WO2013099925A1 - Exosome検出用モノクローナル抗体 - Google Patents
Exosome検出用モノクローナル抗体 Download PDFInfo
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- WO2013099925A1 WO2013099925A1 PCT/JP2012/083612 JP2012083612W WO2013099925A1 WO 2013099925 A1 WO2013099925 A1 WO 2013099925A1 JP 2012083612 W JP2012083612 W JP 2012083612W WO 2013099925 A1 WO2013099925 A1 WO 2013099925A1
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- 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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- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G—PHYSICS
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- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Definitions
- the present invention relates to a set of antibodies for detecting Exosomes. More specifically, a monoclonal antibody or its antibody fragment against a specific antigen (CD9, CD63, CD81) on the surface of Exosome, a set containing the monoclonal antibody or an antibody fragment thereof, an miRNA derived from Exosome using the monoclonal antibody or an antibody fragment thereof , A method for measuring a complex of the set and a biological sample derived from a subject, a method for diagnosing cancer or immune system disease using the set, a kit for performing the method, and the set
- the present invention relates to a method for evaluating the efficacy of an anticancer drug or antiimmune disease drug to be used, and a kit for carrying out the method.
- Exosomes are vesicular granules present in body fluids in vivo. It is known that various membrane proteins exist on the surface of Exosomes as in the case of general cell surfaces. On the other hand, it has also been found that microRNA (miRNA) is contained inside Exosomes in addition to various proteins such as cytokines. Exosomes have been reported to be secreted from various cells such as cells of the immune system and various cancer cells, function as mediators of intercellular communication in vivo, and are related to physiological phenomena, Relevance to such diseases is attracting attention.
- miRNA microRNA
- Non-patent Document 1 Exosomes in circulating blood of ovarian cancer patients are isolated using an antibody of EpCAM, which is a tumor marker, and there is a relationship between the expression level of miRNA derived from the Exosomes and ovarian cancer patients. Has been reported. Therefore, if the quantitative change of Exosomes related to diseases such as cancer can be easily grasped, application of Exosomes to diagnostic agents can be expected.
- EpCAM EpCAM
- CD9, CD63 and CD81 are four-transmembrane membrane proteins belonging to the tetraspanin family and are expressed on many Exosomes.
- Non-Patent Document 2 reports that Exosomes in the plasma of melanoma patients are detected and quantified with an antibody of CD63 or an antibody of tumor-related marker Caveolin-1, and are reported to be elevated compared to normal subjects. Yes.
- Patent Literature 1 a signal derived from Exome in a cancer patient is quantified and analyzed by reacting a plasma sample after centrifugation with a combination of a CD63 antibody and antibodies against various membrane proteins.
- anti-CD9 antibody anti-CD63 antibody
- anti-CD81 antibody Various antibodies are sold as anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody. These commercially available antibodies are one of the antibodies obtained as a result of directly immunizing cells expressing CD9, CD63 or CD81, and are not antibodies obtained by immunization with a specific antigen designed. Therefore, the sensitivity and specificity are not yet sufficient, and further technology development is necessary to detect Exosomes quickly and easily, and accurately and accurately.
- An object of the present invention is to provide a monoclonal antibody or antibody fragment thereof capable of detecting or capturing in vivo Exosomes with good sensitivity and specificity, a set containing the monoclonal antibody or antibody fragment thereof, and the set and subject-derived It is intended to provide a method for measuring a complex with a biological sample, a method for diagnosing cancer or an immune system disease using the set, and a method for evaluating the efficacy of an anticancer agent or an antiimmune disease drug using the set.
- the present invention ⁇ 1> A monoclonal antibody that recognizes amino acids 113 to 195 of the amino acid sequence shown in SEQ ID NO: 1 or an antibody fragment thereof that can detect or capture Exosomes, and amino acid numbers 104 to 104 of the amino acid sequence shown in SEQ ID NO: 2.
- a monoclonal antibody that recognizes 202 or an antibody fragment thereof a monoclonal antibody that recognizes amino acid numbers 36 to 54 of the amino acid sequence shown in SEQ ID NO: 3 or an antibody fragment thereof, and amino acid numbers 113 to 201 of the amino acid sequence shown in SEQ ID NO: 3
- the present invention relates to a monoclonal antibody for Exome detection or capture, which is selected from the group consisting of a monoclonal antibody that recognizes or an antibody fragment thereof.
- ⁇ 2> Recognizes amino acid numbers 113 to 195 of the amino acid sequence shown in SEQ ID NO: 1 which is a monoclonal antibody (CD9-12A12 antibody) produced by the hybridoma deposited under receipt number FERM ABP-11519 or a fragment thereof Monoclonal antibody fragment, Monoclonal antibody (CD63-8A12 antibody) produced by the hybridoma deposited under receipt number FERM ABP-11520 or a fragment thereof, which recognizes amino acid numbers 104 to 202 of the amino acid sequence shown in SEQ ID NO: 2 , Monoclonal antibody (CD63-13C8 antibody) produced by the hybridoma deposited under receipt number FERM ABP-11521 or a fragment thereof, which recognizes amino acid numbers 104 to 202 of the amino acid sequence shown in SEQ ID NO: 2 , Monoclonal antibody (CD81-4G6 antibody) produced by a hybridoma deposited under receipt number NITE A
- ⁇ 3> A combination of the same or two kinds of antibodies or antibody fragments thereof selected from the group consisting of CD9-12A12 antibody or antibody fragment thereof, CD63-8A12 antibody or antibody fragment thereof, and CD63-13C8 antibody or antibody fragment thereof.
