WO2016143805A1 - Method for detecting bladder cancer - Google Patents

Method for detecting bladder cancer Download PDF

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WO2016143805A1
WO2016143805A1 PCT/JP2016/057270 JP2016057270W WO2016143805A1 WO 2016143805 A1 WO2016143805 A1 WO 2016143805A1 JP 2016057270 W JP2016057270 W JP 2016057270W WO 2016143805 A1 WO2016143805 A1 WO 2016143805A1
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monoclonal antibody
antibody
bladder cancer
group
signal intensity
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PCT/JP2016/057270
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French (fr)
Japanese (ja)
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孝広 落谷
祐亮 吉岡
隆之 水谷
辰也 力石
秀郎 佐々木
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テオリアサイエンス株式会社
国立研究開発法人国立がん研究センター
学校法人 聖マリアンナ医科大学
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Priority to JP2017505364A priority Critical patent/JPWO2016143805A1/en
Publication of WO2016143805A1 publication Critical patent/WO2016143805A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

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  • the present invention relates to a method for detecting bladder cancer. More specifically, bladder cancer using a monoclonal antibody or an antibody fragment thereof against a specific antigen (CD9, CD63, CD55, CD82, HLA-DRB1) on the surface of an extracellular endoplasmic reticulum containing exosomes in a sample
  • a specific antigen CD9, CD63, CD55, CD82, HLA-DRB1
  • the present invention relates to a method for detecting the presence of and a kit used in the method.
  • Exosomes are a type of extracellular endoplasmic reticulum that exists in body fluids in vivo. It is known that various membrane proteins exist on the exosome surface as in the general cell surface. In addition, exosomes have been reported to be secreted from various cells, such as cells of the immune system and various cancer cells, functioning as mediators of intercellular communication in vivo and related to physiological phenomena, Relevance to diseases such as cancer is drawing attention.
  • Non-Patent Document 1 discloses that the mRNA expression levels of CD46, CD55, and CD59 are increased in the tumor tissue of bladder cancer patients.
  • CD55 is described as an example of a tumor marker characteristic of the bladder of a bladder cancer patient.
  • Non-Patent Document 2 CD82 (KAI1) mutation rarely occurs, and KAI1 down-regulation and glycosylation are considered to be important as marker information, and prostate cancer, lung cancer, pancreatic cancer It is disclosed that it is involved in the progress.
  • KAI1 is expressed at the protein level in normal bladder cells, but it is shown that expression is suppressed in 3 out of 8 bladder cancer cell lines and greatly promoted in 1 type, The association with bladder cancer is unknown.
  • Patent Document 2 describes that CD82 increases at the gene level (mRNA) in bladder cancer cells that are resistant to chemotherapy using cisplatin.
  • Patent Document 3 discloses a method for diagnosing bladder cancer by targeting a marker that shows an abnormal value compared to normal bladder cells, and HLA-DRB1 is exemplified as a marker that increases the expression level.
  • Cancer can be detected using a membrane protein present in an extracellular vesicle such as an exosome as a biomarker.
  • an extracellular vesicle such as an exosome as a biomarker.
  • it is difficult to obtain sufficient results due to large fluctuations in sensitivity and specificity, etc., and it may be false positive in conventional cancer diagnosis
  • An object of the present invention is to provide a method for detecting bladder cancer by measuring extracellular endoplasmic reticulum, particularly exosomes, and a kit used for the method.
  • the present invention relates to the following [1] to [7].
  • [1] at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody , And at least one selected from the group consisting of antibody fragments thereof, and at least one selected from the group consisting of CD9 and / or CD63 in body fluid samples derived from a subject, CD55, CD82, and HLA-DRB1 Measuring the signal intensity from the extracellular endoplasmic reticulum expressing The presence of bladder cancer in the case where the signal intensity in the subject is recognized to be stronger than the signal intensity in the control, comprising the step of comparing the signal intensity in the subject obtained in the step and the signal intensity in the control Method for detecting bladder cancer, which is an indicator of [2] (A) at least one
  • [4] at least one selected from the group consisting of anti-CD9 monoclonal antibody, anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody And an extracellular endoplasmic reticulum recognized by at least one selected from the group consisting of antibody fragments thereof as a marker for bladder cancer.
  • [5] at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody. And an exosome recognized by at least one selected from the group consisting of antibody fragments thereof as a marker for bladder cancer.
  • bladder cancer can be detected by measuring extracellular vesicles such as exosomes without worrying about false positives, thereby determining the presence or degree of progression of bladder cancer. can do.
  • FIG. 1 is a diagram showing detection results of various markers in urine derived from urine of a bladder cancer patient using an exoscreen method.
  • FIG. 2 is a diagram showing relative results based on the amount of creatinine for the expression of various markers in urinary exosomes of bladder cancer patients using the exoscreen method.
  • FIG. 3 is a graph comparing the detection results (creatinine amount standard) of exosome-derived CD55 and CD82.
  • the present invention is a method for detecting bladder cancer in a subject, wherein a signal intensity derived from an extracellular endoplasmic reticulum in a body fluid sample is measured using a specific monoclonal antibody, and the value is higher than that of a healthy person
  • a large feature is that it is judged that there is a possibility of having bladder cancer when it is large.
  • a step of measuring the signal intensity derived from an extracellular endoplasmic reticulum expressing the antigen in a body fluid sample derived from a subject using a monoclonal antibody or antibody fragment thereof against a specific antigen hereinafter also referred to as step A).
  • step B the step of comparing the signal intensity in the subject obtained in the step with the signal intensity in the control (hereinafter also referred to as step B), wherein the signal intensity in the subject is If it is recognized that it is stronger than the signal intensity, it is an indicator of the presence of bladder cancer.
  • detecting bladder cancer includes detecting the presence or absence of development of bladder cancer and the degree of progression of a disease state.
  • the present inventors focused on membrane proteins present in urinary exosomes, and examined those that are detected in urinary exosomes of bladder cancer patients but difficult to detect in healthy individuals.
  • CEACAM7, DUOX2, HLA-DRB1, ABCB1, ELANE, IFITM2, CD55, and CD82 as antigens specific to exosomes in the urine of patients.
  • the present inventors have previously found that exosomes can be specifically captured using anti-CD9 antibody and anti-CD63 antibody. By combining these with an antibody against the antigen, the measurement value of a healthy person is It was found that good results were obtained that there was little variation and no false positives were detected and that signals were detected only from bladder cancer patients, and the present invention was completed.
  • Step A is a step of measuring a signal derived from an extracellular endoplasmic reticulum expressing the antigen in a body fluid sample derived from a subject using a monoclonal antibody or an antibody fragment thereof against the specific antigen.
  • the extracellular vesicle is an vesicle secreted outside the cell, and examples thereof include exosomes.
  • Examples of the monoclonal antibody or antibody fragment thereof used in Step A include monoclonal antibodies or fragments thereof consisting of two groups. Specifically, an antibody group (A) that specifically captures extracellular vesicles and an antibody group (B) against an antigen specific to the extracellular vesicle secreted from bladder cancer cells.
  • the antibody group (A) may be any antibody that can specifically capture extracellular vesicles, specifically, an anti-CD9 monoclonal antibody or an antibody fragment thereof, an anti-CD63 monoclonal antibody or an antibody fragment thereof. It is sufficient that at least one of these can be used.
  • the antibody group (B) may be any antibody that can specifically capture extracellular vesicles secreted from bladder cancer cells, specifically, an anti-CEACAM7 monoclonal antibody or an antibody fragment thereof, an anti-DUOX2 monoclonal antibody Or an antibody fragment thereof, anti-HLA-DRB1 monoclonal antibody or antibody fragment thereof, anti-ABCB1 monoclonal antibody or antibody fragment thereof, anti-ELANE monoclonal antibody or antibody fragment thereof, anti-IFITM2 monoclonal antibody or antibody fragment thereof, anti-CD55 monoclonal antibody or antibody thereof Fragment, anti-CD82 monoclonal antibody or antibody fragment thereof. It is sufficient that at least one of these can be used.
  • the monoclonal antibody or antibody fragment thereof used in the present invention may be any one that recognizes a specific antigen, and can be prepared according to a known method. That is, an anti-CD9 monoclonal antibody or an antibody fragment thereof recognizes CD9, an anti-CD63 monoclonal antibody or an antibody fragment thereof recognizes CD63, an anti-CEACAM7 monoclonal antibody or an antibody fragment thereof recognizes CEACAM7, and an anti-DUOX2 monoclonal antibody or an antibody thereof
  • the antibody fragment recognizes DUOX2, the anti-HLA-DRB1 monoclonal antibody or an antibody fragment thereof recognizes HLA-DRB1, the anti-ABCB1 monoclonal antibody or an antibody fragment thereof recognizes ABCB1, and the anti-ELANE monoclonal antibody or an antibody fragment thereof is ELANE Anti-IFITM2 monoclonal antibody or antibody fragment thereof recognizes IFITM2, anti-CD55 monoclonal antibody or antibody fragment thereof recognizes CD55, and anti-CD82 monoclonal antibody
  • the anti-CD9 monoclonal antibody, the anti-CD63 monoclonal antibody, and their antibody fragments are hybridomas that produce the monoclonal antibodies.
