WO2019142636A1 - 腎癌の検出方法及び検査薬 - Google Patents
腎癌の検出方法及び検査薬 Download PDFInfo
- Publication number
- WO2019142636A1 WO2019142636A1 PCT/JP2018/047882 JP2018047882W WO2019142636A1 WO 2019142636 A1 WO2019142636 A1 WO 2019142636A1 JP 2018047882 W JP2018047882 W JP 2018047882W WO 2019142636 A1 WO2019142636 A1 WO 2019142636A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tfpi2
- antibody
- renal cancer
- amount
- residue
- 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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- 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
-
- 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
Definitions
- the present invention relates to a method and reagent for detecting renal cancer, which is to be measured using tissue factor pathway inhibitor 2 (TFPI 2).
- TFPI 2 tissue factor pathway inhibitor 2
- kidney cancer The estimated morbidity rate of renal and urinary tract cancers excluding bladder cancer is 19.5 per 100,000 population (National Cancer Research Center, Cancer Control Information Center, 2013), and is increasing in recent years. Renal cancer has a low subjective symptom, so it is often found by chance in a medical checkup or a medical checkup, and it is considered as a cancer for which early detection is difficult.
- Treatment of kidney cancer is basically surgical excision, but as drug therapy, tyrosine kinase inhibitors (TKIs) that inhibit angiogenesis, molecular targeting agents such as mTOR inhibitors, and immune checkpoint inhibitors are appropriately used. ing.
- TKIs tyrosine kinase inhibitors
- Blood tests for kidney cancer include red blood cells, which determine erythrocyte sedimentation rate, CRP (C-reactive protein), IAP (immunosuppressive acidic protein), and the like.
- CRP C-reactive protein
- IAP immunosuppressive acidic protein
- tissue factor pathway inhibitor 2 is a protein identical to placental protein 5 (Placental protein 5; PP5), and is a placenta-derived serine protease inhibitor comprising three Kunitz-type protease inhibitor domains.
- TFPI2 tissue factor pathway inhibitor 2
- PP5 placental protein 5
- TFPI2 tissue factor pathway inhibitor 2
- Non-Patent Document 3 The association between TFPI2 and renal cancer is suggested that the addition of epigallocatechin gallate (ECGC) contained in green tea increases the expression level of TFPI2 in human renal cancer cell lines and suppresses cell proliferation and induces apoptosis.
- ECGC epigallocatechin gallate
- An object of the present invention is to provide a method for detecting renal cancer and a reagent that can be used for the method.
- the present inventors diligently studied and found that TFPI2 is secreted into the culture supernatants of multiple renal cancer cell lines, and blood TFPI2 is significantly improved in renal cancer patients as compared to healthy individuals, and TFPI2 is renal Having considered that cancer can be detected, the present invention has been completed.
- the present invention includes the following aspects.
- a method for detecting renal cancer comprising measuring the amount of TFPI2 in a sample.
- a renal cancer is detected when the measured value of the amount of TFPI 2 exceeds a preset reference value.
- the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
- a renal cancer comprising an antibody that binds to an antigenic determinant within the region from aspartate at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 Reagent for detection.
- the present invention provides a method for detecting renal cancer conveniently and with high accuracy, and a reagent that can be used for the method.
- the figure which showed the amount of TFPI2 in a cell line culture supernatant The first vertical axis represents the measured value of TFPI2, and the second vertical axis represents the number of cells.
- the vertical axis represents blood TFPI2 amount.
- the vertical axis represents sensitivity, and the horizontal axis represents 100% -specificity.
- the vertical axis represents TFPI2 measurements.
- the figure which showed the correlation of a renal cancer tumor diameter and TFPI2 measured value The vertical axis represents TFPI2 measurements and the horizontal axis represents tumor diameter.
- the vertical axis represents TFPI2 measurements.
- the first aspect of the present invention is a method for detecting renal cancer, which comprises measuring the amount of TFPI2 in a sample. This is a method based on the characteristic presence of TFPI2 in a biological sample such as blood of kidney cancer as compared to a healthy sample. The measurement of the amount of TFPI2 in a sample is usually performed in vitro. According to this method, renal cancer can be detected with high sensitivity and specificity, as shown in Examples described later. In addition, the method of the present invention includes steps up to detection of renal cancer and does not include the final judgment on diagnosis of renal cancer. The doctor diagnoses renal cancer and makes a treatment plan with reference to the detection result and the like according to the method of the present invention.
- the TFPI2 to be measured in the present invention is not particularly limited.
- intact TFPI2 hereinafter also referred to as "I-TFPI2”
- NT-TFPI2 TFPI2 processing polypeptide
- SEQ ID NO: 1 shows the amino acid sequence based on human TFPI2 cDNA.
- starting methionine to glycine at residue 22 are signal peptides.
- Insert TFPI2 refers to a peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO: 1.
- NT-TFPI2 refers to a peptide fragment containing Kunitz domain 1 located on the N-terminal side of intact TFPI2.
- NT-TFPI 2 is a peptide comprising at least a sequence from aspartate at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1, or It is a peptide containing an amino acid sequence having 80% or more identity with the sequence. The identity is preferably 90% or more, more preferably 95% or more.
- this polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids in the above sequence are deleted, substituted, inserted and / or added.
- the term “several” refers to preferably 2 to 20, more preferably 2 to 10, and still more preferably 2 to 5.
- it may have other peptide fragments on both sides of the above sequence, it is preferable not to have an antigenic determinant of an antibody that recognizes Kunitz domain 3 of TFPI2.
