for example, a monoclonal antibody that recognizes the GDF15 propeptide, used in the method for detecting castration resistant prostate cancer of the present invention is selected from GPI (glycosyl phosphatidyl inositol) derived from the host expression system. The determination may be performed based on the affinity for the anchored GDF15 propeptide or the secreted GDF15 propeptide.
- 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. It is preferable to use a mammalian cell as a host capable of expressing a protein having a structure close to that of a natural GDF15 propeptide by post-translational modification.
- mammalian cells include human embryonic kidney-derived cells (HEK) 293T cell line, monkey kidney cell COS7 line, Chinese hamster ovary (CHO) cells, or cancer cells isolated from humans. It is done.
- Purification of the antibody used in the castration resistant prostate cancer detection method of the present invention may be appropriately selected from methods for which techniques have been established. As an example, after culturing the hybridoma cells that produce the antibody established by the method described above, the culture supernatant is collected, and if necessary, the antibody is concentrated by ammonium sulfate precipitation, and protein A, protein G, or protein L is fixed. The antibody can be purified by affinity chromatography and / or ion exchange chromatography using a conjugated carrier.
- the labeled antibody used when the antigen-antibody reaction is performed by the sandwich method described above may be performed by labeling the antibody purified by the above-described method with an enzyme such as peroxidase or alkaline phosphatase, and the technology for the labeling is well established. This method can be used.
- a method for detecting GDF15 propeptide and mature GDF15 using mass spectrometry will be specifically described below.
- proteins such as albumin, immunoglobulin, transferrin, etc. that are abundant in blood are removed with Agilent Human 14, etc., and further fractionated by ion exchange, gel filtration, reverse phase HPLC, etc. It is preferable to do.
- Measurements are 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 ionizion-ion time-of-flight mass-spectrometry, MALDI-TOF / MS), surface-enhanced laser-desorption ionization-mass spectrometry, SELDI-MS, and the like.
- the amount of GDF15 propeptide obtained by measurement exceeds the reference value (Cutoff value) calculated from the control.
- the amount of GDF15 propeptide used for the determination may be either a measured value or a converted concentration value.
- concentration value says the value converted from a measured value based on the analytical curve created using GDF15 propeptide as a standard sample. The concentration of the standard sample was determined from the measured value based on the standard peptide calibration curve using mass spectrometry.
- Reference value measures prostate cancer before castration resistance acquisition, other urological cancers, prostate enlargement, or non-castration resistant prostate cancer specimens such as healthy individuals, and castration resistant prostate cancer specimens, respectively Then, it can be appropriately set to a measured value indicating the optimum sensitivity and specificity by analyzing the receiver operating characteristic (ROC) curve.
- the reference value (Cutoff value) of the amount of GDF15 propeptide when plasma is used as a specimen may be set to a concentration of 1.337 ng / mL.
- the third aspect of the present invention is a reagent for detecting castration resistant prostate cancer, which comprises an antibody that recognizes the GDF15 propeptide.
- This aspect can be rephrased as the use of an antibody that recognizes the GDF15 propeptide or a reagent containing the same in the detection of castration resistant prostate cancer.
- the antibody is usually an antibody that binds to an antigenic determinant in the region from leucine at the 30th residue to arginine at the 194th residue of pro-GDF15 represented by SEQ ID NO: 2.
- the GDF15 propeptide to be detected in this embodiment includes intact GDF15 propeptide and / or GDF15 propeptide fragment, and the GDF15 propeptide fragment includes dNT57-GDPP, dNT73-GDPP, and other peptide fragments. It is.
- the reagent of the present invention is used for the sandwich method described above, it is necessary to include two types of antibodies having different epitopes as the antibody.
- the detection reagent of the present invention may further contain a detection reagent for a prostate cancer tumor marker, which contains an antibody that recognizes the prostate cancer tumor marker.
- a detection reagent for a prostate cancer tumor marker which contains an antibody that recognizes the prostate cancer tumor marker.
- tumor markers for prostate cancer include PSA.
- 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 reagent of the present invention can be prepared by the following methods (I) to (III).
- (I) First, of two types of antibodies with different epitopes (hereinafter referred to as “antibody 1” and “antibody 2”) that recognize the GDF15 propeptide used in the sandwich method, antibody 1 is used as an immunoplate or magnetic particle. Etc., and bind to a B / F (Bound / Free) separable carrier.
- the bonding method may be physical bonding using a hydrophobic bond or chemical bonding using a linker reagent that can crosslink between two substances.
- the surface of the carrier is subjected to a blocking treatment with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassay, etc., to be 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.
- label antibody 2 include substances such as peroxidase and alkaline phosphatase, substances that can be detected by a detection device such as a fluorescent substance, a chemiluminescent substance, and a radioisotope, or substances that specifically bind to biotin such as avidin. Etc. are preferred.
- the secondary reagent solution is preferably a buffer capable of satisfactorily performing an antigen-antibody reaction, such as a phosphate buffer or a Tris-HCl buffer.
- the reagent of the present invention thus produced may be lyophilized as necessary.
- an antibody 1 is bound to a carrier and subjected to a blocking treatment, and the labeled antibody 2 is attached to the antibody-immobilized carrier. What is necessary is just to add the buffer solution containing further and to produce a reagent.
- the primary reagent prepared in (II) and the specimen are brought into contact under a constant temperature for a predetermined time.
- the reaction conditions may be a temperature range of 4 ° C. to 40 ° C. and a reaction time of 5 minutes to 180 minutes.
- Unreacted substances are removed by B / F separation, and then contacted with the secondary reagent prepared in (III) at a constant temperature for a certain period of time to form a sandwich complex.
- the reaction conditions may be a temperature range of 4 ° C. to 40 ° C. and a reaction time of 5 minutes to 180 minutes.
- a detection reagent can also be prepared for the mature GDF15 body by the above method.
- the detection reagent for GDF15 mature it can also be applied as a detection reagent for tumor markers of prostate cancer.
- the detection reagent for a prostate cancer tumor marker may be one produced in the same manner as the above-described reagent of the present invention, or may be one commercially available.
- the amount of the reagent component such as an antibody contained in the detection reagent may be appropriately set according to various conditions such as the amount of the sample, the type of the sample, the type of the reagent, and the detection method. Specifically, for example, when measuring the amount of GDF15 propeptide by the sandwich method using 50 ⁇ L of serum or plasma diluted 2.5-fold as a sample as described later, a reaction for reacting 50 ⁇ L of the sample with an antibody
- the amount of antibody bound to the carrier per system may be from 100 ng to 1000 ⁇ g, and the amount of labeled antibody may be from 2 ng to 20 ⁇ g.
- the castration resistant prostate cancer detection reagent of the present invention can be used for detection by a conventional method, and can also be used for detection using an automatic immunodiagnostic apparatus.
- detection using an automatic immunodiagnostic device can be detected without being affected by endogenous measurement interfering factors and competitive enzymes contained in the sample, and the GDF15 propeptide and castration resistance in the sample can be obtained in a short time. This is preferable since the concentration of a tumor marker for prostate cancer can be quantified.
- the method for detecting castration resistant prostate cancer of the present invention and the specimen (test sample) that is the target of the detection reagent of the present invention include blood components such as whole blood, blood cells, serum, and plasma, extracts of cells or tissues, Examples include urine and cerebrospinal fluid. Use of body fluids such as blood components and urine as specimens is preferable because castration-resistant prostate cancer can be detected easily and non-invasively. In consideration of ease of specimen collection and versatility to other test items, blood components Is particularly preferred as a specimen.
- the dilution ratio of the sample may be appropriately selected according to the type and state of the sample to be used from undiluted to 100-fold diluted. For example, in the case of serum or plasma, 50 ⁇ L of a sample diluted 2.5 times is used. Good.
- a method of measuring iGDPP in plasma is more preferable.
- the method for detecting castration resistant prostate cancer of the present invention and the detection reagent of the present invention are also useful for follow-up of CRPC patients after acquiring castration resistance. In particular, it is expected to accurately reflect the disease state, such as fewer false positives than PSA which is an existing prostate cancer marker.