  • 1-1-1 A cell obtained from a cell deposited under the following deposit number in the center of Tsukuba Center 6) can also be used.
  • FERM BP-11519 (monoclonal antibody produced is CD9-12A12 antibody, designated CD9: 12A12, date of acceptance November 8, 2011)
  • FERM BP-11520 (monoclonal antibody produced is CD63-8A12 antibody, designated CD63: 8A12, date of acceptance November 8, 2011)
  • FERM BP-11521 (the monoclonal antibody produced is the CD63-13C8 antibody, designated CD63: 13C8, date of commissioning November 8, 2011)
  • the “monoclonal antibody fragment” is a part of the monoclonal antibody described above, and similarly to the monoclonal antibody, CD9, CD63, CEACAM7, DUOX2, HLA-DRB1, ABCB1, ELANE, IFITM2, CD55, CD82.
  • the fragment having specific binding to the antigen includes Fab, F (ab ′) 2, Fab ′, single chain antibody (scFv), disulfide stabilized antibody (dsFv), dimerization Examples include body V region fragments (Diabodies), CDR-containing peptides, etc. (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996).
  • antibody group (A) and antibody group (B) are not particularly limited as long as monoclonal antibodies belonging to the respective groups or antibody fragments thereof are used in appropriate combinations, and examples include the following combinations. .
  • Aspect 1 An aspect in which an anti-CD9 monoclonal antibody and its antibody fragment are used in combination with an anti-CD55 monoclonal antibody or a fragment thereof
  • Aspect 2 An aspect in which an anti-CD9 monoclonal antibody and its antibody fragment are combined with an anti-CD82 monoclonal antibody or a fragment thereof
  • Aspect 3 An aspect in which an anti-CD63 monoclonal antibody and an antibody fragment thereof are combined with an anti-HLA-DRB1 monoclonal antibody or a fragment thereof
  • an extracellular endoplasmic reticulum is recognized by any one of the monoclonal antibodies or antibody fragments thereof, and the corresponding antigen is obtained by the other monoclonal antibody or the antibody fragment thereof.
  • the signal derived from the extracellular endoplasmic reticulum in which is expressed can be quantified.
  • CD9 and CD55 on the extracellular endoplasmic reticulum are recognized by these monoclonal antibodies or fragments thereof, and signals derived from the extracellular endoplasmic reticulum in which CD9 and CD55 are expressed. Can be quantified. The same applies to other aspects.
  • any one monoclonal antibody or antibody fragment thereof may be used as a solid phase antibody, and the other monoclonal antibody or antibody fragment thereof may be used as a labeled antibody.
  • the solid phase antibody and the labeled antibody may be either the antibody group (A) or the antibody group (B), but are derived from an antigen specific to the extracellular endoplasmic reticulum in a body fluid sample derived from a bladder cancer patient. From the viewpoint of detecting a signal, it is preferable to use the antibody group (A) as a solid phase antibody and the antibody group (B) as a labeled antibody.
  • the preparation of the solid phase antibody and the labeled antibody is not particularly limited, and can be performed according to a known method. Such a combined monoclonal antibody or antibody fragment thereof is suitably used in the sandwich ELISA method and exoscreen method described below.
  • the sample used for measuring the signal intensity derived from the extracellular endoplasmic reticulum is not limited as long as it is a body fluid sample.
  • a body fluid sample For example, blood, serum, plasma, urine, saliva, milk, nasal discharge, cerebral spinal cord Those selected from the group consisting of liquids are exemplified. Of these, urine is preferable.
  • the signal intensity derived from the extracellular endoplasmic reticulum may be measured by any method using the monoclonal antibody or antibody fragment thereof, and can be performed, for example, according to the sandwich ELISA method or the exoscreen method.
  • the sandwich ELISA method first, one type of monoclonal antibody or an antibody fragment thereof is used as a solid phase antibody, and a complex is formed by contacting with a sample containing extracellular vesicles. Thereafter, another monoclonal antibody or an antibody fragment thereof is added thereto after labeling to form a further complex, and the label is detected to derive from the extracellular endoplasmic reticulum expressing the antigen recognized by both antibodies. Signal intensity can be measured.
  • the exoscreen method is an application of AlphaLISA developed by PerkinElmer.
  • two types of antibodies having different epitopes are used.
  • One antibody is biotinylated, and the other is reacted with a sample using an antibody to which Alpha LISA acceptor beads are bound.
  • the biotinylated antibody and donor beads are bound via streptavidin, and the acceptor beads and donor beads are adjacent to each other.
  • the adjacent state within 200 nm
  • singlet oxygen is generated from the donor bead by excitation at 680 nm, and when singlet oxygen reaches the acceptor bead, light of 615 nm can be emitted and detected as a signal.
  • exosomes having a size of about 100 nm can be measured as a sample.
  • the signal intensity derived from the extracellular endoplasmic reticulum containing the target protein in the body fluid sample can be measured.
  • the following step B is performed.
  • Step B is a step of comparing the signal intensity derived from the extracellular endoplasmic reticulum in the subject obtained in Step A and the signal strength in the control, and it is recognized that the signal strength in the subject is stronger than the signal strength in the control. Is an indicator of the presence of bladder cancer.
  • the control person may be any person who does not develop bladder cancer, and includes a healthy person.
  • the signal intensity in the control person is the signal intensity derived from the extracellular endoplasmic reticulum in the body fluid sample derived from the control person, and is measured together with the measurement of the signal intensity derived from the extracellular endoplasmic reticulum of the subject in Step A. Alternatively, it may be measured separately. Alternatively, the signal intensity derived from the extracellular vesicles of a plurality of controls may be measured, and the signal intensity derived from the extracellular vesicles of the controls may be set from the statistics.
  • the body fluid sample derived from the control is preferably the same type of sample as the body fluid sample derived from the subject.
  • the body fluid sample derived from the control is also urine.
  • the method is not particularly limited, and known methods (Steel method, t-test, Wilcoxon test, etc.) can be used. If the analysis shows that the signal intensity from the subject's extracellular endoplasmic reticulum is greater than the signal intensity from the control's extracellular endoplasmic reticulum, the subject has bladder cancer. It is judged that there is a high possibility.
  • the presence or absence of bladder cancer can be determined by detecting extracellular vesicles present in a body fluid sample using the antibody, as one embodiment of the present invention, (A) At least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and an antibody fragment thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and an antibody fragment thereof
  • a method for providing bladder cancer or information on a suspicion of bladder cancer comprising detecting an extracellular vesicle recognized by at least one selected from the group consisting of a body fluid sample derived from a subject. Can be mentioned.
  • the signal intensity derived from the extracellular vesicle of the control person is set to the signal intensity derived from the extracellular vesicle before the operation of the subject, and the signal intensity derived from the extracellular vesicle after the operation is set to the cell of the subject. If the signal intensity derived from the outer endoplasmic reticulum is compared with the signal intensity derived from the outer endoplasmic reticulum, the bladder cancer may be reduced or decreased. It can be judged that it is expensive.
  • the signal intensity derived from the extracellular ER of the control person is set to the signal intensity derived from the extracellular ER before treatment of the subject.
  • the signal intensity derived from the extracellular vesicle after treatment decreased. In this case, it can be determined that there is a high possibility that the treatment is effective for treating bladder cancer.
  • the present invention also measures the signal intensity derived from the extracellular endoplasmic reticulum using the monoclonal antibody or antibody fragment thereof before and after receiving treatment for bladder cancer, and the value after treatment is the value before treatment.
  • the evaluation method characterized by judging that the said treatment has an effect when it is smaller than the above can be provided.
  • a kit for detecting bladder cancer is provided.
  • the kit of the present invention includes any kit capable of detecting extracellular vesicles in a body fluid sample. Specifically, at least one selected from the group consisting of an antibody capable of recognizing an antigen present on the surface of the extracellular endoplasmic reticulum, (A) an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and an antibody fragment thereof; And a kit containing at least one selected from the group consisting of anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody, and antibody fragments thereof.
  • kits can be used as long as they are detection methods using an antibody when detecting extracellular vesicles in a body fluid sample (for example, ELISA method, exoscreen method, etc.). If extracellular vesicles are detected, proteins other than extracellular vesicles may be detected simultaneously by the antibody.
  • kits of the present invention for example, when a signal derived from an extracellular endoplasmic reticulum present in a blood sample of a healthy person and a subject is measured, and there is a significant difference in the signal intensity between the two, the bladder in the subject A determination and / or diagnosis of the onset of cancer can be made.