- the sample (test sample) derived from a patient in the present invention includes whole blood, blood cells, serum, blood components such as plasma, extracts of cells or tissues, urine, cerebrospinal fluid and the like.
- a kidney tissue biopsy sample may be examined, in which case the extract or culture supernatant of the biopsy sample is measured.
- a body fluid such as a blood component or urine as a sample because it can be performed easily and noninvasively, and taking into consideration the ease of sample collection and versatility to other examination items, the blood component is used as a sample Is particularly preferred.
- the dilution factor of the sample may be appropriately selected from non-dilution to 100-fold dilution according to the type and condition of the sample to be used.
- the collection time of the sample in the present invention is not particularly limited.
- follow-up after definitive diagnosis with renal cancer by postoperative pathological examination may be any time, before and after definite diagnosis, before and after treatment start Samples collected at any stage can be subjected to the method of the present invention.
- the amount of TFPI2 obtained by measurement exceeds a preset reference value (Cutoff value).
- the amount of TFPI2 may be any of intact TFPI2, NT-TFPI2, or a total of intact TFPI2 and NT-TFPI2; however, the total of intact TFPI2 and NT-TFPI2 is sufficient for ease of measurement and sufficient It is more preferable from the viewpoint of coexistence of various sensitivity and specificity.
- the reference value used for the determination may be either a measured value or a converted concentration value.
- the converted concentration value is a value converted from the measured value based on a calibration curve prepared using TFPI 2 as a standard sample.
- the reference value (Cutoff value) for determining renal cancer can be appropriately set to a measurement value that indicates optimal sensitivity and specificity by measuring normal subjects and renal cancer, respectively, and analyzing receiver operating characteristics (ROC) curves.
- the reference value of TFPI2 may be set to 219 pg / mL for plasma and 189 pg / mL or 200 pg / mL for serum as shown in the examples below, but it is not limited thereto.
- the amount of NT-TFPI2 or the amount of intact TFPI2 in the sample may be separately measured, or the values may be summed to obtain a total amount. Alternatively, it may be measured by a measurement system capable of measuring the total amount of NT-TFPI2 and intact TFPI2 in a sample at one time. Alternatively, as described later, the amount of NT-TFPI2 may be measured indirectly from the total amount of both measurements and the measured amount of intact TFPI2 alone.
- the method of measuring the amount of NT-TFPI2 and / or the amount of intact TFPI2 is not particularly limited. For example, a method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and / or intact TFPI2 or a method using mass spectrometry can be exemplified.
- A A competitive method using an antibody that recognizes a labeled measurement target and a measurement target, and that the labeled measurement target and a measurement target included in a sample competitively bind to the antibody.
- 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 antibody that recognizes both NT-TFPI2 and intact TFPI2 can be prepared from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the TFPI 2 amino acid sequence shown in SEQ ID NO: 1
- the antibody is preferably an antibody that binds to an antigenic determinant within the region of SEQ ID NO: 1, and more preferably an antibody having an antigen recognition site for Kunitz domain 1 of TFPI2.
- the sandwich method described above is used in this method, usually, the antibody uses two different epitopes.
- (B) A method of measuring the amount of intact TFPI2 alone using an antibody that recognizes intact TFPI2 without recognizing NT-TFPI2 (I-TFPI2 measurement system).
- the antibody that recognizes intact TFPI2 without recognizing NT-TFPI2 is preferably an antibody having an antigen recognition site for Kunitz domain 3 of TFPI2.
- the sandwich method described above usually, the antibody uses two different epitopes, and at least one of them uses an antibody that does not recognize NT-TFPI2 and recognizes intact TFPI2, and another one.
- the type may be an antibody that does not recognize NT-TFPI2 but recognizes intact TFPI2, or an antibody that recognizes both NT-TFPI2 and intact TFPI2.
- the antibody that does not recognize intact TFPI2 and that recognizes NT-TFPI2 include, for example, an antibody that specifically recognizes the peptide sequence of the C-terminal portion of NT-TFPI2.
- the sandwich method described above for example, the antibody is used as a solid phase antibody, and an antibody having a recognition site for Kunitz domain 1 is used as a detection antibody.
- the amount of NT-TFPI2 alone measured by the method of (C) or (D) described above may be used as the criterion, but it is measured by the method of (A)
- the latter is more preferable because sufficient sensitivity and specificity can be obtained by using the total amount of NT-TFPI2 and intact TFPI2 as a criterion for determination, and the ease of obtaining an antibody and the measurement in one step are simple.
- An antibody that recognizes NT-TFPI2 and / or intact TFPI2 is an NT-TFPI2 polypeptide or protein, an intact TFPI2 polypeptide or an oligopeptide consisting of a partial region of TFPI2 protein, an intact or partial region of NT-TFPI2 polypeptide or TFPI2 protein Can be obtained by immunizing an animal using a polynucleotide encoding H. or the like as an immunogen.
- the protein or the oligopeptide or polypeptide may not reflect the three-dimensional structure of TFPI2 in vivo, or its structure may change during preparation.
- the obtained antibody may not have high specificity or avidity for the desired TFPI2 in vivo, and even if a measurement system is constructed using this antibody, it is included in the sample as a result. May not be accurately quantified.
- an expression vector containing a polynucleotide encoding an intact or partial region of TFPI2 polypeptide or intact TFPI2 protein as an immunogen an intact or partial region of TFPI2 polypeptide or intact TFPI2 protein is expressed in the immunized animal And an immune response is elicited, which is more preferable because an antibody having high specificity and avidity (ie, high affinity) to TFPI2 in a sample can be obtained.