- Example 1 Proteome analysis of secreted proteins of urological cancer cell lines
- CRPC cell lines 22Rv, PC3 and LnCap-AI prostate cancer cell lines LnCap, VCap and DU145
- bladder cancer cell lines 5637, T24, TCC and UMUC3 bladder cancer cell lines 5637, T24, TCC and UMUC3, renal cancer cell lines CAKI, ACHN, 786-O and U3 as comparative controls
- Each culture was cultured in an RPMI1640 medium containing 10% fetal calf serum in a ⁇ 150 mm culture dish.
- the medium was added to 20 mL of RPMI1640 medium (serum-free medium) containing 4.0 nM epidermal growth factor (manufactured by Sigma). Exchanged. After further culturing for 2 days, the medium was filtered through a 0.22 ⁇ m filter and lyophilized. The obtained culture supernatant powder was dissolved in 30 mM Tris-HCl buffer (pH 8.5) containing 7 M urea, 2 M thiourea, and 4% CHAPS, and the culture supernatant was desalted and concentrated by acetone precipitation. Redissolved in 10 mM ammonium bicarbonate solution containing 1% RapiGest.
- the obtained protein sample was subjected to reductive alkylation and trypsin digestion according to a conventional method.
- the obtained peptide fragment was measured with a nano LC-LTQ orbitrap mass spectrometer (manufactured by Thermo Fisher Scientific).
- the obtained data was analyzed using Protein Discoverer 1.3 software (manufactured by Thermo Fisher Scientific), and the protein was identified by searching the amino acid sequence on the Swiss-Prot database.
- GDF15 is a propeptide in any prostate cancer cell line except DU145. Part was characteristically detected in the culture supernatant (FIG. 1).
- Example 3 Epitope analysis of various monoclonal antibodies The antigen recognition site of each antibody prepared in Example 2 was expressed as an intact GDF15 propeptide (iGDPP) and N-terminal-deficient GDF15 propeptide fragment (dNT-GDPP) variant-expressing cell. It was identified by the culture supernatant.
- iGDPP intact GDF15 propeptide
- dNT-GDPP N-terminal-deficient GDF15 propeptide fragment
- FIG. 2-1 shows the structures of the various recombinant GDPPs produced. A specific preparation method is shown below.
- Each primer corresponding to iGDPP, dNT37-GDPP, dNT59-GDPP, dNT-77GDPP, and dNT94-GDPP was prepared by using primers designed from cDNA of human GDF15 (GenBank Accession No .: NM_004864) in the combinations shown in Table 1.
- the polynucleotide was amplified by RT-PCR according to a conventional method.
- A ELISA method
- A-1 Rabbit anti-FLAG polyclonal antibody (manufactured by ROCKLAND) was diluted with carbonate buffer (pH 9.8) to 100 ng / well, and solid phase was added to MaxiSorp 96-well plate (manufactured by NUNC). Turned into.
- A-2) After reacting at 4 ° C. overnight, each was washed three times with TBS (Tris-Buffered Saline), and each TBS solution containing 3% bovine serum albumin (BSA; Bovine Serum Albumin) at 250 ⁇ L / well. Added to well and left at room temperature for 2 hours.
- BSA bovine serum albumin
- A-3 Washed 3 times with TBS, added various secretory GDPP solutions, and culture supernatant of 293T cell line not introduced with expression plasmid as a negative control at 50 ⁇ L / well, at room temperature Left for 1 hour.
- Example 4 Immunoprecipitation performance evaluation using various monoclonal antibody-immobilized magnetic beads Preparation of antibody-immobilized magnetic fine particles of 10 types of GDF15 propeptide monoclonal antibody and rabbit anti-FLAG polyclonal antibody (manufactured by ROCKLAND) manufactured in Example 2 Then, proteins in the culture supernatant of the secreted GDF15 propeptide and the prostate cancer cell culture supernatant that specifically bind to each antibody were identified by the following method. Three types of prostate cancer cells, LnCap, PC3 and DU145, were used.
- Example 2 A portion of the purified monoclonal antibody prepared in Example 2 was immobilized on Dynabeads M-280 Tosylactide magnetic particles (manufactured by Invitrogen) according to a conventional method, and then blocked with PBS containing 0.5% BSA to immobilize the antibody. Magnetic fine particles were prepared.
- IP-WB method Immunoprecipitation-Western blot method (IP-WB method)
- RPMI 1640 medium manufactured by Wako Pure Chemical Industries, Ltd.
- 10% fetal bovine serum in a 100% confluent state for 3 days.
- 10 types of prepared antibody-immobilized magnetic microparticles were added to 0.1 mL of the supernatant, and the mixture was reacted at room temperature for 1 hour.
- (2-3) After washing twice with PBST-NP40 (0.1% Tween 20, 1% NP40), washing was performed three times with PBS containing no surfactant.
- Example 5 Identification of monoclonal antibody binding protein by mass spectrometry Three types of antibodies, TS-GDPP02 antibody, TS-GDPP04 antibody, and TS-GDPP08 antibody, confirmed to have high affinity for GDF15 propeptide in Example 4 The IP was revalidated using the antibody-immobilized magnetic fine particles. In addition, the GDF15 propeptide polypeptide that binds to the TS-GDPP02 antibody and the TS-GDPP04 antibody was analyzed by mass spectrometry. The LnCap used in Example 1 was used as the prostate cancer culture supernatant, and a sample was prepared for mass spectrometry as follows.
- the LnCap sample solution was IP-coated with the above three antibody-immobilized magnetic fine particles by the method described in Example 4, and then eluted with SDS-Loading-Buffer according to a conventional method.
- the LnCap supernatant before IP, the LnCap supernatant after IP, and the IP product were developed by SDS-PAGE, and the separated protein was stained by SYPRO-Ruby staining (Invitrogen).
- FIG. 3-1 A Ruby staining image is shown in FIG. 3-1, and peptides identified from IP products having a molecular weight of about 20,000, about 17,000, and about 15,000 of LnCap are shown in FIG. The part shows the area detected by mass spectrometry).
- signals estimated to be GDF15 propeptide were observed at the molecular weight of about 20,000, about 17,000, and about 15,000 for the two types of the TS-GDPP04 antibody and the TS-GDPP08 antibody. .
- the TS-GDPP02 antibody that recognizes the N-terminal region, only a signal having a molecular weight of about 20,000 was detected.
- proteins with molecular weights of about 20,000, about 17,000, and about 15,000 located at 1, 2 and 3 in the Ruby stained image are GDF15 propeptides. Proved.
- the GDF15 propeptide located at 2 and 3 in the Ruby-stained image is an N-terminal deletion type GDF15 propeptide fragment in which the N-terminal to the 57th arginine of SEQ ID NO: 2 and the 73rd leucine are deleted and fragmented, respectively. (DNT57-GDPP and dNT73-GDPP).
- DNT57-GDPP and dNT73-GDPP As for the C-terminal, all GDPPs were detected with good sensitivity up to aspartic acid at residue 167 of SEQ ID NO: 2.
- the C-terminal sequence of GDPP and dNT-GDPP includes at least the sequence up to the 167th aspartic acid of SEQ ID NO: 2.
- Example 6 Preparation of GDF15 propeptide measuring reagent Two types of GDPP measuring reagents were prepared using the anti-GDPP monoclonal antibody prepared in Example 2 and changing the combination of the antibodies. One is a combination of an antibody that recognizes the N-terminal region of GDPP (TS-GDPP02) and an antibody that recognizes the C-terminal region (TS-GDPP04), and detects intact GDPP (iGDPP). The other is a combination of antibodies that recognize the C-terminal region (TS-GDPP04 and TS-GDPP08), and detects both iGDPP and dNT-GDPP. The value detected by the latter is defined as total GDPP (tGDPP). The specific preparation method is described below.
- Anti-GDF15 propeptide monoclonal antibody (TS-GDPP02 and 08) is physically adsorbed at room temperature all day and night so that it becomes 100 ng / carrier on a water-insoluble ferrite carrier, and then 100 mM Tris buffer containing 1% BSA ( Anti-GDF15 propeptide antibody-immobilized carrier was prepared by blocking at 40 ° C. for 4 hours at pH 8.0).