  • Example 1 Detection of bladder cancer marker protein
  • proteomic analysis of the exosomes derived from the urination of 3 bladder cancer patients and the urine of 2 healthy persons was detected in 2 or more patients with bladder cancer and detected in healthy persons Proteins that were not selected were selected. Selected results are shown in Table 1.
  • Example 2 (Detection of bladder cancer) From the results obtained in Example 1, bladder cancer was detected using CD55, CD82, or HLA-DRB1 as an index.
  • Example 3 (Detection of bladder cancer using CD55 and CD82 as an index) Taking the measurement signal of CD9 and CD55 positive exosomes on the horizontal axis and the measurement signal of CD9 and CD82 positive exosomes on the vertical axis, and setting the cut-off value so that all 7 healthy subjects are negative, 8 out of 14 cases An example bladder cancer patient was shown to be positive for at least either exosome (FIG. 3). That is, it was shown that a combination of CD55 and CD82 can be a bladder cancer marker with higher accuracy.
  • the method of the present invention makes it possible to determine whether or not the sample provider has a high possibility of developing bladder cancer. This is useful because the sample provider can take measures to prevent the progression of cancer.

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Abstract

A method for detecting bladder cancer, comprising a step of measuring the intensity of a signal coming from an extracellular vesicle using (A) at least one component selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody and fragments of the antibodies and (B) at least one component selected from the group consisting of an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody and fragments of the antibodies. According to the method of the present invention, it is possible to determine whether or not there is a high probability that bladder cancer is developed in a sample donor. As a result, it becomes possible for the sample donor to take a measure to prevent the progression of the cancer. Therefore, the method of the present invention is useful.

Description

膀胱がんの検出方法Method for detecting bladder cancer
　本発明は、膀胱がんの検出方法に関する。さらに詳しくは、サンプル中のエクソソーム（Ｅｘｏｓｏｍｅ）を含む細胞外小胞体の表面の特定抗原（ＣＤ９、ＣＤ６３、ＣＤ５５、ＣＤ８２、ＨＬＡ－ＤＲＢ１）に対するモノクローナル抗体又はそれらの抗体断片を用いて、膀胱がんの存在を検出するための方法、及び該方法に用いるキットに関する。 The present invention relates to a method for detecting bladder cancer. More specifically, bladder cancer using a monoclonal antibody or an antibody fragment thereof against a specific antigen (CD9, CD63, CD55, CD82, HLA-DRB1) on the surface of an extracellular endoplasmic reticulum containing exosomes in a sample The present invention relates to a method for detecting the presence of and a kit used in the method.
　エクソソームは、生体内の体液中に存在する細胞外小胞体の１種である。エクソソーム表面には、一般的な細胞表面と同様に、種々の膜タンパク質が存在することが知られている。また、エクソソームは、種々の細胞、例えば免疫系の細胞や各種がん細胞から分泌されることが報告されており、生体内の細胞間コミュニケーションの媒介役として機能し生理現象と関連することや、がんなどの疾患との関連性が注目されている。 Exosomes are a type of extracellular endoplasmic reticulum that exists in body fluids in vivo. It is known that various membrane proteins exist on the exosome surface as in the general cell surface. In addition, exosomes have been reported to be secreted from various cells, such as cells of the immune system and various cancer cells, functioning as mediators of intercellular communication in vivo and related to physiological phenomena, Relevance to diseases such as cancer is drawing attention.
　例えば、非特許文献１には、膀胱がん患者の腫瘍組織において、ＣＤ４６、ＣＤ５５、ＣＤ５９のｍＲＮＡ発現量が増加していることが開示されている。また、特許文献１においては、膀胱がん患者の膀胱に特徴的な腫瘍マーカーとして、ＣＤ５５がその一例として記載されている。 For example, Non-Patent Document 1 discloses that the mRNA expression levels of CD46, CD55, and CD59 are increased in the tumor tissue of bladder cancer patients. In Patent Document 1, CD55 is described as an example of a tumor marker characteristic of the bladder of a bladder cancer patient.
　また、非特許文献２には、ＣＤ８２(ＫＡＩ１)の変異はめったに起こらず、ＫＡＩ１のダウンレギュレーションやグリコシル化がマーカー情報としては重要であるとされており、前立腺がん、肺がん、膵臓がんの進行に関与していると開示されている。一方、正常膀胱細胞にはＫＡＩ１がタンパクレベルで発現しているが、膀胱がん細胞株の８種中３種において発現が抑制され、１種では大きく促進されていることが示されており、膀胱がんとの関連性は不明である。特許文献２においては、シスプラチンを用いた化学療法に対して抵抗性を示す膀胱がん細胞において、ＣＤ８２が遺伝子レベル(ｍＲＮＡ)で増加することが記載されている。 Further, in Non-Patent Document 2, CD82 (KAI1) mutation rarely occurs, and KAI1 down-regulation and glycosylation are considered to be important as marker information, and prostate cancer, lung cancer, pancreatic cancer It is disclosed that it is involved in the progress. On the other hand, KAI1 is expressed at the protein level in normal bladder cells, but it is shown that expression is suppressed in 3 out of 8 bladder cancer cell lines and greatly promoted in 1 type, The association with bladder cancer is unknown. Patent Document 2 describes that CD82 increases at the gene level (mRNA) in bladder cancer cells that are resistant to chemotherapy using cisplatin.
　特許文献３においては、正常膀胱細胞に比べて異常値を示すマーカーをターゲットにして膀胱がんを診断する方法が開示されており、発現量が増加するマーカーとしてＨＬＡ-ＤＲＢ１が例示されている。 Patent Document 3 discloses a method for diagnosing bladder cancer by targeting a marker that shows an abnormal value compared to normal bladder cells, and HLA-DRB1 is exemplified as a marker that increases the expression level.
ＷＯ２００７／０９１９０４号公報WO2007 / 091904 ＷＯ２０１４／０８５６６６号公報WO2014 / 085666 ＷＯ２０１２／１７８０８７号公報WO2012 / 178087
　エクソソームなどの細胞外小胞体に存在する膜タンパク質をバイオマーカーとして、がんを検出することは可能である。しかしながら、サンプルの種類や用いる抗体によっては、感度や特異性の変動が大きい等の理由から十分な結果を得ることが困難であり、従前からのがんの診断では、偽陽性となる場合があり、診断の精度に問題があった。よって、細胞外小胞体を検出することで精度よくがんを検出するために、さらなる技術の開発が必要である。 Cancer can be detected using a membrane protein present in an extracellular vesicle such as an exosome as a biomarker. However, depending on the type of sample and the antibody used, it is difficult to obtain sufficient results due to large fluctuations in sensitivity and specificity, etc., and it may be false positive in conventional cancer diagnosis There was a problem with the accuracy of the diagnosis. Therefore, in order to detect cancer accurately by detecting an extracellular endoplasmic reticulum, further technology development is required.
　本発明の課題は、細胞外小胞体、特にエクソソームを測定することで膀胱がんを検出する方法、及び該方法に用いるキットを提供することにある。 An object of the present invention is to provide a method for detecting bladder cancer by measuring extracellular endoplasmic reticulum, particularly exosomes, and a kit used for the method.
　本発明は、下記〔１〕～〔７〕に関する。
〔１〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを用い、被験者に由来する体液試料中のＣＤ９及び／又はＣＤ６３と、ＣＤ５５、ＣＤ８２、及びＨＬＡ－ＤＲＢ１からなる群より選ばれる少なくとも１つとを発現している細胞外小胞体由来のシグナル強度を測定する工程と、
前記工程で得られた被験者におけるシグナル強度と、対照者におけるシグナル強度とを対比する工程
とを含み、前記被験者におけるシグナル強度が対照者におけるシグナル強度より強いと認められる場合が、膀胱がんの存在の指標となる、膀胱がんの検出方法。
〔２〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを用い、被験者に由来する体液試料中のＣＤ９及び／又はＣＤ６３と、ＣＤ５５、ＣＤ８２、及びＨＬＡ－ＤＲＢ１からなる群より選ばれる少なくとも１つとを発現しているエクソソーム由来のシグナル強度を測定する工程と、
前記工程で得られた被験者におけるシグナル強度と、対照者におけるシグナル強度とを対比する工程
とを含み、前記被験者におけるシグナル強度が対照者におけるシグナル強度より強いと認められる場合が、膀胱がんの存在の指標となる、膀胱がんの検出方法。
〔３〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを含有してなる、前記〔１〕又は〔２〕記載の方法に使用するためのキット。
〔４〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識される細胞外小胞体の膀胱がんのマーカーとしての使用。
〔５〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識されるエクソソームの膀胱がんのマーカーとしての使用。
〔６〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識される細胞外小胞体を、被験者に由来する体液試料中から検出することを特徴とする、膀胱がん又は膀胱がんの疑いの情報を提供する方法。
〔７〕　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識されるエクソソームを、被験者に由来する体液試料中から検出することを特徴とする、膀胱がん又は膀胱がんの疑いの情報を提供する方法。 The present invention relates to the following [1] to [7].