- 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.
- TFPI1 which is known as a homologue of TFPI2 in blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without crossing TFPI1.
- the reagent for measuring the amount of TFPI2 is preferably a reagent for measuring the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
- the reagent for measuring the amount of TFPI 2 is preferably an antigenic determinant within a region from asparagine at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1 It is an antibody that binds, more preferably an antibody that recognizes Kunitz domain 1 of TFPI2.
- the present invention relates to a kidney of an antibody which binds to an antigenic determinant within a region from aspartate at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 It can also be referred to as use in the manufacture of a reagent for detecting cancer.
- the present invention also relates to a kidney of an antibody that binds to an antigenic determinant within a region from aspartate at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 It can also be referred to as use in the detection of cancer.
- the antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- the establishment of hybridoma cells producing an antibody that recognizes TFPI2 may be appropriately selected from methods in which techniques are established. As an example, 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 And monocloning of the selected hybridoma cells by limiting dilution to establish a hybridoma cell producing a monoclonal antibody that recognizes TFPI2.
- Selection of a monoclonal antibody that recognizes TFPI2 used in the present invention may be performed based on the affinity to GPI (glycosylphosphatidylinositol) anchored TFPI2 or secreted TFPI2 derived from a host expression system.
- 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 natural TFPI2 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 present invention may be appropriately selected from methods established in the art. As an example, after culturing hybridoma cells producing antibodies established by the above-mentioned method, 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 when performing the antigen-antibody reaction by the above-mentioned sandwich method may be such that the antibody purified by the above-mentioned method may be labeled with an enzyme such as peroxidase or alkaline phosphatase, and the labeling is also well established. It may be done using a method.
- the method of measuring the amount of TFPI 2 using mass spectrometry in the method of the present invention is specifically described below.
- major proteins such as albumin, immunoglobulins and transferrin, which are abundant in blood, are removed by Agilent Human 14 or the like as a pretreatment step, and further divided by ion exchange, gel filtration, reverse phase HPLC or the like. It is preferable to draw. Alternatively, it is also possible to specifically recover only TFPI2 by an immunological procedure using an anti-TFPI2 antibody.
- 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 ionization time-of-flight mass spectrometry) , MALDI-TOF / MS), surface enhanced laser ionization mass spectrometry (SELDI-MS) or the like.
- the antibody contains two kinds of antibodies having different epitopes.
- 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 methods shown in (I) to (III) below.
- (I) First, among two types of antibodies with different epitopes (hereinafter referred to as “antibody 1” and “antibody 2”) that recognize TFPI 2 used in the sandwich method, antibody 1 is used as an immunoplate, magnetic particles, etc. B / F (Bound / Free) It is bound to a 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 performing an antigen-antibody reaction well 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.
- an antibody that binds 20 ⁇ L of the sample to the carrier per reaction system to be reacted with the antibody may be 100 ng to 1000 ⁇ g, and the amount of labeled antibody may be 2 ng to 20 ⁇ g.
- the reagent of the present invention can also be used for manual measurement and can be used for measurement using an automatic immunodiagnostic apparatus.
- measurement using an automatic immunodiagnostic apparatus can be performed without being affected by endogenous measurement interference factors or competing enzymes contained in the sample, and because TFPI2 in the sample can be quantified in a short time. ,preferable.
- the method of detecting renal cancer of the present invention can be applied to a method of treating renal cancer.
- a method of treating renal cancer in a subject comprising (I) a step of receiving identification of a subject as a measured value of TFPI2 amount exceeding a preset reference value, and (ii) the test identified as a measured value of TFPI2 amount exceeds the preset reference value
- a method is provided, comprising the step of applying a treatment to the body.
- the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
- the measurement of the amount of TFPI is preferably from asparagine at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1 It is carried out by an antigen-antibody reaction using an antibody that binds to an antigenic determinant in the region. More preferably, said antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
- the measurement of the amount of TFPI 2 may be performed using mass spectrometry.
- the treatment in the step (ii) includes surgical excision, drug therapy, radiation therapy and the like, and the drug includes, but not particularly limited to, tyrosine kinase inhibitors, mTOR inhibitors, immune checkpoint inhibitors and the like .
- Example 1 Preparation of TFPI2 Measurement Reagent According to the method of Patent Document 3, a TFPI2 measurement reagent was prepared as follows using the TFPI2 antibody obtained by the DNA immunoassay.
- TS-TF04 water-insoluble ferrite carrier
- 100 mM Tris buffer (pH 8.0) containing 1% BSA By carrying out blocking at 53 ° C. for 4 hours, an anti-TFPI2 antibody-immobilized carrier was prepared.
- the anti-TFPI2 monoclonal antibody (TS-TF01) was used with an alkaline phosphatase labeling kit (manufactured by Dojin Chemical Co., Ltd.) to prepare an alkaline phosphatase-labeled anti-TFPI2 antibody.
- Example 2 Evaluation of various cell culture supernatants including renal cancer 293T cells derived from human fetal kidney as normal cells and CAKI1, ACHN and 786-O cells as renal cancer cells are cultured at 37 ° C for 3 days according to a conventional method. The number of cells was counted after collecting various culture supernatants. The medium alone and 20 ⁇ L of various culture supernatants were evaluated using the TFPI2 measurement reagent prepared in Example 1.