- ⁇ 4> a set wherein the solid phase antibody is a CD9-12A12 antibody or an antibody fragment thereof, and the labeled antibody is a CD9-12A12 antibody or an antibody fragment thereof, A set wherein the solid phase antibody is a CD9-12A12 antibody or an antibody fragment thereof, and the labeled antibody is a CD63-13C8 antibody or an antibody fragment thereof; A set in which the solid phase antibody is a CD63-8A12 antibody or an antibody fragment thereof, the labeled antibody is a CD9-12A12 antibody or an antibody fragment thereof, and the solid phase antibody is a CD63-8A12 antibody or an antibody fragment thereof, and the labeled antibody is a CD63-13C8 antibody or A set that is an antibody fragment thereof,
- the set according to ⁇ 3> selected from the group consisting of: ⁇ 5>
- ⁇ 6> an antibody selected from the group consisting of a CD9-12A12 antibody or an antibody fragment thereof, a CD63-8A12 antibody or an antibody fragment thereof, and a CD63-13C8 antibody or an antibody fragment thereof, and a disease-specific membrane protein antibody or A set of monoclonal antibodies for disease-specific Exosomes comprising a combination of the antibody fragments.
- a cancer or immune system disease diagnostic kit comprising the monoclonal antibody or antibody fragment thereof according to any one of ⁇ 3> to ⁇ 5>.
- ⁇ 8> A diagnostic kit for a cancer or immune system disease comprising the monoclonal antibody or antibody fragment thereof according to the set of ⁇ 6>.
- ⁇ 9> A diagnostic kit for cancer or immune system disease, comprising the set according to ⁇ 6>.
- ⁇ 10> A method of detecting a miRNA of the Exosome by contacting a biological sample derived from a subject and the monoclonal antibody according to ⁇ 1> or ⁇ 2> to capture Exosome.
- ⁇ 11> A biological sample derived from a subject is brought into contact with the monoclonal antibody of any of the above ⁇ 3> to ⁇ 5> to form an Exosome complex, and the signal intensity derived from the complex is measured. how to.
- An anti-cancer agent or an anti-immune disease drug comprising a step of determining that the anti-cancer drug or the anti-immune disease drug is highly likely to have a medicinal effect when it is recognized that the signal intensity derived from the complex in a biological sample of Of drug efficacy evaluation.
- the monoclonal antibody for Exosome detection of the present invention can detect and capture CD9, CD63 or CD81 on Exosome in vivo with good sensitivity and specificity. This makes it possible to capture slight changes in Exosomes in blood samples (including changes in the amount of CD9, CD63 or CD81 of membrane proteins on Exosomes in addition to quantitative changes in Exosomes), such as cancer This makes it possible to detect a change in exosome derived from a disease, and has an excellent effect that it can be applied to diagnosis of a disease that causes the change.
- FIG. 1 is a diagram illustrating a concept of a diagnostic method using Exosomes.
- FIG. 2 shows the structure of the tetraspanin family (CD9, CD63 and CD81) (FIG. A), and the small loop peptide conjugate (FIG. B) and large loop Fc fusion protein (FIG. C) used for antibody production.
- FIG. 3 shows sequence information of CD9 and CD63 used as antigens.
- FIG. 4 shows an evaluation method (VLP ELISA) using monoclonal antibody VLPs (Virus-Like-Particles).
- FIG. 5 is a diagram showing a secondary evaluation of anti-CD9 monoclonal antibody.
- FIG. 6 is a diagram showing a secondary evaluation of the anti-CD63 monoclonal antibody.
- FIG. 1 is a diagram illustrating a concept of a diagnostic method using Exosomes.
- FIG. 2 shows the structure of the tetraspanin family (CD9, CD63 and CD81) (FIG.
- FIG. 7 is a diagram showing a standard curve of Exosome ELISA for CD9.
- FIG. 8 is a diagram showing a standard curve of CD63 Exosome ELISA.
- FIG. 9 is a graph comparing the signal intensity derived from Exome in the serum of healthy subjects, breast cancer patients, and colorectal cancer patients using an Exosome ELISA that uses a commercially available anti-CD9 antibody or anti-CD63 antibody of the present invention (self-made). is there.
- FIG. 10 is a diagram in which the signal intensity derived from Exome in the serum of healthy subjects, breast cancer patients, and colon cancer patients was measured by an Exosome ELISA in which the anti-CD9 antibody and anti-CD63 antibody of the present invention were combined.
- FIG. 10 is a diagram in which the signal intensity derived from Exome in the serum of healthy subjects, breast cancer patients, and colon cancer patients was measured by an Exosome ELISA in which the anti-CD9 antibody and anti-CD63 antibody of the present invention were combined.
- FIG. 11 is a diagram in which the signal intensity derived from Exome in the serum of healthy subjects, breast cancer patients, and colorectal cancer patients was measured by an Exosome ELISA in which the anti-CD9 antibody or anti-CD63 antibody of the present invention was combined with an anti-EpCAM antibody. is there.
- FIG. 12 shows the sequence information of CD81 used for the antigen.
- FIG. 13 is a diagram comparing the immunoprecipitation performance of the anti-CD9 antibody of the present invention with a commercially available anti-CD9 antibody.
- FIG. 14 is a diagram comparing the immunoprecipitation performance of the anti-CD63 antibody of the present invention with a commercially available anti-CD63 antibody.
- FIG. 15 is a diagram comparing the immunoprecipitation performance of the anti-CD81 antibody of the present invention with a commercially available anti-CD81 antibody.
- the monoclonal antibody for detecting Exosomes of the present invention has a feature that CD9, CD63 or CD81 on Exosomes in vivo can be detected or captured with good sensitivity and specificity. Therefore, the monoclonal antibody of the present invention may be described as a monoclonal antibody for Exome detection or capture.
- CD9, CD63, and CD81 on Exosomes are respectively a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3. It is.