[1] (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody , And at least one selected from the group consisting of antibody fragments thereof, and at least one selected from the group consisting of CD9 and / or CD63 in body fluid samples derived from a subject, CD55, CD82, and HLA-DRB1 Measuring the signal intensity from the extracellular endoplasmic reticulum expressing
The presence of bladder cancer in the case where the signal intensity in the subject is recognized to be stronger than the signal intensity in the control, comprising the step of comparing the signal intensity in the subject obtained in the step and the signal intensity in the control Method for detecting bladder cancer, which is an indicator of
[2] (A) at least one selected from the group consisting of anti-CD9 monoclonal antibody, anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody , And at least one selected from the group consisting of antibody fragments thereof, and at least one selected from the group consisting of CD9 and / or CD63 in body fluid samples derived from a subject, CD55, CD82, and HLA-DRB1 Measuring the signal intensity derived from exosomes expressing
The presence of bladder cancer in the case where the signal intensity in the subject is recognized to be stronger than the signal intensity in the control, comprising the step of comparing the signal intensity in the subject obtained in the step and the signal intensity in the control Method for detecting bladder cancer, which is an indicator of
[3] (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody. And a kit for use in the method according to [1] or [2] above, comprising at least one selected from the group consisting of antibody fragments thereof.
[4] (A) at least one selected from the group consisting of anti-CD9 monoclonal antibody, anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody And an extracellular endoplasmic reticulum recognized by at least one selected from the group consisting of antibody fragments thereof as a marker for bladder cancer.
[5] (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody. And an exosome recognized by at least one selected from the group consisting of antibody fragments thereof as a marker for bladder cancer.
[6] (A) at least one selected from the group consisting of anti-CD9 monoclonal antibody, anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody And suspicion of bladder cancer or bladder cancer, characterized in that an extracellular vesicle recognized by at least one selected from the group consisting of antibody fragments thereof is detected from a body fluid sample derived from a subject. How to provide information.
[7] (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, and an anti-HLA-DRB1 monoclonal antibody. And exosomes recognized by at least one selected from the group consisting of antibody fragments thereof from a body fluid sample derived from a subject, information on bladder cancer or suspicion of bladder cancer, How to provide.
　本発明の方法により、偽陽性の心配もなく、エクソソーム等の細胞外小胞体を測定することで膀胱がんを検出することができ、これにより、膀胱がんの発症の有無や進行程度を判断することができる。 By the method of the present invention, bladder cancer can be detected by measuring extracellular vesicles such as exosomes without worrying about false positives, thereby determining the presence or degree of progression of bladder cancer. can do.
図１は、エクソスクリーン法を用いた膀胱がん患者尿由来エクソソームにおける各種マーカーの検出結果を示す図である。(a)ビオチン化抗体；ＣＤ５５、アクセプタービーズ結合抗体；ＣＤ９。(b)ビオチン化抗体；ＣＤ８２、アクセプタービーズ結合抗体；ＣＤ９。(c)ビオチン化抗体；ＨＬＡ-ＤＲＢ１、アクセプタービーズ結合抗体；ＣＤ６３。FIG. 1 is a diagram showing detection results of various markers in urine derived from urine of a bladder cancer patient using an exoscreen method. (a) Biotinylated antibody; CD55, acceptor bead-bound antibody; CD9. (b) Biotinylated antibody; CD82, acceptor bead-bound antibody; CD9. (c) Biotinylated antibody; HLA-DRB1, acceptor bead-bound antibody; CD63. 図２は、エクソスクリーン法を用いた膀胱がん患者尿由来エクソソームにおける各種マーカー発現をクレアチニン量に基づいた相対結果を示す図である。(a)ビオチン化抗体；ＣＤ５５、アクセプタービーズ結合抗体；ＣＤ９。(b)ビオチン化抗体；ＣＤ８２、アクセプタービーズ結合抗体；ＣＤ９。(c)ビオチン化抗体；ＨＬＡ-ＤＲＢ１、アクセプタービーズ結合抗体；ＣＤ６３。FIG. 2 is a diagram showing relative results based on the amount of creatinine for the expression of various markers in urinary exosomes of bladder cancer patients using the exoscreen method. (a) Biotinylated antibody; CD55, acceptor bead-bound antibody; CD9. (b) Biotinylated antibody; CD82, acceptor bead-bound antibody; CD9. (c) Biotinylated antibody; HLA-DRB1, acceptor bead-bound antibody; CD63. 図３は、エクソソーム由来ＣＤ５５とＣＤ８２のそれぞれの検出結果(クレアチニン量基準)を対比した図である。FIG. 3 is a graph comparing the detection results (creatinine amount standard) of exosome-derived CD55 and CD82.
　本発明は、被験者における膀胱がんを検出するための方法であって、体液試料中の細胞外小胞体由来のシグナル強度を特定のモノクローナル抗体を用いて測定し、その値が健常人のそれより大きい場合に膀胱がんに罹患している可能性があると判断することを大きな特徴とする。具体的には、特定抗原に対するモノクローナル抗体又はその抗体断片を用い、被験者に由来する体液試料中の該抗原を発現している細胞外小胞体由来のシグナル強度を測定する工程（以降、工程Ａともいう）と、前記工程で得られた被験者におけるシグナル強度と、対照者におけるシグナル強度とを対比する工程（以降、工程Ｂともいう）とを含み、ここで、前記被験者におけるシグナル強度が対照者におけるシグナル強度より強いと認められる場合が、膀胱がんの存在の指標となる。なお、本明細書において、膀胱がんを検出するとは、膀胱がんの発症の有無、病態の進行度を検出することを含む。 The present invention is a method for detecting bladder cancer in a subject, wherein a signal intensity derived from an extracellular endoplasmic reticulum in a body fluid sample is measured using a specific monoclonal antibody, and the value is higher than that of a healthy person A large feature is that it is judged that there is a possibility of having bladder cancer when it is large. Specifically, a step of measuring the signal intensity derived from an extracellular endoplasmic reticulum expressing the antigen in a body fluid sample derived from a subject using a monoclonal antibody or antibody fragment thereof against a specific antigen (hereinafter also referred to as step A). And the step of comparing the signal intensity in the subject obtained in the step with the signal intensity in the control (hereinafter also referred to as step B), wherein the signal intensity in the subject is If it is recognized that it is stronger than the signal intensity, it is an indicator of the presence of bladder cancer. In the present specification, detecting bladder cancer includes detecting the presence or absence of development of bladder cancer and the degree of progression of a disease state.
　本発明者らは、尿中のエクソソームに存在する膜タンパクに着目し、膀胱がん患者の尿中のエクソソームには検出されるものの健常人では検出され難いものを検討することで、膀胱がん患者の尿中のエクソソームに特異的な抗原としてＣＥＡＣＡＭ７、ＤＵＯＸ２、ＨＬＡ－ＤＲＢ１、ＡＢＣＢ１、ＥＬＡＮＥ、ＩＦＩＴＭ２、ＣＤ５５、ＣＤ８２を同定することに成功した。本発明者らは、先に、抗ＣＤ９抗体、抗ＣＤ６３抗体を用いてエクソソームを特異的に捕捉できることを既に見出していたが、これらと前記抗原に対する抗体を組み合わせることにより、健常人の測定値はばらつきが少なく偽陽性が検出されず、膀胱がん患者のみからシグナルが検出されるという良好な結果が得られることを見出し、本発明を完成するに至った。 The present inventors focused on membrane proteins present in urinary exosomes, and examined those that are detected in urinary exosomes of bladder cancer patients but difficult to detect in healthy individuals. We successfully identified CEACAM7, DUOX2, HLA-DRB1, ABCB1, ELANE, IFITM2, CD55, and CD82 as antigens specific to exosomes in the urine of patients. The present inventors have previously found that exosomes can be specifically captured using anti-CD9 antibody and anti-CD63 antibody. By combining these with an antibody against the antigen, the measurement value of a healthy person is It was found that good results were obtained that there was little variation and no false positives were detected and that signals were detected only from bladder cancer patients, and the present invention was completed.
　以下に、本発明における各工程について説明する。 Hereinafter, each process in the present invention will be described.
　工程Ａは、特定抗原に対するモノクローナル抗体又はその抗体断片を用い、被験者に由来する体液試料中の該抗原を発現している細胞外小胞体由来のシグナルを測定する工程である。なお、本明細書において、細胞外小胞体とは、細胞外に分泌された小胞体のことであり、エクソソーム等が例示される。 Step A is a step of measuring a signal derived from an extracellular endoplasmic reticulum expressing the antigen in a body fluid sample derived from a subject using a monoclonal antibody or an antibody fragment thereof against the specific antigen. In the present specification, the extracellular vesicle is an vesicle secreted outside the cell, and examples thereof include exosomes.