- a fully automatic enzyme immunoassay apparatus AIA-2000 manufactured by Tosoh Corp .: manufacturing and sales notification number 13B3X90002000009
- the measurement of TFPI2 by the fully automatic enzyme immunoassay apparatus AIA-2000 was performed according to the following procedure.
- Example 3 Evaluation of Clinical Samples The clinical samples used in the following examples are shown in Table 1. 21 cases of renal cancer plasma were samples collected by the same protocol in Yokohama City University urology department, and were provided with the consent of informed consent and the approval of Yokohama City University ethics committee. Forty-nine healthy human plasmas were purchased from Vidicom Japan. The clinical sample was measured by the method described in Example 2, and a calibration curve was prepared using the commercially available TFPI2 recombinant protein (R & D) as a standard product, and the TFPI2 concentration in the sample was calculated.
- R & D commercially available TFPI2 recombinant protein
- the ROC analysis results are shown in FIG.
- the area under the curve (AUC) is 0.8552, showing that TFPI2 has good renal cancer detection performance.
- the Youden Index maximum value was set as the TFPI2 cutoff value based on the Youden Index ((sensitivity + specificity)-100).
- the TFPI2 cutoff value is 219 pg / mL
- the sensitivity is 85.7%
- the specificity is 75.5%, and it has been shown to have good renal cancer detection performance.
- Example 4 Relationship between histological grade and TFPI 2 Renal cancer sample groups are classified by histological grade (grade: G), and BoxPlot of TFPI 2 measurement values of each group is shown in FIG. There was no significant difference in TFPI2 between low grade grade 2 and high grade 3 and 4. The present results suggest that TFPI2 is not associated with histologic grade, and that expression of TFPI2 is improved even in the early stage of renal cancer.
- TFPI2 standard value and reference standard range in healthy Japanese people
- TFPI2 was measured by the method described in Example 3, and a healthy reference reference range was calculated according to TFPI2 healthy individual reference value (for example, median + 2SD) and CLSI guideline EP28-A3.
- the BoxPlot of the TFPI 2 measurement results are shown in FIG. 7, and the reference values and reference reference ranges calculated from healthy individuals are shown in Table 2.
- the standard value of TFPI2 calculated from the median + 2 SD of healthy people was 200 pg / mL.
- Example 8 Evaluation of clinical samples 52 cases of renal cancer serum (clear cell carcinoma: 40 cases, papillary carcinoma: 4 cases, other renal cancers: 8 cases) were collected by the same protocol at Yokohama City University urology department Were provided with the consent of informed consent and the approval of the Yokohama City University Ethics Committee.
- TFPI2 was measured by the method described in Example 3, and the renal cancer detection performance was evaluated using the healthy human TFPI2 measurement value described in Example 7.
- FIG. 8 shows the results of measuring TFPI2 in BoxPlot. TFPI2 tended to be high in clear cell carcinoma and papillary carcinoma in renal cancer tissue types, and in some other tissue types, it was also high.
- FIG. 8 shows the results of measuring TFPI2 in BoxPlot. TFPI2 tended to be high in clear cell carcinoma and papillary carcinoma in renal cancer tissue types, and in some other tissue types, it was also high.
- FIG. 9 shows the results of ROC curve analysis when healthy individuals and renal cancer patients are compared.
- the area under the curve (AUC) is 0.79, showing that TFPI2 has good renal cancer detection performance.
- the Youden Index ((sensitivity + specificity)-100)
- the Youden Index maximum value is 189 pg / mL is used as a reference value