- An anti-GDF15 propeptide monoclonal antibody (TS-GDPP04) was prepared using an alkaline phosphatase labeling kit (manufactured by Dojindo) to prepare an anti-GDF15 propeptide labeled antibody.
- a GDF15 propeptide measurement reagent was prepared by adding 50 ⁇ L of a solution (Tris buffer containing 3% BSA, pH 8.0) and lyophilization. The prepared GDF15 propeptide measurement reagent was hermetically sealed under nitrogen filling and stored at 4 ° C. until measurement.
- a measurement reagent was prepared using the anti-mGDF15 monoclonal antibodies (TS-mGD18 antibody and TS-mGD20 antibody) prepared in Example 2.
- Example 7 Preparation of GDF15 propeptide standard product Since there is no commercially available product for GDF15 propeptide, secretory iGDPP prepared in Example 3 was used as a standard product. For the mature GDF15, a commercially available recombinant protein (manufactured by R & D system) was used. However, since degradation products are mixed in recombinant iGDPP, accurate quantification cannot be performed as it is. Therefore, using the Strep-II tag (manufactured by IBA) on the N-terminal side of recombinant iGDPP, only the full-length GDF15 propeptide was purified with a commercially available purification kit (manufactured by IBA).
- the purified secreted iGDPP was evaluated by the Western blotting method described in Example 3.
- the western blotting result of the GDPP purified product is shown in FIG. Although the molecular weight was increased due to the tag peptide, only one full-length GDF15 propeptide band was detected using either the N-terminal or C-terminal tag antibody.
- Example 8 Evaluation of GDF15 propeptide standard product
- the full-length GDF15 propeptide prepared in Example 7 was quantified by mass spectrometry according to the following procedure. Using a synthetic peptide labeled with a stable isotope (Heavy peptide, manufactured by Sigma-Aldrich) and an unlabeled synthetic peptide (Light peptide, manufactured by Sigma-Aldrich), a calibration curve based on concentration and L / H signal ratio was prepared. (FIG. 5).
- the purified GDF15 propeptide standard prepared in Example 7 was subjected to immunoprecipitation by the method shown in Example 4 using the magnetic fine particles of the rabbit anti-FLAG polyclonal antibody (manufactured by ROCKLAND) manufactured in Example 4.
- GDF15 propeptide was eluted from the antibody magnetic microparticles with Glycine-HCl (pH 2.5). Further, the elution sample was subjected to mass spectrometry pretreatment by the method shown in Example 5.
- the Heavy peptide was added to the sample after the pretreatment, and LC-MRM (Multiple Reactive Monitoring) was measured with a QTRAP5500 (AB SCIEX) mass spectrometer. The obtained data was analyzed using Skyline® Software. The signal ratio with the Heavy peptide was compared with the calibration curve shown in FIG. 5 to determine the concentration of recombinant iGDPP.
- Example 9 Performance evaluation of GDF15 propeptide measurement reagent
- the recombinant GDPP supernatant prepared in Example 3 as a sample containing GDF15 propeptide and LnCap prepared in Example 5 were each diluted 10-fold with FBS to obtain GDF15
- a total of 3 types of pseudo-analyte samples containing only FBS were prepared as samples not containing propeptide, and the above-mentioned reagents were evaluated by measuring 5 points using the 2 types of GDF15 propeptide measurement reagents prepared in Example 6. .
- a fully automatic enzyme immunoassay apparatus AIA-1800 manufactured by Tosoh Corporation: manufacture and sales notification number 13B3X90002000002
- Measurement with the fully automatic enzyme immunoassay device AIA-1800 (1) 20 ⁇ L of a diluted sample and 80 ⁇ L of a diluent containing a surfactant are automatically dispensed into a container containing the GDF15 propeptide measurement reagent prepared in Example 6, (2) Conduct an antigen-antibody reaction for 10 minutes at a constant temperature of 37 ° C., (3) Wash 8 times with a buffer containing a surfactant, (4) adding 4-methylumbelliferyl phosphate, The measurement value (nmol / (L ⁇ s)) was determined by the concentration of 4-methylumbelliferone produced by alkaline phosphatase per unit time.
- FIG. 6 shows calibration curves prepared for AIA measurement by purifying the purified iGDPP whose concentration was determined in Example 8 and commercially available mGDF15 with FBS, respectively.
- GDPP quantitative values such as clinical evaluation are calculated from this calibration curve.
- Example 10 Measurement of GDF15 propeptide and mature GDF15 in prostate cancer specimen
- Table 3 shows specimen panels (56 serum, 85 plasma) used in this example. This sample is a serum and plasma sample collected by the Yokohama City University Urology Department using the same protocol, and has received informed consent and approval from the Yokohama City University Ethics Committee.
- Measurement was performed using the iGDPP, tGDPP, and mGDF15 measurement reagents prepared in Example 6 using the fully automated enzyme immunoassay device AIA-1800 (manufactured by Tosoh Corporation) as an evaluation device.
- AIA-1800 manufactured by Tosoh Corporation
- Tables 4-1 and 4-2 show the iGDPP and tGDPP measurement results for samples with complete serum and plasma. As shown in the correlation diagrams of FIGS. 7-1 and 7-2, iGDPP decreased in serum compared to plasma, and tGDPP was almost unchanged.
- mGDF15 showed almost the same results for serum and plasma.
- iGDPP and tGDPP show higher castration resistant prostate cancer detection ability when measured in plasma. Since dNT-GDPP is almost absent in plasma, the diagnostic performance of the two is almost the same. However, some of the highly expressed specimens were expected to undergo degradation, and the iGDPP alone in plasma showed slightly higher diagnostic performance. Regarding mGDF15, as shown in FIG. 8-2, diagnostic performance almost unchanged in serum and plasma was confirmed. As a result, a method for measuring iGDPP in plasma is expected to be more desirable for clinical evaluation of CRPC by GDPP.
- FIG. 9 shows the results obtained by classifying and analyzing this panel into three groups of prostatic hypertrophy, prostate cancer before acquisition of castration resistance, and castration resistant prostate cancer.
- FIG. 9 shows results of GDF15 propeptide and GDF15 mature body measurement values and PSA measurement values.
- Table 7 shows the minimum value, the 25th percentile, the median value, the 75th percentile, the maximum value, and the density range in the 95% confidence interval.
- mGDF15 and PSA showed a slightly higher tendency in prostate cancer before acquisition of castration resistance, and a markedly higher value in castration resistant prostate cancer compared to prostate enlargement.
- the overlapping concentration range before and after the castration resistance acquisition is wide, and it cannot be said that the diagnostic performance is high.
- GDPP showed almost no difference between prostatic hypertrophy and prostate cancer before acquisition of castration resistance, and showed a markedly high value only in castration resistant prostate cancer.
- mGDF15 The difference between mGDF15 and GDPP is thought to be due to the difference in secretion mechanism.
- mGDF15 In prostate cancer before acquisition of castration resistance, only mGDF15 is released from pro-GDF15 stored in the extracellular matrix by the action of Furin-like protease, whereas in castration-resistant prostate cancer, the expression level is increased and mGDF15 is increased.
- GDPP that cannot be stored in the extracellular matrix also comes out in the blood.
- FIG. 10 shows the results of receiver operating characteristic (ROC) curve analysis of the iGDPP measurement system, mGDF15 measurement system, and PSA measurement data between the prostate cancer groups before and after the acquisition of castration resistance
- FIG. 10 shows the results of AUC (Area Under the Curve, ROC The area under the curve) and the P value in the significant difference test are shown in Table 8.
- a significant difference in iGDPP measurement value between the prostate cancer groups before and after acquisition of castration resistance showed p ⁇ 0.0001, and a statistically significant difference was observed.
- the iGDPP measurement reagent was useful for detection of castration resistant prostate cancer. It was shown that. Moreover, it was shown that P value and AUC were also exceeded compared with the mGDF15 measurement system and the PSA measurement value.