- CD9, CD63, and CD81 are all membrane proteins belonging to the tetraspan family having a four-transmembrane structure shown in FIG. 2A. These membrane proteins present two types of loop structures, a small loop (referred to as EC1) and a large loop (referred to as EC2), to the outside of the cell.
- the monoclonal antibody of the present invention is specific to these loops. It is characterized by showing general recognition.
- the monoclonal antibody of the present invention is prepared by designing an antigen as follows, it has excellent sensitivity and specificity for the antigen.
- Exome CD9, CD63 or CD81 small loop peptide ie, CD9 polypeptide Arg36-Asn50, CD63 polypeptide Val38-Pro54, CD81 polypeptide Arg36-Ala54 amino acid terminal peptide added cysteine residue, Synthesize according to known methods.
- hapten antigen is prepared through SH group of a peptide using maleimide-ized KLH [Keyhole Lipmet Hemoyanin, Image (registered trademark) Maleimide Activated mcKLH, made by Pierce).
- FIG. 2B shows a schematic diagram of the immunogen
- FIGS. 3A and 12A show the peptide sequences.
- a fusion protein (Fc fusion protein) of an Fc region of rabbit IgG and a large loop peptide of CD9, CD63 or CD81 can be used as an antigen.
- a plasmid vector in which a polynucleotide sequence corresponding to a polypeptide in which Fc is added to the carboxyl terminus of CD9 polypeptide His113-Ile195, CD63 polypeptide Gly104-Asn202, CD81 polypeptide Phe113-Lys201 is introduced.
- FIG. 2C shows a schematic diagram of the immunogen
- FIGS. 3B and 12B show the peptide sequences.
- the Exosome detection monoclonal antibody of the present invention (hereinafter also simply referred to as the monoclonal antibody of the present invention) is not particularly limited, and known methods such as K.I. Watanabe et al. , Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. et al. Clin. Invest. 114 (2004), 898-907.
- a mammal is immunized using the antigen obtained as described above.
- a mammal Generally, a mouse
- the age of the mammal varies depending on the species of animal used and is not particularly limited. In the case of a mouse or rat, it is usually about 4 to 12 weeks old, preferably about 5 to 10 weeks old. These mammals can be selected in consideration of compatibility with plasma cells to be cell-fused for the production of the monoclonal antibody of the present invention.
- the antigen is used as an immunogen mixed with an adjuvant to enhance the immune response.
- an adjuvant there is no limitation and a well-known thing can be used.
- the adjuvant and the antigen can be mixed according to a method known in the art for the adjuvant to be used.
- Mammal immunization is performed according to methods known in the art.
- the immunogen is administered by injection injection into a mammal subcutaneously, intradermally, intravenously and / or intraperitoneally.
- administration of the immunogen is repeated several times after the first immunization, and the administration interval can be adjusted as appropriate. Since the immune response varies depending on the type and strain of the mammal to be immunized, the immunization schedule and the dose of the immunogen can be appropriately set according to the animal to be used.
- desired antibody-producing cells can be prepared in the immunized mammal.
- Such antibody-producing cells are preferably splenocytes isolated 3 to 5 days after the final administration of the immunogen.
- boost additional injection of immunogen
- the amount of immunogen administered in boost is preferably about 4 to 5 times the amount of immunogen initially administered, but can be increased or decreased as appropriate using this as a guide.
- the obtained antibody-producing cells are fused with myeloma-derived cells (myeloma cells) to prepare hybridomas.
- the myeloma cells are preferably compatible with the mammal from which the antibody-producing cells to be fused are derived. Examples thereof include myeloma cells such as mouse myeloma P3U1, X63-Ag8.653.
- the cell fusion method a method known in the art can be used, and examples thereof include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device.
- PEG polyethylene glycol
- Sendai virus a method using Sendai virus
- electrofusion device a method using an electrofusion device.
- the obtained hybridoma can be separated by culturing in a selective medium according to a known method.
- the culture supernatant is collected and an antibody titer assay can be performed based on a known method, for example, the ELISA method described later. .
- hybridoma producing the desired monoclonal antibody can be obtained.
- the hybridoma can be subcultured in a normal medium and can be stored semipermanently in liquid nitrogen.
- Desired monoclonal antibodies can be prepared in large quantities by in vivo and in vitro culture methods.
- the in vitro culture method can be performed by culturing the hybridoma in an appropriate serum medium or serum-free medium, and the desired monoclonal antibody is produced in the medium. According to this culture method, a desired antibody having a relatively high purity can be obtained as a culture supernatant.
- the in vivo culture method can be carried out by inoculating the hybridoma with a hybridoma, for example, an intraperitoneal cavity of a mammal that is compatible with the hybridoma, such as a mouse, and proliferating, and the desired antibody can be recovered in large amounts as mouse ascites. .
- the obtained culture supernatant and ascites fluid such as mouse can be used as a crude antibody solution as they are.
- These are purified by conventional methods, for example, by appropriately combining DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, etc. to obtain purified antibodies. be able to.
- each monoclonal antibody can be represented by an antigen protein and a clone number, and can be represented, for example, as a CD9-12A12 antibody.
- the Patent Microorganism Depositary Center As a hybridoma producing the above monoclonal antibody, the Patent Microorganism Depositary Center, Product Evaluation Technology Organization (1-1-1 East Tsukuba, Tsukuba, Ibaraki Prefecture, or Kazusa, Kisarazu, Chiba Prefecture) The cells deposited under the following receipt number can be used for sickle feet 2-5-8).
- FERM ABP-11519 (the monoclonal antibody produced is CD9-12A12 antibody, designated CD9: 12A12, date of receipt November 8, 2011)
- FERM ABP-11520 (the monoclonal antibody produced is CD63-8A12 antibody, designated CD63: 8A12, date of receipt November 8, 2011)
- FERM ABP-11521 (monoclonal antibody produced is CD63-13C8 antibody, designated CD63: 13C8, date of receipt November 8, 2011).