- the discrimination performance between renal cancer patients and healthy people is 59.6% , 94.6% specificity. From the above results, it was shown that TFPI 2 has good renal cancer detection performance by setting a suitable reference value in serum as well as plasma shown in Example 3.
- a method for detecting a renal cancer patient by a simple and relatively low burden blood test This is expected to contribute to renal cancer medical care that relies on imaging diagnosis without the presence of effective tumor markers, and is very useful in industry.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Gastroenterology & Hepatology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biophysics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
Description
腎癌の治療は原則外科的切除であるが、薬物療法として血管新生を阻害するチロシンキナーゼ阻害剤(TKI)、mTOR阻害剤などの分子標的薬、並びに、免疫チェックポイント阻害剤等が適宜用いられている。
[1]検体において、TFPI2量を測定することを含む、腎癌を検出する方法。
[2]前記TFPI2量の測定値が、予め設定した基準値を超えた場合に、腎癌が検出されたとする、[1]に記載の方法。
[3]前記TFPI2量が、TFPI2プロセシングポリペプチド量及びインタクトTFPI2量の合計である、[1]又は[2]に記載の方法。
[4]前記TFPI2量の測定が、配列番号1のアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われるものである、[1]~[3]のいずれかに記載の方法。
[5]前記抗体が、TFPI2のクニッツドメイン1を認識する抗体である、[4]に記載の方法。
[6]質量分析法を用いて測定を行う、[1]~[3]のいずれかに記載の方法。
[7]配列番号1に示すアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体を含む、腎癌を検出するための試薬。
本発明の第一の態様は、腎癌を検出する方法であり、検体においてTFPI2量を測定することを含む。これは、健常な検体と比べて、腎癌の血液等の生体試料中に特徴的にTFPI2が存在することに基づく方法である。検体におけるTFPI2量の測定は、通常インビトロ(in vitro)で行われる。この方法により、後述する実施例が示す通り、高い感度と特異度で腎癌を検出することができる。
なお、本発明の方法は、腎癌を検出する段階までを含むものであり、腎癌の診断に関する最終的な判断行為は含まれない。医師は、本発明の方法による検出結果等を参照して、腎癌を診断したり治療方針を立てたりする。
「インタクトTFPI2」とは、配列番号1のアミノ酸配列の23残基目から235残基目で表されるペプチドをいう。
また、「NT-TFPI2」は、特許文献3に記載されるように、インタクトTFPI2のN末端側に位置するクニッツドメイン1を含むペプチド断片をいう。より具体的には、NT-TFPI2は、配列番号1のアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの配列を少なくとも含むペプチド、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ペプチドである。前記同一性は、好ましくは90%以上、より好ましくは95%以上である。また、このポリペプチドは、前記配列において1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなるポリペプチドであってもよい。なお、数個とは、好ましくは2~20個、より好ましくは2~10個、さらに好ましくは2~5個をいう。また、前記配列の両側に他のペプチドフラグメントを有していてもよいが、TFPI2のクニッツドメイン3を認識する抗体の抗原決定基を有しないことが好ましい。
判定に用いる基準値は、測定値もしくは換算濃度値のいずれでもよい。なお、換算濃度値は、TFPI2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。
本発明において、検体中のNT-TFPI2量又はインタクトTFPI2量を個別に測定してもよく、またその値を合計して合計量としてもよい。また、検体中のNT-TFPI2とインタクトTFPI2の合計量を一度に測定できる測定系で測定してもよい。あるいは、後述するように、両方の測定による合計量とインタクトTFPI2単独の測定量とから間接的にNT-TFPI2量を測定してもよい。
本発明の方法において、NT-TFPI2量及び/又はインタクトTFPI2量を測定する方法は特に制限されない。例えば、NT-TFPI2及び/又はインタクトTFPI2を認識する抗体を用いる抗原抗体反応を利用した方法や、質量分析法を利用した方法が例示できる。
(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。
(A)NT-TFPI2とインタクトTFPI2の両方を認識する抗体を用いて、NT-TFPI2及びインタクトTFPI2の合計量を測定する方法(NT+I-TFPI2測定系)。なお、前記NT-TFPI2とインタクトTFPI2の両方を認識する抗体は、配列番号1で表されるTFPI2アミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体であることが好ましく、TFPI2のクニッツドメイン1に対する抗原認識部位を有する抗体であることがさらに好ましい。また、この方法で前述したサンドイッチ法を用いる場合は、通常、前記抗体はエピトープの異なる2種類を用いる。
(D)インタクトTFPI2を認識せずNT-TFPI2を認識する抗体を用いて、NT-TFPI2単独の量を測定する方法。なお、前記インタクトTFPI2を認識せずNT-TFPI2を認識する抗体は、例えば、NT-TFPI2のC末端部分のペプチド配列を特異的に認識する抗体が挙げられる。前述したサンドイッチ法を用いる場合は、例えば、当該抗体を固相抗体とし、クニッツドメイン1に対する認識部位を有する抗体を検出抗体とする。
一方、免疫原としてTFPI2ポリペプチド又はインタクトTFPI2タンパク質のインタクトまたは部分領域をコードするポリヌクレオチドを含む発現ベクターを用いることで、免疫動物の体内でTFPI2ポリペプチド又はインタクトTFPI2タンパク質のインタクトまたは部分領域が発現され免疫応答が惹起されるため、検体中のTFPI2に対して高い特異性及び結合力(すなわち高親和性)を有した抗体が得られるためより好ましい。
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。
したがって、本発明は、配列番号1に示すアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体の、腎癌を検出するための試薬の製造における使用ともいうことができる。
また、本発明は、配列番号1に示すアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体の、腎癌の検出における使用ともいうことができる。
TFPI2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、TFPI2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、前記宿主としては特に限定はなく、当業者がタンパク質の発現に通常用いる、大腸菌や酵母などの微生物細胞、昆虫細胞、動物細胞の中から適宜選択すればよいが、ジスルフィド結合もしくは糖鎖付加といった翻訳後修飾により、天然型のTFPI2に近い構造を有するタンパク質の発現が可能な、哺乳細胞を宿主として用いると好ましい。哺乳細胞の一例としては、従来用いられている、ヒト胎児腎臓由来細胞(HEK)293T細胞株、サル腎臓細胞COS7株、チャイニーズハムスター卵巣(CHO)細胞またはヒトから単離された癌細胞などが挙げられる。