- castration resistance when each reference value (Cutoff value) of iGDPP and mGDF15 is a value obtained from the ROC analysis, and the reference value of PSA is 20 ng / mL, which is around a general reference value.
- Table 9 shows the sensitivity and specificity between the prostate cancer groups before and after sex acquisition.
- Table 10 shows the positive rates of all clinical specimens described in Example 10 when iGDPP and mGDF15 are the above reference values and PSA is a general reference value of 20 ng / mL.
- the iGDPP measurement reagent has a low false positive rate and can identify castration-resistant prostate cancer as positive with a high probability, indicating that it has a performance as a castration-resistant prostate cancer diagnostic marker.
- iGDPP has the lowest false positive rate and false negative rate, and has the highest correct diagnosis rate. Moreover, a false negative rate can be reduced by combining iGDPP and PSA. Future studies may allow further CRPC characterization for iGDPP high and PSA high.
- Table 12 shows the correlation coefficients between iGDPP and various parameters for each disease group. There was almost no correlation between age or PSA and iGDPP, suggesting that it reflects completely different events. On the other hand, although mGDF15 and iGDPP have a correlation in CRPC, the correlation is characteristically lowered in PCa before obtaining castration resistance. This is presumably because GDPP is secreted by CRPC due to the difference in secretion mechanism. There is no high correlation with EOD (Extent of Disease) or BSI (Bone Scan Index), which is an index of bone metastasis. Although mGDF15 has been reported as a marker for bone metastasis, since it tends to be high even in PCa without bone metastasis, it cannot be said that it always reflects the presence or absence of bone metastasis.
- Example 11 Evaluation of CRPC patient plasma specimen during follow-up period after obtaining castration resistance iGDPP measurement results, PSA value, non-steroidal anti-steroid properties of 10 CRPC patient plasma specimens obtained after castration resistance and follow-up Table 13 shows the administration status and pathological conditions of inflammatory drugs (NSAIDS).
- NSAIDS inflammatory drugs
- FIG. 11 shows the iGDPP measurement results in the NSAIDS administration group and the non-administration group using 9 cases excluding CRPC-05 in which malignant progression was observed.
- the present invention provides a method and a reagent for detecting GDF15 propeptide, which is a novel detection marker for castration resistant prostate cancer.
- acquisition of castration resistance which is difficult to discriminate only by measuring PSA as a conventional detection marker, can be easily and accurately detected by blood diagnosis or the like.
- These are very useful industrially because they facilitate the diagnosis of castration-resistant prostate cancer and enable the examination of treatment at an early stage. If it is possible to identify castration resistance by simple means such as blood tests in prostate cancer that requires treatment changes due to cancer progression, screening for castration resistant prostate cancer and the expectation of the most therapeutic effect It is expected to contribute to identifying the timing of possible therapeutic drug changes. Furthermore, in the future, it is expected that prostate cancer chemotherapy including new therapeutic agents will contribute to the companion diagnosis.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
Description
しかし、これらの知見はいずれもGDF15成熟体に関するものであり、GDF15プロペプチドは細胞外マトリクスに局在するものと考えられていた(非特許文献2)。網羅的プロテオーム解析のデータ(PeptideAtras、Protein Name:ENSP00000252809、Build:Human Plasma Non-Glyco 2015-09)からは、血中GDF15プロペプチドの存在は示唆されているものの、GDF15プロペプチドの分解物が存在することは今日まで知られていなかった。また、当該タンパク質を測定対象として疾患を検出することもその効果も不明であった。
これまでCRPC患者には抗癌剤治療が実施されてきたが、近年、日本でもCRPCを対象とした新規治療薬3種(ホルモン治療薬2種、抗癌剤1種)が保険適用となり、治療薬の使用順序や抗癌剤治療へ移行するタイミングの鑑別が重要視されている。
しかしながら、血中PSA濃度とCRPCの病態とは必ずしも一致しておらず、去勢抵抗性の獲得をPSAの測定のみで判別することは困難である。そのため、他の検査方法を含めて包括的に経過観察が行われているのが現状である。また、グリソンスコアが高い患者や病期の進んだ患者では比較的去勢抵抗性の獲得が早いとされているが、PSAによる明確な診断ができず、人によって数ヶ月~10年と去勢抵抗性獲得期間の幅が広く骨転移など病態が悪化して判明する場合が多いため、血液診断による簡便かつより正確な去勢抵抗性獲得の鑑別方法が切望されている。
さらに、鋭意検討の結果、前立腺腫瘍及び前立腺肥大において、GDF15プロペプチドを認識する抗体を用いたイムノアッセイにより、血液中のGDF15プロペプチドは去勢抵抗性獲得後の前立腺癌検体で顕著な増加を示すという知見を得て、GDF15プロペプチドが去勢抵抗性前立腺癌の検出マーカーとなり得ることを見出し、本発明を完成させた。
[1]検体において、インタクトGDF15プロペプチド量を測定することを含む、去勢抵抗性前立腺癌を検出する方法。
[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、去勢抵抗性前立腺癌を検出する方法。
[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、去勢抵抗性前立腺癌を検出する方法。
[4]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。
(A)以下の(a)及び(b)の特徴を有する、GDF15プロペプチド断片。
(a)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)還元SDS-PAGEにより分子量約17,000に分画される。
(B)以下の(c)及び(d)の特徴を有する、GDF15プロペプチド断片。
(c)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。
(d)還元SDS-PAGEにより分子量約15,000に分画される。