- NITE ABP-1480 (the monoclonal antibody produced is CD81-4G6 antibody, indicated CD81-4G6, date of receipt December 12, 2012)
- NITE ABP-1481 (monoclonal antibody produced is CD81-6D12 antibody, designated CD81-6D12, date of receipt December 12, 2012)
- NITE ABP-1482 (monoclonal antibody produced is CD81-12C4 antibody, designated CD81-12C4, date of receipt December 12, 2012)
- the “monoclonal antibody fragment” means a fragment that is a part of the above-described monoclonal antibody of the present invention and has a specific binding property to CD9, CD63, or CD81 like the monoclonal antibody.
- the fragment having specific binding property to CD9, CD63 or CD81 is Fab, F (ab ′) 2 , Fab ′, single chain antibody (scFv), disulfide stabilized antibody (dsFv) Examples include dimerized V region fragments (Diabodies), peptides containing CDRs, etc. (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996) ).
- the monoclonal antibody or antibody fragment thereof of the present invention shows specific recognition for CD9, CD63 or CD81 on the surface of Exosome, it is suitable for the evaluation method described below for substances expressing CD9, CD63 or CD81. Used for.
- the present invention also provides a set of monoclonal antibodies comprising at least one monoclonal antibody or antibody fragment thereof of the present invention. By using such a set, it is possible to capture Exosomes in a sample with high sensitivity, so that, for example, quantitative accuracy by the sandwich ELISA method is improved.
- the set includes the following two types depending on the type of antibody combined with the monoclonal antibody of the present invention.
- Aspect 1 A set comprising two or more of the monoclonal antibodies of the present invention or antibody fragments thereof
- Aspect 2 A set comprising the monoclonal antibodies of the present invention or antibody fragments thereof and disease-specific membrane protein antibodies or antibody fragments thereof
- the set of Embodiment 1 is not particularly limited as long as it contains two or more of the monoclonal antibodies or antibody fragments thereof of the present invention, and includes an embodiment including a plurality of the same type of antibodies or antibody fragments thereof.
- an antibody selected from the group consisting of a CD9-12A12 antibody or an antibody fragment thereof, a CD63-8A12 antibody or an antibody fragment thereof, and a CD63-13C8 antibody or an antibody fragment thereof, a CD9-12A12 antibody or an antibody fragment thereof examples include a combination of a CD63-8A12 antibody or an antibody fragment thereof and an antibody selected from the group consisting of a CD63-13C8 antibody or an antibody fragment thereof. In this case, one is used as a solid phase antibody and the other is used as a labeled antibody. be able to.
- the preparation of the solid phase antibody and the labeled antibody that is, the solid phase immobilization and labeling of the monoclonal antibody or antibody fragment thereof shown in Table 1 is not particularly limited and can be performed according to a known method.
- the combination of the solid phase antibody and the labeled antibody can be evaluated by examining, for example, specific binding properties and quantitative properties of Exosomes according to a known ELISA method.
- Specific binding can be evaluated from the signal intensity obtained when the Exosome in a sample to which a known amount of Exosome is added is measured by the sandwich EILSA method described in Example 4 described later. A combination that provides a sufficient signal intensity is judged as a good combination. Furthermore, an Exosome addition / recovery test can be performed on a blood sample, and whether the addition / recovery rate is good or bad can be used to determine the selection of the combination. For example, if the addition recovery rate is about 85 to 115%, it is determined that the combination is good.
- the signal intensity increases depending on the Exome concentration in a concentration range of at least 100 ng / mL, preferably 50 to 25000 ng / mL.
- a combination that is recognized and that yields a standard curve with good linearity can be determined as a good combination.
- Table 2 below shows combinations that were determined to be good combinations from the above.
- the set of embodiment 2 includes the monoclonal antibody or antibody fragment thereof of the present invention and a disease-specific membrane protein antibody or antibody fragment thereof.
- an antibody selected from the group consisting of a CD9-12A12 antibody or antibody fragment thereof, a CD63-8A12 antibody or antibody fragment thereof, and a CD63-13C8 antibody or antibody fragment thereof, and a disease-specific membrane protein antibody or antibody thereof.
- the set which combined the fragment is mentioned.
- either one can be used for Exome solid phase (also called Exosome immobilization), and the other can be used for Exosome labeling. From the viewpoint of fixing Exosomes and determining whether they are specific to the disease.
- the monoclonal antibody of the present invention is used for Exosome solid phase
- the disease-specific membrane protein antibody is used for Exosome labeling.
- the disease-specific membrane protein antibody means an antibody against a disease-specific membrane protein present on the surface of Exosomes. Examples include Ep-CAM, EGFR, CD276, CD55, CD71, EphA2, PSMA, Integrin, HER2, and HER3.
- VLPs in which CD9 or CD63 and type I membrane protein (for example, TEM7) are forcibly expressed are added to an anti-mouse IgG antibody solid phase plate, and each anti-CD test antibody and labeled type I membrane protein are added.
- An antibody for example, HRP-labeled anti-TEM7 antibody Fab ′
- the plate is washed and the remaining VLPs are quantified by detecting the label of the antibody (in the above example, the HRP activity labeled with the antibody is detected). It can be evaluated that the more the remaining VLPs, the better the binding of the test antibody.
- the monoclonal antibody of the present invention is preferably used.
- isolation can be performed by using streptavidin solid phase magnetic beads.
- the monoclonal antibody of the present invention or an antibody fragment thereof is biotinylated according to a known method.
- Exosomes and biotinylated antibodies are mixed and reacted overnight at 4 ° C.
- streptavidin solid-phase magnetic beads are added and reacted at 4 ° C. for another 2 hours, followed by separation using a magnet to recover Exosomes bound to the biotinylated antibody.