　工程Ａで用いるモノクローナル抗体又はその抗体断片としては、２種類の群からなるモノクローナル抗体又はその断片が挙げられる。具体的には、細胞外小胞体を特異的に捕捉する抗体群（Ａ）及び膀胱がん細胞から分泌される細胞外小胞体に特異的な抗原に対する抗体群（Ｂ）である。 Examples of the monoclonal antibody or antibody fragment thereof used in Step A include monoclonal antibodies or fragments thereof consisting of two groups. Specifically, an antibody group (A) that specifically captures extracellular vesicles and an antibody group (B) against an antigen specific to the extracellular vesicle secreted from bladder cancer cells.
　抗体群（Ａ）としては、細胞外小胞体を特異的に捕捉できる抗体であればよく、具体的には、抗ＣＤ９モノクローナル抗体又はその抗体断片、抗ＣＤ６３モノクローナル抗体又はその抗体断片である。これらは少なくとも１種を用いることができればよい。 The antibody group (A) may be any antibody that can specifically capture extracellular vesicles, specifically, an anti-CD9 monoclonal antibody or an antibody fragment thereof, an anti-CD63 monoclonal antibody or an antibody fragment thereof. It is sufficient that at least one of these can be used.
　抗体群（Ｂ）としては、膀胱がん細胞から分泌される細胞外小胞体を特異的に捕捉できる抗体であればよく、具体的には、抗ＣＥＡＣＡＭ７モノクローナル抗体又はその抗体断片、抗ＤＵＯＸ２モノクローナル抗体又はその抗体断片、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体又はその抗体断片、抗ＡＢＣＢ１モノクローナル抗体又はその抗体断片、抗ＥＬＡＮＥモノクローナル抗体又はその抗体断片、抗ＩＦＩＴＭ２モノクローナル抗体又はその抗体断片、抗ＣＤ５５モノクローナル抗体又はその抗体断片、抗ＣＤ８２モノクローナル抗体又はその抗体断片である。これらは少なくとも１種を用いることができればよい。 The antibody group (B) may be any antibody that can specifically capture extracellular vesicles secreted from bladder cancer cells, specifically, an anti-CEACAM7 monoclonal antibody or an antibody fragment thereof, an anti-DUOX2 monoclonal antibody Or an antibody fragment thereof, anti-HLA-DRB1 monoclonal antibody or antibody fragment thereof, anti-ABCB1 monoclonal antibody or antibody fragment thereof, anti-ELANE monoclonal antibody or antibody fragment thereof, anti-IFITM2 monoclonal antibody or antibody fragment thereof, anti-CD55 monoclonal antibody or antibody thereof Fragment, anti-CD82 monoclonal antibody or antibody fragment thereof. It is sufficient that at least one of these can be used.
　本発明で用いるモノクローナル抗体又はその抗体断片は、それぞれ特定抗原を認識するものであればよく、公知の方法に従って調製することができる。即ち、抗ＣＤ９モノクローナル抗体又はその抗体断片はＣＤ９を認識し、抗ＣＤ６３モノクローナル抗体又はその抗体断片はＣＤ６３を認識し、抗ＣＥＡＣＡＭ７モノクローナル抗体又はその抗体断片はＣＥＡＣＡＭ７を認識し、抗ＤＵＯＸ２モノクローナル抗体又はその抗体断片はＤＵＯＸ２を認識し、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体又はその抗体断片はＨＬＡ－ＤＲＢ１を認識し、抗ＡＢＣＢ１モノクローナル抗体又はその抗体断片はＡＢＣＢ１を認識し、抗ＥＬＡＮＥモノクローナル抗体又はその抗体断片はＥＬＡＮＥを認識し、抗ＩＦＩＴＭ２モノクローナル抗体又はその抗体断片はＩＦＩＴＭ２を認識し、抗ＣＤ５５モノクローナル抗体又はその抗体断片はＣＤ５５を認識し、抗ＣＤ８２モノクローナル抗体又はその抗体断片はＣＤ８２を認識するものであり、哺乳動物の免疫応答により調製してもよく、各抗原の配列情報に基づいて調製してもよい。 The monoclonal antibody or antibody fragment thereof used in the present invention may be any one that recognizes a specific antigen, and can be prepared according to a known method. That is, an anti-CD9 monoclonal antibody or an antibody fragment thereof recognizes CD9, an anti-CD63 monoclonal antibody or an antibody fragment thereof recognizes CD63, an anti-CEACAM7 monoclonal antibody or an antibody fragment thereof recognizes CEACAM7, and an anti-DUOX2 monoclonal antibody or an antibody thereof The antibody fragment recognizes DUOX2, the anti-HLA-DRB1 monoclonal antibody or an antibody fragment thereof recognizes HLA-DRB1, the anti-ABCB1 monoclonal antibody or an antibody fragment thereof recognizes ABCB1, and the anti-ELANE monoclonal antibody or an antibody fragment thereof is ELANE Anti-IFITM2 monoclonal antibody or antibody fragment thereof recognizes IFITM2, anti-CD55 monoclonal antibody or antibody fragment thereof recognizes CD55, and anti-CD82 monoclonal antibody or antibody thereof Piece is intended to recognize CD82, it may be prepared by the immune response of mammals may be prepared based on the sequence information of each antigen.
　また、本発明では、抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片としては、前記モノクローナル抗体を産生するハイブリドーマとして、独立行政法人　製品評価技術基盤機構　特許微生物寄託センター(茨城県つくば市東１－１－１　つくばセンター中央第６)に下記受託番号のもとで寄託された細胞より得られるものを使用することもできる。
ＦＥＲＭ　ＢＰ－１１５１９(産生されるモノクローナル抗体がＣＤ９－１２Ａ１２抗体、表示ＣＤ９：１２Ａ１２、受託日２０１１年１１月８日）
ＦＥＲＭ　ＢＰ－１１５２０（産生されるモノクローナル抗体がＣＤ６３－８Ａ１２抗体、表示ＣＤ６３：８Ａ１２、受託日２０１１年１１月８日）
ＦＥＲＭ　ＢＰ－１１５２１（産生されるモノクローナル抗体がＣＤ６３－１３Ｃ８抗体、表示ＣＤ６３：１３Ｃ８、受託日２０１１年１１月８日） In the present invention, the anti-CD9 monoclonal antibody, the anti-CD63 monoclonal antibody, and their antibody fragments are hybridomas that produce the monoclonal antibodies. 1-1-1 A cell obtained from a cell deposited under the following deposit number in the center of Tsukuba Center 6) can also be used.
FERM BP-11519 (monoclonal antibody produced is CD9-12A12 antibody, designated CD9: 12A12, date of acceptance November 8, 2011)
FERM BP-11520 (monoclonal antibody produced is CD63-8A12 antibody, designated CD63: 8A12, date of acceptance November 8, 2011)
FERM BP-11521 (the monoclonal antibody produced is the CD63-13C8 antibody, designated CD63: 13C8, date of commissioning November 8, 2011)
　本発明において「モノクローナル抗体断片」とは、前述するモノクローナル抗体の一部であって、当該モノクローナル抗体と同様にＣＤ９や、ＣＤ６３、ＣＥＡＣＡＭ７、ＤＵＯＸ２、ＨＬＡ－ＤＲＢ１、ＡＢＣＢ１、ＥＬＡＮＥ、ＩＦＩＴＭ２、ＣＤ５５、ＣＤ８２に特異的な結合性を有する断片を意味する。前記抗原に対して特異的結合性を有する断片とは、具体的には、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、一本鎖抗体（ｓｃＦｖ）、ジスルフィド安定化抗体（ｄｓＦｖ）、２量化体Ｖ領域断片（Ｄｉａｂｏｄｙ）、ＣＤＲを含むペプチド等を挙げることができる（エキスパート・オピニオン・オン・テラピューティック・パテンツ、第６巻、第５号、第４４１～４５６頁、１９９６年）。 In the present invention, the “monoclonal antibody fragment” is a part of the monoclonal antibody described above, and similarly to the monoclonal antibody, CD9, CD63, CEACAM7, DUOX2, HLA-DRB1, ABCB1, ELANE, IFITM2, CD55, CD82. Means a fragment having specific binding properties. Specifically, the fragment having specific binding to the antigen includes Fab, F (ab ′) 2, Fab ′, single chain antibody (scFv), disulfide stabilized antibody (dsFv), dimerization Examples include body V region fragments (Diabodies), CDR-containing peptides, etc. (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996).