[5]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]~[4]の何れかに記載の方法。
[6]質量分析法を用いて前記測定を行う、[1]~[5]の何れかに記載の方法。
[7]GDF15プロペプチドを認識する抗体を含む、去勢抵抗性前立腺癌を検出するための試薬。
[8]以下の(a)及び(b)の特徴を有する、GDF15プロペプチド断片。
(a)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)還元SDS-PAGEにより分子量約17,000に分画される。
[9] 以下の(c)及び(d)の特徴を有する、GDF15プロペプチド断片。
(c)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。
(d)還元SDS-PAGEにより分子量約15,000に分画される。
また、本発明の試薬はGDF15プロペプチドを検出するものであり、GDF15発現制御はp53下流に位置しているため、既存の前立腺癌治療薬、特にタキサン系抗癌剤の治療効果を反映することが推測される。したがって、本発明の試薬は、前立腺癌の治療におけるコンパニオン診断薬にもなり得る。
本発明の第一の態様は、GDF15プロペプチドの分解物であるGDF15プロペプチド断片である。
GDF15の発現・分泌様式から、GDF15成熟体分泌後もGDF15プロペプチドは細胞外マトリックスに局在していると考えられてきた。本発明者らが、PCa及びCRPC細胞株培養上清のプロテオーム解析を行ったところ、培養上清中にはGDF15成熟体のみならずGDF15プロペプチド及びその分解物が存在することが分かった。さらに、本発明者らは、GDF15プロペプチド及びその分解物が血中に分泌されることを見出した。発現量が増加するにつれてGDF15プロペプチドも血中へ分泌されるようになり、GDF15プロペプチドはその過程で何らかの特徴的なプロセシングを経て、その分解物としてGDF15プロペプチド断片が存在するものと推察される。なお、これまでGDF15プロペプチドの分解物(断片)の存在は知られていなかった。
1つ目は、GDPPにおいて配列番号2の58残基目のリジンのN末端側でプロセシングが生じて57残基目までが欠損した、GDF15プロペプチドのC末端側断片である(以降、「dNT57-GDPP」とも記す)。より具体的には、配列番号2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配列を含む、または、前記配列と80%以上の同一性を有するアミノ酸配列を含むものである。
2つ目は、GDPPにおいて配列番号2の74残基目のグルタミン酸のN末端側でプロセシングが生じて73残基目までが欠損した、GDF15プロペプチドのC末端側断片である(以降、「dNT73-GDPP」とも記す)。より具体的には、配列番号2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配列を含む、または、前記配列と80%以上の同一性を有するアミノ酸配列を含むものである。
また、還元SDS-PAGEにより、GDF15プロペプチドは分子量20,000付近に、dNT57-GDPPは分子量17,000付近に、dNT73-GDPPは分子量15,000付近に検出される。より具体的には、例えば10~20質量%のグラジエントのポリアクリルアミドゲルを用いて常法に従い還元条件下でSDS-PAGEを実施した場合に、分子量マーカー、好ましくはプレシジョンPlusプロテインプレステインドスタンダード(BIO-RAD社製)の分子量20,000、17,000及び15,000に相当するバンドとほぼ同じ位置で検出される。
本発明の第二の態様は、去勢抵抗性前立腺癌(CRPC)を検出する方法であり、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、去勢抵抗性獲得前のPCaと比べて、CRPC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(PSA)やGDF15成熟体単独を測定した場合に比べて、去勢抵抗性前立腺癌を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。
(d)、(e)の方法が簡便かつ汎用性が高いが、多検体を処理する上では(d)の方法が試薬及び装置に関する技術が十分確立されている点でより好ましい。
免疫に用いる動物は、抗体産生能を有するものであれば特に限定はなく、マウス、ラット、ウサギなど通常免疫に用いる哺乳動物でもよいし、ニワトリなど鳥類を用いてもよい。
GDF15プロペプチドを認識する抗体を産生するハイブリドーマ細胞の樹立は、技術が確立された方法の中から適宜選択して行えばよい。一例として、前述した方法で免疫した動物からB細胞を採取し、前記B細胞とミエローマ細胞とを電気的にまたはポリエチレングリコール存在下で融合させ、HAT培地により所望の抗体を産生するハイブリドーマ細胞の選択を行ない、選択したハイブリドーマ細胞を限界希釈法によりモノクローン化を行なうことで、GDF15プロペプチドを認識するモノクローナル抗体を産生するハイブリドーマ細胞を樹立することができる。
なお、前述したサンドイッチ法で抗原抗体反応を行なう際に用いる標識した抗体は、前述した方法で精製した抗体をペルオキシダーゼやアルカリ性ホスファターゼなどの酵素等で標識すればよく、その標識も技術が十分確立された方法を用いて行なえばよい。
検体が血液である場合は、前処理工程として血液に多く含まれるアルブミン、イムノグロブリン、トランスフェリン等のタンパク質をAgilent Human 14等で除去した後、イオン交換、ゲル濾過または逆相HPLC等でさらに分画することが好ましい。
判定に用いるGDF15プロペプチド量は、測定値もしくは換算濃度値の何れでもよい。なお、換算濃度値は、GDF15プロペプチドを標準試料として作成された検量線に基づいて測定値から換算される値をいう。標準試料の濃度決定は、質量分析を用いた標準ペプチドの検量線に基づいて測定値から換算される値とした。
基準値(Cutoff値)は、去勢抵抗性獲得前の前立腺癌、その他の泌尿器癌、前立腺肥大、又は健常人などの去勢抵抗性前立腺癌ではない検体と、去勢抵抗性前立腺癌検体とをそれぞれ測定し、受信者動作特性(ROC)曲線解析により最適な感度と特異度を示す測定値に適宜設定することができる。例えば、具体的には、血漿を検体として用いた際のGDF15プロペプチド量の基準値(Cutoff値)は、濃度1.337ng/mLに設定してもよい。
本発明の第三の態様は、GDF15プロペプチドを認識する抗体を含む、去勢抵抗性前立腺癌を検出するための試薬である。本態様は、去勢抵抗性前立腺癌の検出における、GDF15プロペプチドを認識する抗体又はこれを含む試薬の使用、とも言い換えることができる。
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194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。
本態様において検出対象であるGDF15プロペプチドには、インタクトGDF15プロペプチド及び/又はGDF15プロペプチド断片が含まれ、GDF15プロペプチド断片には、dNT57-GDPP、dNT73-GDPP、及びその他のペプチド断片が含まれる。
本発明の試薬を前述したサンドイッチ法に利用する場合は、前記抗体としてエピトープの異なる2種類の抗体を含むことが必要である。
本発明の試薬に含まれる抗体は、抗体そのものであってもよく、標識されていてもよく、固相に固定化されていてもよい。
(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。
なお、1ステップサンドイッチ法の場合は、前述した(I)~(II)同様に担体に抗体1を結合させブロッキング処理を行なったものを作製し、前記抗体固定化担体に、標識した抗体2を含む緩衝液をさらに添加して試薬を作製すればよい。
(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。
本発明の去勢抵抗性前立腺癌検出試薬は、用手法での検出にも利用可能であり、自動免疫診断装置を用いた検出にも利用可能である。特に自動免疫診断装置を用いた検出は、検体中に含まれる内在性の測定妨害因子や競合酵素の影響を受けることなく検出が可能で、かつ短時間に検体中のGDF15プロペプチド並びに去勢抵抗性前立腺癌の腫瘍マーカーの濃度が定量可能であるため、好ましい。
なお、GDPP量を測定する方法により去勢抵抗性前立腺癌を臨床評価する場合は、後述の実施例に示されるように、血漿中のiGDPPを測定する方法がより好ましい。
また本発明の去勢抵抗性前立腺癌を検出する方法及び本発明の検出試薬は、去勢抵抗性獲得後のCRPC患者の経過観察にも有用である。特に既存の前立腺癌マーカーであるPSAと比べて偽陽性が少ない等、病態を正確に反映することが期待されるものである。
去勢抵抗性前立腺癌が特徴的に産生するタンパク質を探索するために、まず培養泌尿器癌細胞株の分泌タンパク質の網羅的解析を行った。CRPC細胞株22Rv、PC3及びLnCap-AI、比較対照として前立腺癌細胞株LnCap、VCap及びDU145、膀胱癌細胞株5637、T24、TCC及びUMUC3、腎癌細胞株CAKI、ACHN、786-O及びU3を、それぞれφ150mm培養皿に10%ウシ胎児血清を含むRPMI1640培地中にて培養し、24時間後、培地を4.0nMの上皮増殖因子(シグマ社製)を含むRPMI1640培地(無血清培地)20mLに交換した。さらに2日間培養後、培地を0.22μmのフィルターにて濾過し凍結乾燥した。得られた培養上清の粉末を7M尿素、2Mチオ尿素、4%CHAPSを含む30mMトリス塩酸緩衝液(pH8.5)で溶解し、アセトン沈殿により培養上清を脱塩濃縮し、界面活性剤RapiGest1%を含む10 mM重炭酸アンモニウム溶液にて再び溶解した。得られたタンパク質試料は、常法に従い、還元アルキル化、トリプシン消化を行った。得られたペプチド断片をナノLC-LTQオービトラップ質量分析装置(サーモフィッシャーサイエンティフィック社製)により測定した。得られたデータはProtein Discoverer 1.3ソフトウェア(サーモフィッシャーサイエンティフィック社製)を用いて解析し、Swiss-Protデータベース上のアミノ酸配列に対して検索をかけてタンパク質を同定した。
既知の方法(DNA免疫:特開2013-061321号公報)により、GDF15プロペプチド及びGDF15成熟体を認識するモノクローナル抗体を10種類ずつ作製した。
実施例2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。
各種組換えGDPPの発現評価及び精製工程のため、5’末端にFLAGタグ及びStrepII-tagを、3’末端にBNP(脳性ナトリウム利尿ペプチド)のC末端側7アミノ酸からなるBNCペプチド(特開2009-240300号公報)をコードするオリゴヌクレオチドをさらに挿入した、分泌型iGDPP及び4種のdNT-GDPPを発現可能なプラスミドを調製した。図2-1に、作製した各種組換えGDPPの構造を示す。具体的な調製方法を以下に示す。