- Exosome-derived miRNA detection method Exosomes are secreted from various cells, such as cells of the immune system and various cancer cells, so if it is possible to detect miRNAs derived from Exosomes, it is possible to determine physiological phenomena and various diseases by analyzing them. Become.
- a biological sample derived from a subject is contacted with the monoclonal antibody of the present invention or an antibody fragment thereof, Exosomes are isolated according to the Exosome capture and separation method, and miRNA is known from the recovered Exosomes. Detect according to the method.
- the detected miRNA can be analyzed according to a known method, whereby it can be determined that the patient is suffering from a physiological phenomenon analysis or a specific disease.
- the biological sample is not particularly limited as long as it is a biological sample selected from the group consisting of blood, serum, and plasma.
- Immunoassays are performed using the set of monoclonal antibodies of the present invention.
- Immunoassays include enzyme immunoassay (EIA), enzyme immunometric assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay, immunoblot, Western blot
- EIA enzyme immunoassay
- ELISA enzyme immunometric assay
- FFA fluorescence immunoassay
- RIA radioimmunoassay
- luminescence immunoassay immunoblot
- Western blot Western blot
- the ELISA method is preferable because the antibody can be easily detected with high sensitivity.
- the ELISA method includes a general competitive method, a sandwich method, and the like.
- the monoclonal antibody of the present invention or an antibody fragment thereof is used for the solid phase antibody in the sandwich method or both the solid phase antibody and the labeled antibody.
- the sandwich method is preferred because it can be used.
- a test sample containing Exosomes is contacted to form a complex.
- the other monoclonal antibody or antibody fragment thereof of the present invention is modified, and in the case of aspect 2, the disease-specific membrane protein antibody or antibody fragment thereof is modified.
- the labeled antibody is added to form a further complex to detect the label, so that the signal amount derived from Exosomes contained in the sample is detected in the first aspect, and the disease-specific Exosomes included in the sample in the second aspect. The amount can be measured individually.
- the present invention also provides a method of measuring the abundance of the complex by bringing a biological sample derived from a subject into contact with a monoclonal antibody contained in the set of monoclonal antibodies of the present invention to form a complex.
- a biological sample derived from a subject into contact with a monoclonal antibody contained in the set of monoclonal antibodies of the present invention to form a complex.
- I will provide a.
- the monoclonal antibody or antibody fragment thereof of the present invention when immobilizing the monoclonal antibody or antibody fragment thereof of the present invention, it may be immobilized directly or via a known medium such as streptavidin.
- the present invention also provides a diagnostic method (onset prediction method) for cancer or immune system diseases.
- the set of aspect 1 of the present invention can detect Exosomes distributed in a living body with good sensitivity and specificity.
- the set of aspect 2 can detect disease-specific Exosomes with good sensitivity. Exosomes are secreted from various cells, such as cells of the immune system and various cancer cells. Therefore, it is considered that the amount of Exosomes in the living body is increased when cancer or immune system diseases develop. .
- cancer cells and the like may change on the cell surface as compared to normal cells, and the expression level of membrane proteins such as CD9 and CD63 may be increased.
- diagnosis of cancer or immune system disease can be made by using the signal amount derived from the Exosomes as an index, and in the case of aspect 2, it is also possible to specify cancer or immune system disease.
- the method can also be used as a diagnostic method (measurement-related factor measurement method or determination method).
- Cancers or immune system diseases that can be diagnosed as onset include cancers such as colorectal cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, kidney cancer, skin cancer, And inflammatory diseases such as rheumatism, osteoarthritis, nephropathy (diabetic nephropathy, glomerulonephritis), pancreatitis, hepatitis, allergy and the like.
- cytotoxic T cell CTL
- Step (I) A step of contacting a biological sample derived from a subject with the monoclonal antibody of the set of the present invention to form an Exosome complex, and measuring a signal intensity derived from the complex;
- step (I) of the prediction method For the measurement of the signal intensity derived from the complex in step (I) of the prediction method, a method well known to those skilled in the art can be used as long as it is a method using the set of monoclonal antibodies of the present invention. Is preferred.
- step (II) the signal intensity obtained as described above is compared by performing a statistical analysis based on the signal from the control person.
- a statistical analysis method There is no limitation in particular as an analysis method, A well-known method can be used.
- the control means the average of the same age and same sex as the subject who does not develop cancer or immune system disease, and the signal intensity in the control is measured together with the signal intensity in the subject.
- a statistical value obtained from a separately measured value may be used.
- kits of the present invention include those containing the set of monoclonal antibodies of the present invention. If the detection method uses the set of monoclonal antibodies of the present invention when detecting Exosomes in a sample, the kit is used. be able to. Since the set of monoclonal antibodies of the present invention can detect in vivo Exosomes with good sensitivity and specificity, the use of the kit can greatly contribute to the diagnosis of cancer or immune system diseases. .
- Exosomes are secreted from various cells, such as immune system cells and various cancer cells. Therefore, changes in blood Exosomes before and after administration of anticancer drugs or antiimmune disease drugs (in addition to increase / decrease in abundance, (Including fluctuations in the amount of membrane protein), it is considered possible to evaluate the drug efficacy in patients.
- an antibody against a membrane protein specific for cancer cells or an antibody fragment thereof and the monoclonal antibody or antibody fragment thereof of the present invention it can be expected to improve the specificity of cancer diagnosis or specify the type of cancer. Therefore, it is considered possible to develop diagnostic agents more specific to cancer diseases.
- the drug efficacy evaluation method is as follows: Step (A): Forming an Exosome complex by contacting a biological sample derived from a subject before and after administration of an anticancer drug or anti-immune disease drug with a monoclonal antibody included in the set of monoclonal antibodies of the present invention. Step (B) of measuring the signal intensity derived from the complex: the signal intensity derived from the complex in the biological sample derived from the subject after administration of the anticancer drug or the anti-immune disease drug is the anticancer drug.