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主要タンパク質を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。または、抗TFPI2抗体を用いた免疫的手法によりTFPI2のみを特異的に回収することも可能である。
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好ましい。
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。
まず、本発明の試薬は、以下の(I)から(III)に示す方法で作製することができる。
(I)まず、サンドイッチ法で用いる、TFPI2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。
(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緩衝液などが好ましい。このようにして作製した本発明の試薬は必要に応じ凍結乾燥させてもよい。
(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。
本発明の試薬に含まれる抗体等の試薬成分の量は、検体量、検体の種類、試薬の種類、測定の手法等の諸条件に応じて適宜設定すればよい。具体的には、例えば、後述するように検体として血清や血漿を20μL使用して、サンドイッチ法によりTFPI2量の測定を行う場合、当該検体20μLを抗体と反応させる反応系当たり、担体へ結合させる抗体量が100ngから1000μgであってよく、標識抗体量が2ngから20μgであってよい。
すなわち、本発明により、被験体における腎癌を治療する方法であって、
(i)TFPI2量の測定値が予め設定した基準値を超えるものとして被験体の同定を受ける工程、及び
(ii)TFPI2量の測定値が予め設定した基準値を超えるものとして同定された前記被験体に対して、治療を施す工程を含む、方法が提供される。
前記腎癌を治療する方法の好ましい態様において、前記TFPI2量は、TFPI2プロセシングポリペプチド量及びインタクトTFPI2量の合計である。
また、前記工程(i)の同定において、TFPI2量の測定は、好ましくは、配列番号1のアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われる。より好ましくは、前記抗体は、TFPI2のクニッツドメイン1を認識する抗体である。
また、前記工程(i)の同定において、TFPI2量の測定は、質量分析法を用いて行われてもよい。
前記工程(ii)の治療としては、外科的切除、薬物療法、放射線療法等が挙げられ、前記薬物としてはチロシンキナーゼ阻害剤、mTOR阻害剤、免疫チェックポイント阻害剤等が挙げられるが特に限定されない。
特許文献3の方法に従い、DNA免疫法により得られたTFPI2抗体を用いてTFPI2測定試薬を以下の通り調製した。
(2)抗TFPI2モノクローナル抗体(TS-TF01)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、アルカリフォスファターゼ標識抗TFPI2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)で調製したアルカリフォスファターゼ標識抗体を1μg/mL含む緩衝液(3%BSAを含むトリス緩衝液、pH8.0)100μLを添加し、凍結乾燥を実施することで、TFPI2測定試薬を作製した。なお、作製したTFPI2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。
正常細胞としてヒト胎児腎由来の293T細胞、腎癌細胞としてCAKI1、ACHN及び786-O細胞を常法に従い37℃で3日間培養し、各種培養上清を回収した後に細胞数を計測した。培地のみと各種培養上清20μLを実施例1で調製したTFPI2測定試薬で評価した。評価用装置は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)を用いた。全自動エンザイムイムノアッセイ装置AIA-2000によるTFPI2の測定は、以下の手順で行った。
(2)37℃恒温下で10分間の抗原抗体反応を行ない、
(3)B/F分離後、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し、
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(TFPI2 intensity、nmol/(L・s))とした。
評価結果を図1に示す。TFPI2は293T細胞と比較して3種の腎癌細胞培養上清にて明瞭に高値を示し、腎癌細胞が培養上清中にTFPI2を分泌していることが明らかとなった。
以下の実施例で使用した臨床検体を表1に示す。腎癌血漿21例は横浜市立大学泌尿器科にて同一プロトコルにて収集された検体であり、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提供された。健常人血漿49例はビジコムジャパン社から購入した。
実施例2記載の方法で臨床検体を測定し、市販TFPI2組み換えタンパク(R&D社)を標準品として検量線を作成し、検体中のTFPI2濃度を算出した。
腎癌検体群を組織学的悪性度(グレード:G)で分類し、各群のTFPI2測定値のBoxPlotを図4に示す。TFPI2は低グレードであるグレード2と高グレードである3及び4で有意差は認められなかった。本結果より、TFPI2は組織学的悪性度との関連性はなく、腎癌の初期であってもTFPI2の発現が向上していることが示唆された。
腎癌腫瘍直径とTFPI2の相関を解析した結果を図5に示す。TFPI2と腫瘍直径は低い正の相関は認められたものの、統計学的な有意差は認められなかった(スピアマン順位相関係数、r=0.27、p=0.2365)。
腎癌検体群を転移の有無で2群に分類し、各群のTFPI2測定値のBoxPlotを図6に示す。2群間のTFPI2測定値に統計的有意差は認められず(マン=ホイットニーのU検定、p=0.5906)、TFPI2と転移に明瞭な関係性は認められなかった。
男性健常人血清102例はインフォームドコンセントの承諾を受けて収集された社内ボランティア検体であり、女性健常人血清120例は横浜市大にて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提供された。実施例3に記載した方法でTFPI2を測定し、TFPI2の健常人基準値(例として中央値+2SD)およびCLSIガイドラインEP28-A3に従って健常人参考基準範囲を算出した。
TFPI2測定結果のBoxPlotを図7に、健常人から算出した基準値および参考基準範囲を表2に示す。本検討の結果、日本人健常人においてTFPI2測定値に性差はなく、健常人の中央値+2SDから算出したTFPI2の基準値は200pg/mLとなった。
腎癌血清52例(淡明細胞癌:40例、乳頭状癌:4例、その他腎癌:8例)は横浜市立大学泌尿器科にて同一プロトコルにて収集された検体であり、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提供された。
実施例3記載の方法でTFPI2を測定し、実施例7記載の健常人TFPI2測定値を用いて腎癌検出性能を評価した。
図8は、TFPI2測定値をBoxPlotにて示した結果である。TFPI2は腎癌組織型において淡明細胞癌および乳頭状癌で高値傾向を示し、その他に区分した組織型でも一部で高値を示した。
図9は、健常人と腎癌患者を比較した際のROC曲線解析結果である。曲線下面積(AUC)は0.79であり、TFPI2は良好な腎癌検出性能を有することが示された。Youden Index((感度+特異度)-100)に基づいて、Youden Index最大値である189pg/mLを基準値とした場合、腎がん患者と健常人との識別性能は、感度59.6%、特異度94.6%であった。
以上の結果より、実施例3に示す血漿と同様に、血清においてもTFPI2は適した基準値を設定することで、良好な腎癌検出性能を有することが示された。
Claims (7)
- 検体において、TFPI2量を測定することを含む、腎癌を検出する方法。
- 前記TFPI2量の測定値が、予め設定した基準値を超えた場合に、腎癌が検出されたとする、請求項1に記載の方法。
- 前記TFPI2量が、TFPI2プロセシングポリペプチド量及びインタクトTFPI2量の合計である、請求項1又は請求項2に記載の方法。
- 前記TFPI2量の測定が、配列番号1のアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われるものである、請求項1~3のいずれか一項に記載の方法。