(1)ヒトGDF15のcDNA(GenBank Accessesion No.:NM_004864)から設計したプライマーを表1に示す組み合わせで用いて、iGDPP、dNT37-GDPP、dNT59-GDPP、dNT-77GDPP及びdNT94-GDPPに相当する各ポリヌクレオチドを、常法に従いRT-PCR法により増幅した。
(3-1)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。
(3-2)各種分泌型GDPP溶液を試料として用いて、(A)酵素免疫測定法(ELISA法)、及び(B)ウエスタンブロット(WB)法を行った。
(A-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。
(A-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。
(A-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。
(A-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。
(A-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。
(B-1)(3-1)で得られた各種分泌型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。
(B-2)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。
(B-3)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。
各種分泌型GDPP溶液(分泌型GDPP培養上清)では、分子量約27,000付近、約20,000付近、及び約18,000付近に明瞭なバンドが検出され、N末端にFLAGタグ、C末端にBNCタグを有する分泌型GDPPが培養上清中に産生されていることが確認された。
上記のELISA法で、上記5種の組換えGDPP培養上清と各モノクローナル抗体の反応を評価した。図2-2に示すELISA解析の結果から判明した各抗体の抗原認識部位を表2に示す。
実施例2で作製した10種類のGDF15プロペプチドモノクローナル抗体及びウサギ抗FLAGポリクローナル抗体(ROCKLAND社製)の抗体固定化磁性微粒子を調製し、各抗体と特異的に結合する分泌型GDF15プロペプチドの培養上清及び前立腺癌細胞培養上清中のタンパク質を以下の方法により同定した。なお、前立腺癌細胞としてはLnCap、PC3及びDU145の3種類を用いた。
(2-1)前記3種の癌細胞を、10%ウシ胎児血清を含むRPMI1640培地(和光純薬社製)にて100%コンフルエント状態で3日間培養した。
(2-2)培養上清を遠心後、上清0.1mLに対し、調製した10種の抗体固定化磁性微粒子を各々添加し、室温で1時間撹拌して反応させた。
(2-3)PBST-NP40(0.1% Tween 20、1% NP40)で2回洗浄後、界面活性剤を含まないPBSで3回洗浄した。
(2-4)各抗体固定化磁性微粒子に結合したタンパク質を実施例3(B)記載のウエスタンブロットにより解析した。なお、分子量マーカーはFull-Range Rainbow Molecular Weight Markers(GE社製)を、SDS-PAGE用ゲルは10~20%グラジェントゲル(マリソル社製)用いた。ウエスタンブロットの検出用抗体は、抗BNC抗体をアルカリフォスファターゼ標識キット(同仁化学社製)にて標識したものを使用した。
実施例4でGDF15プロペプチドに親和性の高いことが確認されたTS-GDPP02抗体、TS-GDPP04抗体、及びTS-GDPP08抗体の3種類の抗体固定化磁性微粒子を用いたIPの再検証を行った。また、TS-GDPP02抗体及びTS-GDPP04抗体に結合するGDF15プロペプチドのポリペプチドを質量分析法により解析した。前立腺癌培養上清としては実施例1で使用したLnCapを用い、質量分析用に以下の通りサンプルを調製した。
LnCap IP産物の質量分析データより、Ruby染色像で1,2及び3に位置する分子量約20,000付近、約17,000付近、及び約15,000付近のタンパク質は、GDF15プロペプチドであることが証明された。また、Ruby染色像で2及び3に位置するGDF15プロペプチドは、それぞれN末端から配列番号2の57番目のアルギニンまで及び73番目のロイシンまでが欠損し断片化したN末端欠損型GDF15プロペプチド断片(dNT57-GDPP、及びdNT73-GDPP)であることが判明した。C末端に関しては、いずれのGDPPも配列番号2の167残基目のアスパラギン酸までが感度よく検出された。dNT57-GDPPは170残基目のロイシンまで検出されていたが、測定間のバラつきによる可能性もあり、特にGDPPのC末端領域はアルギニンリッチで質量分析による正確な末端の同定は困難であると推察される。この結果から、GDPP及びdNT-GDPPのC末端配列には、少なくとも配列番号2の167残基目のアスパラギン酸までの配列が含まれると考える。
実施例2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。
また、GDF15成熟体(mGDF15)についても同様に、実施例2で調製した抗mGDF15モノクローナル抗体(TS-mGD18抗体とTS-mGD20抗体)を使用して測定試薬を作製した。
GDF15プロペプチドには市販品が存在しないため、実施例3で調製した分泌型iGDPPを標準品として使用した。GDF15成熟体に関しては、市販の組換えタンパク質(R&D system社製)を使用した。
しかし、組換えiGDPPの中にも分解物が混在しているため、そのままでは正確な定量ができない。そこで、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、実施例3記載のウェスタンブロッティング法により評価した。GDPP精製品のウェスタンブロッティング結果を図4に示す。タグペプチドのために分子量が大きくなっているが、N末及びC末どちらのタグ抗体を用いても、全長GDF15プロペプチドのバンドが1本のみ検出された。
実施例7で調製した全長GDF15プロペプチドについて、以下の手順で質量分析による定量を実施した。
安定同位体標識された合成ペプチド(Heavyペプチド、Sigma-Aldrich社製)と未標識の合成ペプチド(Lightペプチド、Sigma-Aldrich社製)を用い、濃度とL/Hシグナル比による検量線を作成した(図5)。
実施例7で調製した精製GDF15プロペプチド標準品について、実施例4で調製したウサギ抗FLAGポリクローナル抗体(ROCKLAND社製)の磁性微粒子を用い、実施例4に示す方法で免疫沈降を実施した。常法に従い、Glycine-HCl(pH2.5)で抗体磁性微粒子からGDF15プロペプチドを溶出した。さらに、その溶出サンプルを実施例5に示す方法で質量分析測定前処理を実施した。
GDF15プロペプチドを含むサンプルとして実施例3で作製した組換えGDPP上清、及び実施例5で調製したLnCap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、実施例6で作製した2種のGDF15プロペプチド測定試薬を用いて5点測定することで前記試薬を評価した。
(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6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。
また、実施例8で濃度決定された精製iGDPP及び市販のmGDF15をそれぞれFBSで希釈し、AIA測定用に作成した検量線を図6に示す。以下、臨床評価等のGDPP定量値は、この検量線から算出されている。
本実施例で使用した検体パネル(血清56例、血漿85例)を表3に示す。本検体は横浜市立大泌尿器科にて同一プロトコルにて収集された血清及び血漿検体であり、インフォームドコンセントの承諾及び横浜市立大学倫理委員会の承認を受けている。
表4-1及び表4-2に、血清及び血漿がそろった検体についてのiGDPP及びtGDPP測定結果を示す。図7-1及び図7-2の相関図が示すように、iGDPPは血漿に比べて血清で減少し、tGDPPはほぼ変わらなかった。PCa及びCRPC群におけるiGDPP及びtGDPP血清血漿間差を、図7-3に示す。PCa群においてはiGDPP及びtGDPPともに血清血漿間の差がほぼないのに対し、CRPC群ではtGDPPこそ変化はないが、血清中のiGDPPが血漿中に比べて減少傾向にある。この結果から、血清中ではiGDPPの分解が生じており、高濃度で存在するCRPC群においてその影響が大きく出ていることが推察された。血漿中のiGDPPとtGDPPの定量値がほぼ一致していることから、血漿中では分解がほとんど起こっていないことが示された。なお、今回使用した血漿検体はEDTA血漿であるため、GDPPの分解に関与するプロテアーゼはキレート剤により失活するものであると推察される。
mGDF15に関しては、図8-2に示す通り、血清及び血漿でほぼ変わらない診断性能を確認した。これらの結果、GDPPによるCRPCの臨床評価については、血漿中のiGDPPを測定する方法がより望ましいと見込まれる。
iGDPP、mGDF15の測定値及びPSA測定値を図9に示す。mGDF15及びPSAは前立腺癌全般で高値傾向を示すが、GDPPは去勢抵抗性前立腺癌で特徴的に高値を示した。
本パネルを前立腺肥大、去勢抵抗性獲得前の前立腺癌、去勢抵抗性前立腺癌の3群に分類し解析した結果を図9に、GDF15プロペプチド及びGDF15成熟体測定値及びPSA測定値の各群における最小値、25パーセンタイル、中央値、75パーセンタイル、最大値、95%信頼区間における濃度範囲を表7に示す。mGDF15およびPSAは、前立腺肥大に比べ去勢抵抗性獲得前の前立腺癌でやや高値傾向、去勢抵抗性前立腺癌で顕著な高値を示した。しかし去勢抵抗性獲得の前後で重なる濃度域が広く、診断性能が高いとは言えない。一方、GDPPは前立腺肥大と去勢抵抗性獲得前の前立腺癌でほぼ差がなく、去勢抵抗性前立腺癌でのみ顕著な高値を示した。
また、iGDPPとPSAを組み合わせることにより偽陰性率を低下させることができる。今後の検討により、iGDPP高値の場合とPSA高値の場合でさらなるCRPCの特徴付けが可能となり得る。
年齢やPSAとiGDPPとの間にはほぼ相関がなく、全く別の事象を反映していることが示唆された。一方、mGDF15とiGDPPとは、CRPCで相関があるものの、去勢抵抗性獲得前のPCaで特徴的に相関が低下する。これは分泌機構の違いにより、CRPC特徴的にGDPPが分泌されるためと推察される。
骨転移の指標であるEOD(Extent of Disease)やBSI(Bone Scan Index)とも、高い相関は見られない。mGDF15は骨転移のマーカーとしての報告があるものの、骨転移のないPCaにおいても高値傾向にあるため、必ずしも骨転移の有無を反映しているとは言えない結果となった。