　これらの抗体群（Ａ）と抗体群（Ｂ）は、それぞれの群に属するモノクローナル抗体又はその抗体断片を適宜組み合わせて用いるのであれば特に限定はなく、例えば、以下の組み合わせを用いる態様が挙げられる。
態様１：抗ＣＤ９モノクローナル抗体及びその抗体断片と抗ＣＤ５５モノクローナル抗体又はその断片とを組み合わせて用いる態様
態様２：抗ＣＤ９モノクローナル抗体及びその抗体断片と抗ＣＤ８２モノクローナル抗体又はその断片とを組み合わせて用いる態様
態様３：抗ＣＤ６３モノクローナル抗体及びその抗体断片と抗ＨＬＡ－ＤＲＢ１モノクローナル抗体又はその断片とを組み合わせて用いる態様 These antibody group (A) and antibody group (B) are not particularly limited as long as monoclonal antibodies belonging to the respective groups or antibody fragments thereof are used in appropriate combinations, and examples include the following combinations. .
Aspect 1: An aspect in which an anti-CD9 monoclonal antibody and its antibody fragment are used in combination with an anti-CD55 monoclonal antibody or a fragment thereof Aspect 2: An aspect in which an anti-CD9 monoclonal antibody and its antibody fragment are combined with an anti-CD82 monoclonal antibody or a fragment thereof Aspect 3: An aspect in which an anti-CD63 monoclonal antibody and an antibody fragment thereof are combined with an anti-HLA-DRB1 monoclonal antibody or a fragment thereof
　これらの組み合わせたモノクローナル抗体又はその抗体断片を用いる方法としては、いずれか一方のモノクローナル抗体又はその抗体断片により細胞外小胞体を認識し、そして、もう一方のモノクローナル抗体又はその抗体断片により対応する抗原が発現している細胞外小胞体由来のシグナルを定量することができる。具体的には、例えば、態様１では、細胞外小胞体上のＣＤ９とＣＤ５５とを、これらのモノクローナル抗体又はその断片により認識して、ＣＤ９とＣＤ５５が発現している細胞外小胞体由来のシグナルを定量することができる。他の態様についても同様である。 As a method of using these combined monoclonal antibodies or antibody fragments thereof, an extracellular endoplasmic reticulum is recognized by any one of the monoclonal antibodies or antibody fragments thereof, and the corresponding antigen is obtained by the other monoclonal antibody or the antibody fragment thereof. The signal derived from the extracellular endoplasmic reticulum in which is expressed can be quantified. Specifically, for example, in aspect 1, CD9 and CD55 on the extracellular endoplasmic reticulum are recognized by these monoclonal antibodies or fragments thereof, and signals derived from the extracellular endoplasmic reticulum in which CD9 and CD55 are expressed. Can be quantified. The same applies to other aspects.
　また、前記組み合わせたモノクローナル抗体又はその抗体断片を用いる方法としては、いずれか一方のモノクローナル抗体又はその抗体断片を固相抗体として、もう一方のモノクローナル抗体又はその抗体断片を標識抗体として用いてもよい。固相抗体と標識抗体は抗体群（Ａ）と抗体群（Ｂ）のいずれであってもよいが、膀胱がん患者に由来する体液試料中の細胞外小胞体に特異的な抗原に由来するシグナルを検出する観点から、抗体群（Ａ）を固相抗体として、抗体群（Ｂ）を標識抗体として用いることが好ましい。固相抗体及び標識抗体の調製は、特に限定はなく、公知の方法に従って行うことができる。かかる組み合わせのモノクローナル抗体又はその抗体断片は、後述のサンドイッチＥＬＩＳＡ法、エクソスクリーン法に好適に用いられる。 In addition, as a method of using the combined monoclonal antibody or antibody fragment thereof, any one monoclonal antibody or antibody fragment thereof may be used as a solid phase antibody, and the other monoclonal antibody or antibody fragment thereof may be used as a labeled antibody. . The solid phase antibody and the labeled antibody may be either the antibody group (A) or the antibody group (B), but are derived from an antigen specific to the extracellular endoplasmic reticulum in a body fluid sample derived from a bladder cancer patient. From the viewpoint of detecting a signal, it is preferable to use the antibody group (A) as a solid phase antibody and the antibody group (B) as a labeled antibody. The preparation of the solid phase antibody and the labeled antibody is not particularly limited, and can be performed according to a known method. Such a combined monoclonal antibody or antibody fragment thereof is suitably used in the sandwich ELISA method and exoscreen method described below.
　本発明における、細胞外小胞体由来のシグナル強度の測定用に供されるサンプルとしては、体液試料であれば限定はなく、例えば、血液、血清、血漿、尿、唾液、乳汁、鼻汁、脳脊髄液からなる群より選択されるものが例示される。なかでも、尿が好ましい。 In the present invention, the sample used for measuring the signal intensity derived from the extracellular endoplasmic reticulum is not limited as long as it is a body fluid sample. For example, blood, serum, plasma, urine, saliva, milk, nasal discharge, cerebral spinal cord Those selected from the group consisting of liquids are exemplified. Of these, urine is preferable.
　細胞外小胞体由来のシグナル強度の測定は、前記モノクローナル抗体又はその抗体断片を用いる方法であればよく、例えば、サンドイッチＥＬＩＳＡ法、エクソスクリーン法に従って行なうことができる。 The signal intensity derived from the extracellular endoplasmic reticulum may be measured by any method using the monoclonal antibody or antibody fragment thereof, and can be performed, for example, according to the sandwich ELISA method or the exoscreen method.
　サンドイッチＥＬＩＳＡ法では、具体的には、先ず、一種類のモノクローナル抗体又はその抗体断片を固相抗体とし、細胞外小胞体を含有するサンプルと接触させて複合体を形成させる。その後、そこに、別のモノクローナル抗体又はその抗体断片を標識後添加して、さらなる複合体を形成させて標識を検出することにより、両抗体が認識する抗原を発現している細胞外小胞体由来のシグナル強度を測定することができる。 Specifically, in the sandwich ELISA method, first, one type of monoclonal antibody or an antibody fragment thereof is used as a solid phase antibody, and a complex is formed by contacting with a sample containing extracellular vesicles. Thereafter, another monoclonal antibody or an antibody fragment thereof is added thereto after labeling to form a further complex, and the label is detected to derive from the extracellular endoplasmic reticulum expressing the antigen recognized by both antibodies. Signal intensity can be measured.
　エクソスクリーン法はＰｅｒｋｉｎＥｌｍｅｒ社が開発したＡｌｐｈａＬＩＳＡを応用したものである。本方法はエピトープの異なる２種類の抗体を用いて、片方の抗体はビオチン化を、もう片方にはＡｌｐｈａＬＩＳＡアクセプタービーズを結合させた抗体を用いてサンプルと反応させる。その後、ストレプトアビジンが結合したドナービーズを添加することで、ビオチン化抗体とドナービーズがストレプトアビジンを介して結合し、アクセプタービーズとドナービーズが隣接する。隣接した状態で(２００ｎｍ以内)、６８０ｎｍ励起により、ドナービーズから一重項酸素が発生し、アクセプタービーズに一重項酸素が到達すると６１５ｎｍの光を発してシグナルとして検出することができる。この方法を応用することで、１００ｎｍ程度の大きさであるエクソソームをサンプルとして測定することができる。 The exoscreen method is an application of AlphaLISA developed by PerkinElmer. In this method, two types of antibodies having different epitopes are used. One antibody is biotinylated, and the other is reacted with a sample using an antibody to which Alpha LISA acceptor beads are bound. Thereafter, by adding donor beads to which streptavidin is bound, the biotinylated antibody and donor beads are bound via streptavidin, and the acceptor beads and donor beads are adjacent to each other. In the adjacent state (within 200 nm), singlet oxygen is generated from the donor bead by excitation at 680 nm, and when singlet oxygen reaches the acceptor bead, light of 615 nm can be emitted and detected as a signal. By applying this method, exosomes having a size of about 100 nm can be measured as a sample.
　かくして、体液試料中の標的タンパク質を含む細胞外小胞体由来のシグナル強度を測定することができる。得られた細胞外小胞体由来のシグナル強度を用いて、次の工程Ｂを行う。 Thus, the signal intensity derived from the extracellular endoplasmic reticulum containing the target protein in the body fluid sample can be measured. Using the obtained signal intensity derived from the extracellular endoplasmic reticulum, the following step B is performed.
　工程Ｂは、工程Ａで得られた被験者における細胞外小胞体由来のシグナル強度と、対照者におけるシグナル強度とを対比する工程であり、前記被験者におけるシグナル強度が対照者におけるシグナル強度より強いと認められる場合が、膀胱がんの存在の指標となる。なお、本明細書において、対照者とは、膀胱がんを発症していない者であれば良く、健常人が挙げられる。 Step B is a step of comparing the signal intensity derived from the extracellular endoplasmic reticulum in the subject obtained in Step A and the signal strength in the control, and it is recognized that the signal strength in the subject is stronger than the signal strength in the control. Is an indicator of the presence of bladder cancer. In the present specification, the control person may be any person who does not develop bladder cancer, and includes a healthy person.