- the anti-cancer agent or the anti-immune disease drug is likely to have a medicinal effect.
- step (B) the signal intensity obtained as described above is compared by performing a statistical analysis based on the signal in the biological sample before administration of the anticancer drug or antiimmune disease drug.
- a statistical analysis method There is no limitation in particular as an analysis method, A well-known method can be used.
- the subsequent determination is made, for example, when the amount of signal in the biological sample after administration of the anticancer drug or antiimmune disease drug is small compared to the amount of signal in the biological sample before administration, the anticancer agent or antiimmune disease drug It is judged that there is a high possibility of having an effect of suppressing cancer or immune system diseases.
- kits for anticancer drugs or antiimmune diseases In another aspect of the present invention, a kit for evaluating the efficacy of an anticancer agent or an anti-immune disease drug is provided.
- kits of the present invention include those containing the set of monoclonal antibodies of the present invention. If the detection method uses the set of monoclonal antibodies of the present invention when detecting Exosomes in a sample, the kit is used. be able to. Since the set of monoclonal antibodies of the present invention can detect in vivo Exosomes with good sensitivity and specificity, the use of the kit greatly contributes to the evaluation of the efficacy of anticancer drugs or antiimmune diseases drugs. Can do.
- Example 1 [Preparation of anti-CD9 and CD63 monoclonal antibodies] (Preparation of antigen) CD9 and CD63 protein partial peptides, ie, two peptides having a cysteine residue added to each amino terminus of CD9 polypeptide Arg36-Asn50 and CD63 polypeptide Val38-Pro54 were synthesized by Sigma-Aldrich. A hapten antigen was prepared via the SH group of a peptide using KLH (keyhole limpet hemocyanin, Image (registered trademark) Maleimide Activated mcKLH, manufactured by Thermo Scientific Co., Ltd.) obtained by maleidizing these.
- KLH keyhole limpet hemocyanin, Image (registered trademark) Maleimide Activated mcKLH, manufactured by Thermo Scientific Co., Ltd.
- FIG. 2A schematically shows the structure of the tetraspanin family (CD9, CD63)
- FIG. 2B schematically
- Fc fusion protein is transiently expressed by introducing a plasmid vector into which a polynucleotide sequence having Fc added to the carboxyl terminus of the large loop of each CD antigen is introduced into Freestyle 293-F Cells (manufactured by Invitrogen). Then, it was prepared by purification using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.).
- Emulsions as an immunogen were prepared by mixing CD9 and CD63 hapten antigens and Fc fusion protein with equal amounts of complete adjuvant for the first immunization and incomplete adjuvant for the second and subsequent immunizations.
- Monoclonal antibodies are available from K. Watanabe et al. , Vasohibin as an endothelium-derived negative feedback back regulator of engineering, J. Clin. Invest. 114 (2004), 898-907. That is, for each administration, 50 ⁇ g of hapten antigen or 10 ⁇ g of Fc fusion protein was subcutaneously and intraperitoneally divided into 5-week-old female A / J mice. Thereafter, after the 4th or 5th immunization, the spleen was extracted from the mouse that had undergone the final booster (4 days before cell fusion) to prepare spleen cells. Cell fusion between spleen cells and myeloma cells (P3U1) Watanabe et al. , Vasohibin as an endothelium-derived negative feedback back regulator of engineering, J. Clin. Invest. 114 (2004), 898-907.
- P3U1 myeloma cells
- the antibody titer (primary evaluation) of antiserum and hybridoma supernatant was evaluated by the ELISA method described below. Specifically, antiserum or hybridoma supernatant is added to a 96-well microplate on which a goat anti-mouse IgG antibody is solid-phased, and biotinylated CD9, CD63 protein and HRP-labeled streptavidin are mixed and stirred, and then at room temperature for 2 hours or 4 hours. The reaction was allowed to proceed overnight at 0 ° C.
- the plate is washed three times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), 100 ⁇ L of TMB reagent is added, and after stirring, the mixture is allowed to stand for 15 to 20 minutes. The reaction was stopped by adding 50 ⁇ L. Absorbance at 450 nm was measured with ARVO MX (manufactured by Perkin Elmer), and a case where a signal intensity exceeding 3 times the signal obtained when antiserum or hybridoma was not added was judged as positive.
- a washing solution saline containing 0.01% Tween 20 and 0.1% ProClin 150
- Each of the obtained monoclonal antibodies was purified from a serum-free medium of antibody-producing hybridoma or ascites obtained by administering the hybridoma to mice using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.). As described above, all the monoclonal antibodies described below were prepared.
- VLPs virus-like particles in which CD9 or CD63 and type I membrane protein TEM7 were co-expressed were prepared using Invitrogen's MembranePro functional protein expression kit. Specifically, a plasmid vector in which CD9 or CD63 and the TEM7 gene were incorporated into pEF V5-His TA Vector Kit was transfected into 293FT cells (Invitrogen) using Lipofectamine 2000. Precipitation Mix reagent was added to the culture supernatant after transfection to precipitate VLPs. Expression of TEM7 and each CD antigen in VLPs was confirmed by Western blotting (WB) (results not shown).
- WB Western blotting
- VLPs sandwich ELISA was performed using the VLPs thus prepared as shown in FIG. Specifically, the VLPs were added to an anti-mouse IgG antibody solid phase plate, and each anti-CD antibody and HRP-labeled anti-TEM7 antibody Fab 'were added and reacted at 4 ° C overnight. As a comparative control, a well to which a commercially available anti-CD antibody was added was also prepared. After the overnight reaction, 100 ⁇ L of TMB solution was added after washing with the washing solution, and after stirring, the mixture was allowed to stand for 15 to 20 minutes, and 50 ⁇ L of 1N sulfuric acid solution was added to stop the reaction. Absorbance at 450 nm was measured with ARVO MX (manufactured by Perkin Elmer).