去勢抵抗性を獲得し経過観察中のCRPC患者血漿検体10例のiGDPPの測定結果とPSA値、非ステロイド性抗炎症薬(NSAIDS)の投与状況および病態を表13に示す。
CRPC-01およびCRPC-05を除く8例は、PSAおよびiGDPPのいずれもが低値を示し、がんの進展は見られず良好な管理下と診断された。しかし、PSAがいずれも高値であったCRPC-05(PSA高値/iGDPP高値)とCRPC-01(PSA高値/iGDPP低値)の2例を比較すると、前者は骨転移等の悪性進展が認められたのに対し、後者はPSAは高値ながらも悪性進展は認められず良好な管理下にあると診断されていた。本結果より、iGDPPはPSAに比べてCRPCの悪性進展を正確に反映するマーカーとなりうることが示唆された。
悪性進展が認められたCRPC-05を除く9例を用いてNSAIDS投与群および非投与群におけるiGDPPの測定結果を図11に示す。本検討ではMann-Whitney検定にて群間に有意差が認められなかったことから(p=0.9643)、NSAIDS投与と血中iGDPP量は関連しない可能性が示唆された。
癌の進行によって治療薬の変更が必要な前立腺癌において、去勢抵抗性の獲得を血液検査等の簡便な手段で同定することが可能となれば、去勢抵抗性前立腺癌スクリーニング及び最も治療効果の期待できる治療薬変更時期の特定への貢献が期待される。さらに、将来的には新規治療薬を含む前立腺癌化学療法のコンパニオン診断への貢献が期待される。
Claims (9)
- 検体において、インタクト増殖分化因子15(GDF15)プロペプチド量を測定することを含む、去勢抵抗性前立腺癌を検出する方法。
- 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、去勢抵抗性前立腺癌を検出する方法。
- 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、去勢抵抗性前立腺癌を検出する方法。
- 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(a)及び(b)の特徴を有する、GDF15プロペプチド断片。
(a)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)還元SDS-PAGEにより分子量約17,000に分画される。
(B)以下の(c)及び(d)の特徴を有する、GDF15プロペプチド断片。
(c)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。
(d)還元SDS-PAGEにより分子量約15,000に分画される。 - 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1~4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、去勢抵抗性前立腺癌を検出するための試薬。
- 以下の(a)及び(b)の特徴を有する、GDF15プロペプチド断片。
(a)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)還元SDS-PAGEにより分子量約17,000に分画される。 - 以下の(c)及び(d)の特徴を有する、GDF15プロペプチド断片。
(c)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。
(d)還元SDS-PAGEにより分子量約15,000に分画される。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2018503075A JP6829444B2 (ja) | 2016-02-29 | 2017-02-23 | 去勢抵抗性前立腺癌を検出する方法及び検出試薬 |
CN201780014137.4A CN108700573B (zh) | 2016-02-29 | 2017-02-23 | 检测去势抵抗性前列腺癌的方法和检测试剂 |
EP17759780.4A EP3425392A4 (en) | 2016-02-29 | 2017-02-23 | METHOD FOR DETECTING CASTRATION-RESISTANT PROSTATE CANCER AND REAGENT REAGENT |
US16/080,577 US11604194B2 (en) | 2016-02-29 | 2017-02-23 | Method for detecting castration-resistant prostate cancer and detection reagent |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016037135 | 2016-02-29 | ||
JP2016-037135 | 2016-02-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017150314A1 true WO2017150314A1 (ja) | 2017-09-08 |
Family
ID=59743886
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2017/006705 WO2017150314A1 (ja) | 2016-02-29 | 2017-02-23 | 去勢抵抗性前立腺癌を検出する方法及び検出試薬 |
Country Status (5)
Country | Link |
---|---|
US (1) | US11604194B2 (ja) |
EP (1) | EP3425392A4 (ja) |
JP (1) | JP6829444B2 (ja) |
CN (1) | CN108700573B (ja) |
WO (1) | WO2017150314A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019044602A1 (ja) * | 2017-08-30 | 2019-03-07 | 東ソー株式会社 | 癌を検出する方法及び検出試薬 |
WO2021100621A1 (ja) | 2019-11-22 | 2021-05-27 | 国立大学法人大阪大学 | がんの骨転移を検出する方法及び検出試薬 |
WO2023085424A1 (ja) * | 2021-11-12 | 2023-05-19 | 国立大学法人熊本大学 | 前立腺がんの新規マーカー |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003532079A (ja) | 2000-04-20 | 2003-10-28 | セイント ヴィンセンツ ホスピタル シドニー リミテッド | マクロファージ抑制サイトカイン−1(mic−1)が関連する診断アッセイおよび治療方法 |
US20090004181A1 (en) * | 2004-04-13 | 2009-01-01 | St Vincent's Hospitalsydney Limited | Method for Modulating Appetite |
JP2009240300A (ja) | 2008-03-14 | 2009-10-22 | Tosoh Corp | 遺伝子組換え抗体の製造方法 |
JP2009545735A (ja) | 2006-08-04 | 2009-12-24 | メディツィニッシュ ホホシュール ハノーバー | Gdf−15に基づく心臓インターベンションの危険性を評価するための手段および方法 |
JP2010528275A (ja) | 2007-05-24 | 2010-08-19 | エフ.ホフマン−ラ ロシュ アーゲー | Gdf−15及びナトリウム利尿ペプチドを用いた、心房細動を有する患者の心不全を評価するための手段及び方法 |
JP2011501136A (ja) * | 2007-10-22 | 2011-01-06 | セントビンセンツ ホスピタル シドニー リミテッド | 予後判定の方法 |
JP2011509403A (ja) | 2008-01-08 | 2011-03-24 | エフ.ホフマン−ラ ロシュ アーゲー | Gdf−15に基づいて救急室を受診する患者のリスクを評価するための手段及び方法 |
JP2011102461A (ja) | 2011-02-16 | 2011-05-26 | Toray Ind Inc | 多層基材、プリフォームおよびプリフォーム製造方法 |
JP2011523051A (ja) | 2008-05-20 | 2011-08-04 | エフ.ホフマン−ラ ロシュ アーゲー | 1型糖尿病におけるバイオマーカーとしてのgdf−15 |
JP2012515335A (ja) | 2009-01-16 | 2012-07-05 | エフ.ホフマン−ラ ロシュ アーゲー | 線維症と肝硬変を識別する手段及び方法 |
JP2013061321A (ja) | 2011-08-19 | 2013-04-04 | Yokohama City Univ | 組織因子経路阻害因子2(tfpi2)測定による卵巣明細胞腺癌の検査方法および検査薬 |
JP2015108077A (ja) | 2013-12-05 | 2015-06-11 | アイシン化工株式会社 | 構造用接着剤組成物 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5224308B2 (ja) | 2010-02-22 | 2013-07-03 | 公立大学法人横浜市立大学 | 卵巣明細胞腺癌に特異的に発現しているタンパク質とその応用 |
CN103533951B (zh) | 2011-04-08 | 2017-04-19 | 安姆根有限公司 | 使用生长分化因子15(gdf‑15)治疗或改善代谢障碍的方法 |
LT2900263T (lt) * | 2012-09-26 | 2019-10-10 | Julius-Maximilians-Universität Würzburg | Monokloniniai antikūnai, atpažįstantys augimo ir diferenciacijos faktorių 15 (gdf-15) |
WO2015108077A1 (ja) | 2014-01-15 | 2015-07-23 | 地方独立行政法人東京都健康長寿医療センター | ミトコンドリア病診断用バイオマーカーとしてのgdf15 |
-
2017
- 2017-02-23 CN CN201780014137.4A patent/CN108700573B/zh active Active
- 2017-02-23 JP JP2018503075A patent/JP6829444B2/ja active Active
- 2017-02-23 EP EP17759780.4A patent/EP3425392A4/en active Pending