　対照者におけるシグナル強度は、対照者に由来する体液試料中の細胞外小胞体由来のシグナル強度であり、工程Ａで被験者の細胞外小胞体由来のシグナル強度を測定する際に併せて測定しても、別途測定してもよい。また、複数の対照者の細胞外小胞体由来のシグナル強度を測定し、その統計から対照者の細胞外小胞体由来のシグナル強度を設定してもよい。 The signal intensity in the control person is the signal intensity derived from the extracellular endoplasmic reticulum in the body fluid sample derived from the control person, and is measured together with the measurement of the signal intensity derived from the extracellular endoplasmic reticulum of the subject in Step A. Alternatively, it may be measured separately. Alternatively, the signal intensity derived from the extracellular vesicles of a plurality of controls may be measured, and the signal intensity derived from the extracellular vesicles of the controls may be set from the statistics.
　対照者由来の体液試料としては、被験者由来の体液試料と同種の試料であることが好ましく、例えば、被験者由来の体液試料が尿である場合、対照者由来の体液試料も尿である。 The body fluid sample derived from the control is preferably the same type of sample as the body fluid sample derived from the subject. For example, when the body fluid sample derived from the subject is urine, the body fluid sample derived from the control is also urine.
　被験者のシグナル強度と対照者のシグナル強度とを対比するには、被験者の細胞外小胞体由来のシグナル強度と対照者の細胞外小胞体由来のシグナル強度とを対比して解析すればよく、かかる方法としては、特に限定はなく、公知の方法(Steel法、t検定、Wilcoxon検定等)を用いることができる。前記解析により対照者の細胞外小胞体由来のシグナル強度に対する被験者の細胞外小胞体由来のシグナル強度がより有意に増加していることが示された場合は、被験者に膀胱がんが存在している可能性が高いと判断される。 In order to compare the signal intensity of the subject with the signal intensity of the control person, it is only necessary to analyze the signal intensity derived from the extracellular endoplasmic reticulum of the subject and the signal intensity derived from the control person's extracellular endoplasmic reticulum. The method is not particularly limited, and known methods (Steel method, t-test, Wilcoxon test, etc.) can be used. If the analysis shows that the signal intensity from the subject's extracellular endoplasmic reticulum is greater than the signal intensity from the control's extracellular endoplasmic reticulum, the subject has bladder cancer. It is judged that there is a high possibility.
　なお、前記抗体を用いて体液試料中に存在する細胞外小胞体を検出することにより、膀胱がんの存在可能性の有無を判断することができることから、本発明の一態様として、（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識される細胞外小胞体を、被験者に由来する体液試料中から検出することを特徴とする、膀胱がん又は膀胱がんの疑いの情報を提供する方法を挙げることができる。 Since the presence or absence of bladder cancer can be determined by detecting extracellular vesicles present in a body fluid sample using the antibody, as one embodiment of the present invention, (A) At least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and an antibody fragment thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and an antibody fragment thereof A method for providing bladder cancer or information on a suspicion of bladder cancer, comprising detecting an extracellular vesicle recognized by at least one selected from the group consisting of a body fluid sample derived from a subject. Can be mentioned.
　また、前記解析において、対照者の細胞外小胞体由来のシグナル強度を被験者の術前の細胞外小胞体由来のシグナル強度に設定し、術後の細胞外小胞体由来のシグナル強度を被験者の細胞外小胞体由来のシグナル強度として対比することで、術後の細胞外小胞体由来のシグナル強度が減少していることが示された場合は、膀胱がんが縮小又は減退している可能性が高いと判断することができる。 In the above analysis, the signal intensity derived from the extracellular vesicle of the control person is set to the signal intensity derived from the extracellular vesicle before the operation of the subject, and the signal intensity derived from the extracellular vesicle after the operation is set to the cell of the subject. If the signal intensity derived from the outer endoplasmic reticulum is compared with the signal intensity derived from the outer endoplasmic reticulum, the bladder cancer may be reduced or decreased. It can be judged that it is expensive.
　またさらに、前記解析において、例えば、被験者が膀胱がんと診断された場合においては、対照者の細胞外小胞体由来のシグナル強度を被験者の治療前の細胞外小胞体由来のシグナル強度に設定し、治療後の細胞外小胞体由来のシグナル強度を被験者の細胞外小胞体由来のシグナル強度として対比することで、治療後の細胞外小胞体由来のシグナル強度が減少していることが示された場合は、当該治療が膀胱がんの治療に有効である可能性が高いと判断することができる。従って、本発明はまた、膀胱がんの治療を受ける前と受けた後において、前記モノクローナル抗体又はその抗体断片を用いて細胞外小胞体由来のシグナル強度を測定し、治療後の値が治療前のそれより小さい場合に、当該治療が効果を有すると判断することを特徴とする評価方法を提供することができる。 Furthermore, in the above analysis, for example, when the subject is diagnosed with bladder cancer, the signal intensity derived from the extracellular ER of the control person is set to the signal intensity derived from the extracellular ER before treatment of the subject. By comparing the signal intensity derived from the extracellular vesicle after treatment as the signal intensity derived from the extracellular vesicle of the subject, it was shown that the signal intensity derived from the extracellular vesicle after treatment decreased. In this case, it can be determined that there is a high possibility that the treatment is effective for treating bladder cancer. Therefore, the present invention also measures the signal intensity derived from the extracellular endoplasmic reticulum using the monoclonal antibody or antibody fragment thereof before and after receiving treatment for bladder cancer, and the value after treatment is the value before treatment. The evaluation method characterized by judging that the said treatment has an effect when it is smaller than the above can be provided.
　また、本発明の別の態様では、膀胱がんを検出するためのキットが提供される。 In another aspect of the present invention, a kit for detecting bladder cancer is provided.
　本発明のキットには、体液試料中の細胞外小胞体を検出することができるものであれば全て含まれる。具体的には、前記細胞外小胞体表面に存在する抗原を認識できる抗体、（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを含有するキットが挙げられる。 The kit of the present invention includes any kit capable of detecting extracellular vesicles in a body fluid sample. Specifically, at least one selected from the group consisting of an antibody capable of recognizing an antigen present on the surface of the extracellular endoplasmic reticulum, (A) an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and an antibody fragment thereof; And a kit containing at least one selected from the group consisting of anti-CD55 monoclonal antibody, anti-CD82 monoclonal antibody, anti-HLA-DRB1 monoclonal antibody, and antibody fragments thereof.
　これらのキットは、体液試料中の細胞外小胞体を検出する際に抗体を用いる検出方法であれば(例えば、ＥＬＩＳＡ法、エクソスクリーン法等)、用いることができる。なお、細胞外小胞体が検出されるのであれば、細胞外小胞体以外のタンパク質も前記抗体により同時に検出されることがあってもよい。 These kits can be used as long as they are detection methods using an antibody when detecting extracellular vesicles in a body fluid sample (for example, ELISA method, exoscreen method, etc.). If extracellular vesicles are detected, proteins other than extracellular vesicles may be detected simultaneously by the antibody.
　本発明のキットを用いて、例えば、健常人と被験者の血液サンプル中に存在する細胞外小胞体由来のシグナルを測定して、両者のシグナル強度に有意差が生じた場合には、被験者における膀胱がんの発症の決定及び／又は診断を行うことができる。 Using the kit of the present invention, for example, when a signal derived from an extracellular endoplasmic reticulum present in a blood sample of a healthy person and a subject is measured, and there is a significant difference in the signal intensity between the two, the bladder in the subject A determination and / or diagnosis of the onset of cancer can be made.
　以下、本発明を実施例に基づいて説明するが、実施例は本発明をより良く理解するために例示するものであって、本発明の範囲がこれらの実施例に限定されることを意図するものではない。 Hereinafter, the present invention will be described based on examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.
実施例１（膀胱がんマーカータンパク質の検出）
　膀胱がん患者の尿に由来するエクソソームを用いたプロテオーム解析を行った。具体的には、３人の膀胱がん患者の導尿と２人の健常人の導尿に由来するエクソソームをプロテオーム解析し、２人以上の膀胱がんの患者で検出され、健常人では検出されなかったタンパク質を選択した。選択された結果を表１に示す。 Example 1 (Detection of bladder cancer marker protein)
Proteome analysis using exosomes derived from urine of bladder cancer patients was performed. Specifically, proteomic analysis of the exosomes derived from the urination of 3 bladder cancer patients and the urine of 2 healthy persons was detected in 2 or more patients with bladder cancer and detected in healthy persons Proteins that were not selected were selected. Selected results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例２（膀胱がんの検出）
　実施例１で得られた結果より、ＣＤ５５、ＣＤ８２、又はＨＬＡ－ＤＲＢ１を指標として、膀胱がんを検出した。 Example 2 (Detection of bladder cancer)
From the results obtained in Example 1, bladder cancer was detected using CD55, CD82, or HLA-DRB1 as an index.