- 前記抗体が、TFPI2のクニッツドメイン1を認識する抗体である、請求項4に記載の方法。
- 質量分析法を用いて測定を行う、請求項1~3のいずれか一項に記載の方法。
- 配列番号1に示すアミノ酸配列の23残基目のアスパラギン酸から131残基目のヒスチジン又は130残基目のシステインまでの領域内の抗原決定基に結合する抗体を含む、腎癌を検出するための試薬。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201880086569.0A CN111656196A (zh) | 2018-01-16 | 2018-12-26 | 肾癌的检测方法和检查试剂 |
EP18901702.3A EP3742166A4 (en) | 2018-01-16 | 2018-12-26 | KIDNEY CANCER DETECTION METHOD AND TEST DRUG |
CA3088342A CA3088342A1 (en) | 2018-01-16 | 2018-12-26 | Renal cancer detection method and test drug |
BR112020014205-0A BR112020014205A2 (pt) | 2018-01-16 | 2018-12-26 | Método e reagente para detecção de câncer renal |
JP2019566393A JP7449530B2 (ja) | 2018-01-16 | 2018-12-26 | 腎癌の検出方法及び検査薬 |
AU2018402956A AU2018402956A1 (en) | 2018-01-16 | 2018-12-26 | Renal cancer detection method and test drug |
US16/962,352 US20210063400A1 (en) | 2018-01-16 | 2018-12-26 | Renal cancer detection method and test drug |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2018-004737 | 2018-01-16 | ||
JP2018004737 | 2018-01-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019142636A1 true WO2019142636A1 (ja) | 2019-07-25 |
Family
ID=67301706
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2018/047882 WO2019142636A1 (ja) | 2018-01-16 | 2018-12-26 | 腎癌の検出方法及び検査薬 |
Country Status (8)
Country | Link |
---|---|
US (1) | US20210063400A1 (ja) |
EP (1) | EP3742166A4 (ja) |
JP (1) | JP7449530B2 (ja) |
CN (1) | CN111656196A (ja) |
AU (1) | AU2018402956A1 (ja) |
BR (1) | BR112020014205A2 (ja) |
CA (1) | CA3088342A1 (ja) |
WO (1) | WO2019142636A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021246153A1 (ja) | 2020-06-03 | 2021-12-09 | 公立大学法人横浜市立大学 | 膵臓がんの検出方法及び検出試薬 |
WO2022255401A1 (ja) * | 2021-06-03 | 2022-12-08 | 国立大学法人 東京大学 | Erk-mapk経路の異常な活性化に伴い発現する疾患マーカー |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5224309B2 (ja) | 1973-09-05 | 1977-06-30 | ||
US20130065782A1 (en) * | 2011-08-22 | 2013-03-14 | Somalogic, Inc. | Renal Cell Carcinoma Biomarkers and Uses Thereof |
WO2016084912A1 (ja) | 2014-11-27 | 2016-06-02 | 公立大学法人横浜市立大学 | 卵巣明細胞腺癌の検査方法及び検査薬 |
JP6074676B2 (ja) | 2011-08-19 | 2017-02-08 | 公立大学法人横浜市立大学 | 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 |
CN107723368A (zh) * | 2017-11-28 | 2018-02-23 | 杭州可帮基因科技有限公司 | 一组用于肾细胞癌分子分型的基因及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005024603A2 (en) * | 2003-09-10 | 2005-03-17 | The Board Of Regents Of The University Of Texas System | Methods for detecting, diagnosing and treating human renal cell carcinoma |
US8790869B2 (en) * | 2009-03-20 | 2014-07-29 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Renal cell carcinoma biomarkers |
-
2018
- 2018-12-26 JP JP2019566393A patent/JP7449530B2/ja active Active
- 2018-12-26 US US16/962,352 patent/US20210063400A1/en active Pending
- 2018-12-26 WO PCT/JP2018/047882 patent/WO2019142636A1/ja unknown
- 2018-12-26 EP EP18901702.3A patent/EP3742166A4/en active Pending
- 2018-12-26 AU AU2018402956A patent/AU2018402956A1/en active Pending
- 2018-12-26 BR BR112020014205-0A patent/BR112020014205A2/pt unknown
- 2018-12-26 CA CA3088342A patent/CA3088342A1/en active Pending
- 2018-12-26 CN CN201880086569.0A patent/CN111656196A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5224309B2 (ja) | 1973-09-05 | 1977-06-30 | ||
JP6074676B2 (ja) | 2011-08-19 | 2017-02-08 | 公立大学法人横浜市立大学 | 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 |
US20130065782A1 (en) * | 2011-08-22 | 2013-03-14 | Somalogic, Inc. | Renal Cell Carcinoma Biomarkers and Uses Thereof |
WO2016084912A1 (ja) | 2014-11-27 | 2016-06-02 | 公立大学法人横浜市立大学 | 卵巣明細胞腺癌の検査方法及び検査薬 |
CN107723368A (zh) * | 2017-11-28 | 2018-02-23 | 杭州可帮基因科技有限公司 | 一组用于肾细胞癌分子分型的基因及其应用 |
Non-Patent Citations (8)
Title |
---|