- 2017-02-23 WO PCT/JP2017/006705 patent/WO2017150314A1/ja active Application Filing
- 2017-02-23 US US16/080,577 patent/US11604194B2/en active Active
Patent Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003532079A (ja) | 2000-04-20 | 2003-10-28 | セイント ヴィンセンツ ホスピタル シドニー リミテッド | マクロファージ抑制サイトカイン−1(mic−1)が関連する診断アッセイおよび治療方法 |
US20090004181A1 (en) * | 2004-04-13 | 2009-01-01 | St Vincent's Hospitalsydney Limited | Method for Modulating Appetite |
JP2011190262A (ja) | 2004-04-13 | 2011-09-29 | St Vincent's Hospital Sydney Ltd | 食欲を調節する方法 |
JP2009545735A (ja) | 2006-08-04 | 2009-12-24 | メディツィニッシュ ホホシュール ハノーバー | Gdf−15に基づく心臓インターベンションの危険性を評価するための手段および方法 |
JP2010528275A (ja) | 2007-05-24 | 2010-08-19 | エフ.ホフマン−ラ ロシュ アーゲー | Gdf−15及びナトリウム利尿ペプチドを用いた、心房細動を有する患者の心不全を評価するための手段及び方法 |
JP2011501136A (ja) * | 2007-10-22 | 2011-01-06 | セントビンセンツ ホスピタル シドニー リミテッド | 予後判定の方法 |
JP2013174614A (ja) | 2007-10-22 | 2013-09-05 | St Vincent's Hospital Sydney Ltd | 予後判定の方法 |
JP2011509403A (ja) | 2008-01-08 | 2011-03-24 | エフ.ホフマン−ラ ロシュ アーゲー | Gdf−15に基づいて救急室を受診する患者のリスクを評価するための手段及び方法 |
JP2009240300A (ja) | 2008-03-14 | 2009-10-22 | Tosoh Corp | 遺伝子組換え抗体の製造方法 |
JP2011523051A (ja) | 2008-05-20 | 2011-08-04 | エフ.ホフマン−ラ ロシュ アーゲー | 1型糖尿病におけるバイオマーカーとしてのgdf−15 |
JP2012515335A (ja) | 2009-01-16 | 2012-07-05 | エフ.ホフマン−ラ ロシュ アーゲー | 線維症と肝硬変を識別する手段及び方法 |
JP2011102461A (ja) | 2011-02-16 | 2011-05-26 | Toray Ind Inc | 多層基材、プリフォームおよびプリフォーム製造方法 |
JP2013061321A (ja) | 2011-08-19 | 2013-04-04 | Yokohama City Univ | 組織因子経路阻害因子2(tfpi2)測定による卵巣明細胞腺癌の検査方法および検査薬 |
JP2015108077A (ja) | 2013-12-05 | 2015-06-11 | アイシン化工株式会社 | 構造用接着剤組成物 |
Non-Patent Citations (14)
Title |
---|
"GenBank", Database accession no. NM_004864 |
AGING CELL, vol. 9, 2010, pages 1057 - 1064 |
BAUSKIN, A R ET AL.: "The Propeptide Mediates Formation of Stromal Stores of PROMIC-1: Role in Determining Prostate Cancer Outcome", CANCER RES., vol. 65, no. 6, 15 March 2005 (2005-03-15), pages 2330 - 2336, XP055540445 * |
BIOCHEMICAL PHARMACOLOGY, vol. 85, 2013, pages 597 - 606 |
CANCER EPIDEMIOL BIOMARKERS PREV., vol. 16, 2007, pages 532 - 537 |
CANCER RES., vol. 65, no. 6, 2005, pages 2330 - 2336 |
CLIN CANCER RES., vol. 12, 2006, pages 442 - 446 |
CLIN CANCER RES., vol. 12, no. 1, 2006, pages 89 - 96 |
CLIN CANCER RES., vol. 15, no. 21, 2009, pages 6658 - 6664 |
CLIN CANCER RES., vol. 17, 2011, pages 4825 - 4833 |
CLIN CANCER RES., vol. 9, 2003, pages 2642 - 2650 |
MAGADOUX, L ET AL.: "Emerging targets to monitor and overcome docetaxel resistance in castration resistant prostate cancer(Review", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 45, 2014, pages 919 - 928, XP055540448 * |
PROSTATE CANCER PROSTATIC DIS, vol. 15, no. 4, 2012, pages 320 - 328 |
See also references of EP3425392A4 |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019044602A1 (ja) * | 2017-08-30 | 2019-03-07 | 東ソー株式会社 | 癌を検出する方法及び検出試薬 |
US20200256872A1 (en) * | 2017-08-30 | 2020-08-13 | Tosoh Corporation | Methods for detecting cancers, and detecting reagent |
US11913955B2 (en) * | 2017-08-30 | 2024-02-27 | Tosoh Corporation | Methods for detecting cancers, and detecting reagent |
WO2021100621A1 (ja) | 2019-11-22 | 2021-05-27 | 国立大学法人大阪大学 | がんの骨転移を検出する方法及び検出試薬 |
WO2023085424A1 (ja) * | 2021-11-12 | 2023-05-19 | 国立大学法人熊本大学 | 前立腺がんの新規マーカー |
Also Published As
Publication number | Publication date |
---|---|
JP6829444B2 (ja) | 2021-02-10 |
US11604194B2 (en) | 2023-03-14 |
JPWO2017150314A1 (ja) | 2019-02-07 |
CN108700573B (zh) | 2021-06-11 |
EP3425392A1 (en) | 2019-01-09 |
US20210190786A1 (en) | 2021-06-24 |
EP3425392A4 (en) | 2020-02-05 |
CN108700573A (zh) | 2018-10-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20100240088A1 (en) | Peptide Markers for Diagnosis of Angiogenesis | |
US11193936B2 (en) | Method and reagent for detecting ovarian clear cell adenocarcinoma | |
US20200124604A1 (en) | Biomarker for detecting colorectal cancer | |
JP6074676B2 (ja) | 組織因子経路阻害因子2(tfpi2)測定による卵巣明細胞腺癌の検査方法および検査薬 | |
WO2017150314A1 (ja) | 去勢抵抗性前立腺癌を検出する方法及び検出試薬 | |
JP2024056971A (ja) | APP669-xのN末端を特異的に認識する抗体、及び免疫測定法 | |
JP7127422B2 (ja) | 癌を検出する方法及び検出試薬 | |
WO2008054763A2 (en) | Biomarkers for breast cancer | |
JP7546255B2 (ja) | 癌を検出する方法および検出試薬 | |
WO2021100621A1 (ja) | がんの骨転移を検出する方法及び検出試薬 | |
KR101819939B1 (ko) | 유방암 진단용 단백질 마커 베타-투 마이크로글로불린 및 이를 검출하는 방법 | |
Lang | An LC-MS/MS-based approach for analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using the biomarker prostate-specific antigen (PSA) as example | |
SE1650619A1 (en) | Novel binding assay |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 2018503075 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2017759780 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2017759780 Country of ref document: EP Effective date: 20181001 |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17759780 Country of ref document: EP Kind code of ref document: A1 |