　具体的には、１４例の膀胱がん患者の導尿及び７例の健常人の導尿を用いて、Ｔｏｔａｌ Ｅｘｏｓｏｍｅ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（ライフテクノロジーズ社）により７．５倍に濃縮したエクソソーム溶液５μＬを調製後、ビオチン化した抗ＣＤ５５モノクローナル抗体（ＡＢＣＡＭ社製、クローン１４３?３０）、ビオチン化した抗ＣＤ８２モノクローナル抗体（ダイアクローン社製、クローンＢ-Ｌ２）、ビオチン化した抗ＨＬＡ-ＤＲＢ１モノクローナル抗体（ミリポア社製、クローンＤＪ９）とアクセプタービーズが結合した抗ＣＤ９モノクローナル抗体（受託番号ＦＥＲＭ　ＢＰ－１１５１９として寄託されているハイブリドーマにより産出されるモノクローナル抗体）もしくはアクセプタービーズが結合した抗ＣＤ６３モノクローナル抗体（受託番号 ＢＰ－１１５２０として寄託されているハイブリドーマにより産出されるモノクローナル抗体）を用いて、エクソスクリーン法により、ＣＤ９とＣＤ５５、ＣＤ９とＣＤ８２、ＣＤ６３とＨＬＡ-ＤＲＢ１が発現しているエクソソーム由来のシグナルを測定した(図１ａ、ｂ、ｃ)。 Specifically, 5 μL of an exosome solution concentrated 7.5 times was prepared by Total Exosome Isolation７．５Kit (Life Technologies) using 14 bladder cancer patients and 7 healthy persons. Then, biotinylated anti-CD55 monoclonal antibody (ABCAM, clone 143-30), biotinylated anti-CD82 monoclonal antibody (Diaclone, clone B-L2), biotinylated anti-HLA-DRB1 monoclonal antibody (Millipore) Anti-CD9 monoclonal antibody (monoclonal antibody produced by a hybridoma deposited under accession number FERM BP-11519) or acceptor beads bound to clone DJ9) and acceptor beads Derived from exosomes expressing CD9 and CD55, CD9 and CD82, CD63 and HLA-DRB1 by the exoscreen method using a null antibody (monoclonal antibody produced by the hybridoma deposited under accession number BP-11520) Was measured (FIGS. 1a, b, c).
　また、尿中成分を補正するため、各種測定シグナルをエクソソーム濃縮前の尿中クレアチニン濃度にて補正した(図２ａ、ｂ、ｃ)。なお、クレアチニン濃度は改良型ヤッフェ法を用いて測定した。 In addition, in order to correct urinary components, various measurement signals were corrected with the urinary creatinine concentration before exosome concentration (FIGS. 2a, b and c). The creatinine concentration was measured using an improved Jaffe method.
　結果、いずれの抗体の組み合わせにおいても、膀胱がん患者１４例中６例程度は健常人のそれよりも高いシグナルを示すことが明らかになった。 As a result, it was revealed that in any combination of antibodies, about 6 out of 14 bladder cancer patients showed a signal higher than that of healthy persons.
実施例３（ＣＤ５５及びＣＤ８２を指標にした膀胱がんの検出）
　ＣＤ９とＣＤ５５陽性エクソソームの測定シグナルを横軸に、ＣＤ９とＣＤ８２陽性エクソソームの測定シグナルを縦軸に取り、７例全ての健常人が陰性になるようにカットオフ値を定めると、１４例中８例の膀胱がん患者が少なくともどちらかのエクソソームが陽性であることが示された（図３）。すなわち、ＣＤ５５とＣＤ８２を組み合わせることで、より精度の高い膀胱がんマーカーとなりうることが示された。 Example 3 (Detection of bladder cancer using CD55 and CD82 as an index)
Taking the measurement signal of CD9 and CD55 positive exosomes on the horizontal axis and the measurement signal of CD9 and CD82 positive exosomes on the vertical axis, and setting the cut-off value so that all 7 healthy subjects are negative, 8 out of 14 cases An example bladder cancer patient was shown to be positive for at least either exosome (FIG. 3). That is, it was shown that a combination of CD55 and CD82 can be a bladder cancer marker with higher accuracy.
　本発明の方法により、サンプル提供者が膀胱がんを発症している可能性が高いか否かを判定することができる。これにより、サンプル提供者は癌の進行を阻止する手段を講じることができるため、有用である。 The method of the present invention makes it possible to determine whether or not the sample provider has a high possibility of developing bladder cancer. This is useful because the sample provider can take measures to prevent the progression of cancer.

Claims (8)

  1. 　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを用い、被験者に由来する体液試料中のＣＤ９及び／又はＣＤ６３と、ＣＤ５５、ＣＤ８２、及びＨＬＡ－ＤＲＢ１からなる群より選ばれる少なくとも１つとを発現している細胞外小胞体由来のシグナル強度を測定する工程と、
    前記工程で得られた被験者におけるシグナル強度と、対照者におけるシグナル強度とを対比する工程
    とを含み、前記被験者におけるシグナル強度が対照者におけるシグナル強度より強いと認められる場合が、膀胱がんの存在の指標となる、膀胱がんの検出方法。     (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and them And expressing at least one selected from the group consisting of CD55, CD82, and HLA-DRB1 in a body fluid sample derived from a subject using at least one selected from the group consisting of the antibody fragments of Measuring the signal intensity derived from the extracellular endoplasmic reticulum,
    The presence of bladder cancer in the case where the signal intensity in the subject is recognized to be stronger than the signal intensity in the control, comprising the step of comparing the signal intensity in the subject obtained in the step and the signal intensity in the control Method for detecting bladder cancer, which is an indicator of
  2. 　体液試料中の細胞外小胞体由来のシグナル強度を測定する工程が、エクソスクリーン法又はサンドイッチＥＬＩＳＡ法を使用する請求項１に記載の方法。 The method according to claim 1, wherein the step of measuring the signal intensity derived from the extracellular endoplasmic reticulum in the body fluid sample uses an exoscreen method or a sandwich ELISA method.
  3. 　体液試料が尿である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the body fluid sample is urine.
  4. 　抗ＣＤ９モノクローナル抗体又はその抗体断片が、受託番号ＦＥＲＭ　ＢＰ－１１５１９として寄託されているハイブリドーマにより産出されるモノクローナル抗体又はその抗体断片である請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the anti-CD9 monoclonal antibody or an antibody fragment thereof is a monoclonal antibody produced by a hybridoma deposited under accession number FERM BP-11519 or an antibody fragment thereof.
  5. 　抗ＣＤ６３モノクローナル抗体又はその抗体断片が、受託番号ＦＥＲＭ　ＢＰ－１１５２０として寄託されているハイブリドーマにより産出されるモノクローナル抗体、受託番号ＦＥＲＭ　ＢＰ－１１５２１として寄託されているハイブリドーマにより産出されるモノクローナル抗体、又はそれらの抗体断片である請求項１～４のいずれかに記載の方法。 An anti-CD63 monoclonal antibody or an antibody fragment thereof is a monoclonal antibody produced by a hybridoma deposited under accession number FERM BP-11520, a monoclonal antibody produced by a hybridoma deposited under accession number FERM BP-11521, or these The method according to any one of claims 1 to 4, which is an antibody fragment.
  6. 　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとを含有してなる、請求項１～５のいずれかに記載の方法に使用するためのキット。 (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and them A kit for use in the method according to any one of claims 1 to 5, comprising at least one selected from the group consisting of antibody fragments.
  7. 　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識される細胞外小胞体の膀胱がんのマーカーとしての使用。 (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and them Use of an extracellular endoplasmic reticulum recognized by at least one selected from the group consisting of the antibody fragments as a marker for bladder cancer.
  8. 　（Ａ）抗ＣＤ９モノクローナル抗体、抗ＣＤ６３モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つと、（Ｂ）抗ＣＤ５５モノクローナル抗体、抗ＣＤ８２モノクローナル抗体、抗ＨＬＡ－ＤＲＢ１モノクローナル抗体、及びそれらの抗体断片からなる群より選ばれる少なくとも１つとによって認識される細胞外小胞体を、被験者に由来する体液試料中から検出することを特徴とする、膀胱がん又は膀胱がんの疑いの情報を提供する方法。 (A) at least one selected from the group consisting of an anti-CD9 monoclonal antibody, an anti-CD63 monoclonal antibody, and antibody fragments thereof; and (B) an anti-CD55 monoclonal antibody, an anti-CD82 monoclonal antibody, an anti-HLA-DRB1 monoclonal antibody, and them And detecting vesicular cancer or suspicion of bladder cancer, characterized in that an extracellular vesicle recognized by at least one selected from the group consisting of antibody fragments is detected from a body fluid sample derived from a subject. How to provide.
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