ANONYMOUS: "Expression of TFPI2", 26 August 2017 (2017-08-26), pages 1, XP055628024, Retrieved from the Internet <URL:https://web.archive.org/web/20170826002830/https://www.proteinatlas.org/ENSG00000105825-TFPI2/pathology> [retrieved on 20190219] * |
BIN GU , QIANG DING , GUOWEI XIA , ZUJUN FANG : "EGCG inhibits growth and induces apoptosis in renal cell carcinoma through TFPI-2 overexpression", ONCOLOGY REPORTS, vol. 21, no. 3, 1 March 2009 (2009-03-01), pages 635 - 640, XP055628029, ISSN: 1021-335X, DOI: 10.3892/or_00000266 * |
BIN GU ; QIANG DING ; ZUJUN FANG ; CUOWEI XIA: "Expression and methylation status of tissue factor pathway inhibitor-2 in renal cell carcinoma", CHINESE JOURNAL OF UROLOGY, vol. 29, no. Z1, 1 January 2008 (2008-01-01), pages 12 - 15, XP009522535, ISSN: 1000-6702, DOI: 10.3321/j.issn:1000-6702.2008.z1.003 * |
J. PROTEOME RES., vol. 12, no. 10, 2013, pages 4340 - 4350 |
NAREK Z WOJTUKIEWICZ , EWA SIERKO , LECH ZIMMOCH , LESZEK KOZLOWSKI , WALTER KISEL : "Immunohistochemical localization of tissue factor pathway inhibitor-2 in human tumor tissue", THROMBOSIS AND HAEMOSTASIS, vol. 90, no. 1, 31 July 2003 (2003-07-31), pages 140 - 146, XP008131680, ISSN: 0340-6245 * |
ONCOLOGY REPORTS, vol. 21.3, 2009, pages 635 - 640 |
PLOS ONE, vol. 11.10, 2016, pages e0165609 |
See also references of EP3742166A4 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021246153A1 (ja) | 2020-06-03 | 2021-12-09 | 公立大学法人横浜市立大学 | 膵臓がんの検出方法及び検出試薬 |
WO2022255401A1 (ja) * | 2021-06-03 | 2022-12-08 | 国立大学法人 東京大学 | Erk-mapk経路の異常な活性化に伴い発現する疾患マーカー |
Also Published As
Publication number | Publication date |
---|---|
CA3088342A1 (en) | 2019-07-25 |
US20210063400A1 (en) | 2021-03-04 |
EP3742166A4 (en) | 2021-08-25 |
AU2018402956A1 (en) | 2020-08-27 |
JPWO2019142636A1 (ja) | 2021-02-04 |
AU2018402956A2 (en) | 2020-09-03 |
CN111656196A (zh) | 2020-09-11 |
JP7449530B2 (ja) | 2024-03-14 |
EP3742166A1 (en) | 2020-11-25 |
BR112020014205A2 (pt) | 2020-12-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6096813B2 (ja) | 乳癌診断用マルチバイオマーカーセット、その検出方法、及びそれに対する抗体を含む乳癌診断キット | |
WO2015182580A1 (ja) | 大腸がんの転移検出方法 | |
WO2010101047A1 (ja) | 子宮内膜症の判定方法、および子宮内膜症の診断用キット | |
US20160349272A1 (en) | Assessing neuronal damage from blood samples | |
WO2019142636A1 (ja) | 腎癌の検出方法及び検査薬 | |
KR101495225B1 (ko) | Ast 양의 측정을 통한 간 질환 진단, 예후 또는 모니터링 키트 및 방법 | |
CN107110848B (zh) | 以脱氧羟腐胺缩赖氨酸合酶基因作为指标使用的动脉硬化及癌的检测方法 | |
CN111051889B (zh) | 检测癌症的方法和检测试剂 | |
JP6760562B2 (ja) | 卵巣明細胞癌患者の予後を予測するための情報提供方法 | |
WO2021246153A1 (ja) | 膵臓がんの検出方法及び検出試薬 | |
WO2021100621A1 (ja) | がんの骨転移を検出する方法及び検出試薬 | |
WO2023013673A1 (ja) | 悪性腫瘍関連血栓塞栓症の検出方法及び検出試薬 | |
WO2021172000A1 (ja) | 卵巣悪性腫瘍の検出方法及び検出試薬 | |
WO2018222069A1 (ru) | Белковый маркер для диагностики рака предстательной железы, способ измерения количества маркера и алгоритм интерпретации результата | |
EP2799877A1 (en) | Process for diagnosing a human subject with diseases affecting the kidneys, or at risk of acquiring diseases affecting the kidneys | |
KR101702115B1 (ko) | 대장암에 대한 신규 바이오마커 및 그의 용도 | |
JP2022076410A (ja) | 悪性末梢神経鞘腫瘍の診断マーカー | |
WO2013059105A2 (en) | Predictive biomarkers for breast cancer | |
WO2016005916A1 (en) | A method and kit for the detection of a biomarker of thyroid cancer in biological samples | |
KR20150030046A (ko) | 보체인자 i 단백질에 특이적으로 결합하는 폴리펩타이드 또는 항체를 포함하는 췌장암 진단용 조성물 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18901702 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2019566393 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 3088342 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2018402956 Country of ref document: AU Date of ref document: 20181226 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2018901702 Country of ref document: EP Effective date: 20200817 |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112020014205 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112020014205 Country of ref document: BR Kind code of ref document: A2 Effective date: 20200710 |