- the antibody which recognizes a three-dimensional structure by VLP ELISA was each selected as a commercially available antibody, the antibody (clone IVA50) manufactured by Abnova was used for the anti-CD9 antibody, and the antibody manufactured by BD (clone H5C6) was used for the anti-CD63 antibody.
- the absorbance results are shown in FIGS. 5 and 6, respectively.
- the CD9-12A12 antibody, the CD63-8A12 antibody, and the CD63-13C8 antibody are three-dimensionally exosomes with a reactivity equivalent to or higher than that of a commercially available antibody, that is, a commercially available antibody that recognizes a three-dimensional structure usable for FACS. It was found that it was possible to catch it.
- Example 3 Combination of anti-CD9 and anti-CD63 monoclonal antibodies
- solid-phase antibodies and labeled antibodies are prepared for all the monoclonal antibodies obtained in Example 1, and are used for measuring blood Exosomes.
- the sandwich ELISA was performed.
- a 96-well microplate on which all the monoclonal antibodies were solid-phased was prepared. That is, 100 ⁇ L / well of each antibody solution prepared to 10 ⁇ g / mL with phosphate buffered saline (PBS) was added to a 96-well microplate and allowed to react overnight. Thereafter, the reaction solution is removed, and after washing 3 times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), 2% Block Ace (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) is 200 ⁇ L / well. Blocking was performed by adding and allowing to stand overnight at 4 ° C.
- PBS phosphate buffered saline
- an antibody suitable for use as a solid phase antibody in combination with a specific labeled antibody is generally suitable for use as a solid phase antibody in combination with another labeled antibody. Yes.
- an antibody suitable for use as a labeled antibody in combination with a specific solid phase antibody should generally be suitable for use as a labeled antibody in combination with other solid phase antibodies.
- Tables 5 and 6 summarize the present invention and commercially available antibodies. Regarding antibody suitability, from the above results, very strong antibodies (optimum antibodies) are indicated with “ ⁇ ”, usable antibodies with “ ⁇ ”, weak but reactive antibodies with “ ⁇ ”, and unusable antibodies. Was evaluated as “ ⁇ ”.
- Example 4 [Construction of Exosome ELISA Method Using Anti-CD9 and Anti-CD63]
- an ELISA method was constructed using a labeled antibody prepared by directly labeling the antibody.
- alkaline phosphatase (ALP) labeling of an antibody suitable for detection was performed.
- the EpCAM antibody is reduced by using the Alkaline Phosphatase Labeling Kit-SH (ALP labeling reagent, manufactured by Dojin) to reduce the SH group.
- ALP labeling was performed via
- Fab ′ was prepared, enzyme labeling was performed using the hinge method. That is, 50 ⁇ g of pepsin was reacted with 1 mg of antibody and digested to prepare F (ab ′) 2, followed by purification by gel filtration using a HPLC TSKgel G2000SWXL column (manufactured by Tosoh Corporation). Next, Fab 'was prepared by reduction with 10 mM 2-mercaptoethylamine and the above column. On the other hand, ALP was maleimidized with Sulfo-HMCS and purified with a PD-10 column (manufactured by GE Healthcare). An equimolar Fab 'and maleimidated ALP were mixed and reacted at 4 ° C overnight, and then purified by the above TSKgel G2000SWXL column of HPLC to prepare a labeled antibody.
- a CD9-12A12 antibody solid phase plate was previously loaded with an assay buffer (50 mM Tris-HCl buffered saline containing 1 mM magnesium chloride, 0.1 mM calcium chloride, 0.5% BSA, 1% BSA). 75 ⁇ L of water (pH 7.4) was added, 25 ⁇ L of Exosome standard solution (0 to 25 ⁇ g / mL) was added, and the mixture was reacted at room temperature for 2 hours while shaking.
- an assay buffer 50 mM Tris-HCl buffered saline containing 1 mM magnesium chloride, 0.1 mM calcium chloride, 0.5% BSA, 1% BSA.
- 75 ⁇ L of water (pH 7.4) was added, 25 ⁇ L of Exosome standard solution (0 to 25 ⁇ g / mL) was added, and the mixture was reacted at room temperature for 2 hours while shaking.
- biotinylated CD63-8A12 antibody was reacted in advance at a concentration of 1 ⁇ g / mL at 4 ° C. overnight.
- 75 ⁇ L of the above assay buffer was added, 25 ⁇ L of Exosome standard solution (0-25 ⁇ g / mL) was added, and the mixture was allowed to react at room temperature for 2 hours with shaking.
- 12.5 ng / mL ALP-labeled CD63-13C8 antibody 100 ⁇ L was added, and after stirring, the mixture was allowed to stand at room temperature for 3 hours. Thereafter, the reaction solution was removed, and after washing with the washing solution four times, 100 ⁇ L of the chemiluminescence substrate solution was added, and after stirring, the amount of chemiluminescence in each well was measured. The result is shown in FIG.
- an addition recovery test was performed on the Exosome ELISA using the combination of the anti-CD9 antibody of the present invention and the combination of the anti-CD63 antibody, and the binding specificity was evaluated. Specifically, as a result of examining the addition and recovery of C32 melanoma cell-derived Exosomes to serum samples, the recovery rate was 87.9 to 113.4% when anti-CD9 antibody was used, and 96. when anti-CD63 antibody was used. A good addition recovery rate of 2 to 99.3% was obtained. On the other hand, in the Exosome ELISA using the anti-CD9 commercially available antibody, the recovery rate was 32.1 to 41.8%, and a sufficient addition recovery rate was not obtained. The same commercially available antibody as in Example 2 was used.
- Example 5 [Detection of Exosomes in Blood Samples of Cancer Patients with Anti-CD9 and Anti-CD63 Antibodies]
- the results are shown in FIG. In addition, the comparison between groups was performed by t test.
- an Exosome ELISA in which an anti-CD9 antibody and an anti-CD63 antibody were combined was constructed, and the Exosomes in serum of healthy subjects, breast cancer patients, and colon cancer patients were similarly measured.
- solid phase antibody: CD9-12A12 antibody, labeled antibody: CD63-8A12 antibody combination, solid phase antibody: CD63-13C8 antibody, labeled antibody: CD9-12A12 antibody combination were used. The results are shown in FIG.
- the labeled antibody an ALP directly labeled antibody was used.
- Example 6 Diagnostic application of Exosome ELISA in combination with antibody against disease-specific membrane protein
- the above-mentioned anti-CD9 and anti-CD63 antibodies were combined with antibodies against disease-specific membrane proteins to examine the possibility of quantifying Exosomes related to cancer.
- EpCAM antibody was selected as a membrane protein antibody related to cancer, and an antibody (clone AUA1) manufactured by AbCAM was used.
- EpCAM is previously used in breast cancer cell lines (ZR75-1, T47D, MCF7, BT474, MDA-MB-468), colon cancer cell lines (HT29, SW48, SW480, HCT116), etc. It was confirmed that lysate and Exosome in the culture supernatant were strongly expressed. On the other hand, it was confirmed that no expression was observed in normal breast cells 184A1 (both results are not shown).
- an Exosome ELISA using an anti-EpCAM antibody the EpCAM antibody was subjected to ALP labeling using Alkaline Phosphatase Labeling Kit-SH (ALP labeling reagent, manufactured by Dojin).
- ALP labeling reagent manufactured by Dojin.
- a blood sample was quantified by performing an Exosome ELISA for the colon cancer cell line HCT116-derived Exosome. The results are shown in FIG. In addition, the comparison between groups was performed by t test.
- Example 7 [Preparation of anti-CD81 monoclonal antibody] (Preparation of antigen) Furthermore, in order to produce an anti-CD81 antibody, a small loop peptide of CD81, that is, a peptide in which a cysteine residue was added to the amino terminus of Arg36-Ala54 of the CD81 polypeptide was synthesized by Sigma-Aldrich. A hapten antigen was prepared via the SH group of the peptide using KLH obtained by maleidation of this (Keyhole Limet Hemocyanin, Image (registered trademark) Maleimide Activated mcKLH, manufactured by Thermo Scientific).
- FIG. 2A schematically shows the structure of the tetraspanin family (CD9, CD63 and CD81), and FIG. 2B schematically shows the antigen.
- FIG. 12A shows the peptide sequence.
- a fusion protein of the Fc region of rabbit IgG and the large loop portion of CD81 was also prepared as an antigen (Fc fusion protein).
- FIG. 2C and FIG. 12B show antigen information. That is, the Fc fusion protein is transiently expressed by introducing a plasmid vector into which a polynucleotide sequence having Fc added to the carboxyl terminus of the large loop of the CD antigen is introduced into Freestyle 293-F Cells (manufactured by Invitrogen). Then, it was prepared by purification using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.).
- An emulsion as an immunogen was prepared by mixing CD81 hapten antigen and Fc fusion protein with equal amounts of complete adjuvant for the first immunization and incomplete adjuvant for the second and subsequent immunizations.
- Monoclonal antibodies are available from K. Watanabe et al. , Vasohibin as an endothelium-derived negative feedback back regulator of engineering, J. Clin. Invest. 114 (2004), 898-907. That is, for each administration, 50 ⁇ g of hapten antigen or 10 ⁇ g of Fc fusion protein was subcutaneously and intraperitoneally divided into 5-week-old female A / J mice. Thereafter, after the 4th or 5th immunization, the spleen was extracted from the mouse that had undergone the final booster (4 days before cell fusion) to prepare spleen cells. Cell fusion between spleen cells and myeloma cells (P3U1) Watanabe et al. , Vasohibin as an endothelium-derived negative feedback back regulator of engineering, J. Clin. Invest. 114 (2004), 898-907.
- P3U1 myeloma cells
- the antibody titer (primary evaluation) of antiserum and hybridoma supernatant was evaluated by the ELISA method described below. Specifically, antiserum or hybridoma supernatant is added to a 96-well microplate on which a goat anti-mouse IgG antibody is solid-phased, and biotinylated CD9, CD63 protein and HRP-labeled streptavidin are mixed and stirred, and then at room temperature for 2 hours or 4 hours. The reaction was allowed to proceed overnight at 0 ° C.
- the plate is washed three times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), 100 ⁇ L of TMB reagent is added, and after stirring, the mixture is allowed to stand for 15 to 20 minutes. The reaction was stopped by adding 50 ⁇ L. Absorbance at 450 nm was measured with ARVO MX (manufactured by Perkin Elmer), and a case where a signal intensity exceeding 3 times the signal obtained when antiserum or hybridoma was not added was judged as positive.
- a washing solution saline containing 0.01% Tween 20 and 0.1% ProClin 150
- Each of the obtained monoclonal antibodies was purified from a serum-free medium of antibody-producing hybridoma or ascites obtained by administering the hybridoma to mice using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.). As described above, all the anti-CD81 monoclonal antibodies described below were prepared.
- Example 8 Capture by immunoprecipitation of Exosomes with anti-CD9, anti-CD63 and anti-CD81 antibodies.
- an antibody having better immunoprecipitation ability than any three commercially available antibodies was selected in advance and used.
- the anti-CD9 antibody is an Abnova antibody (IVA50)
- the anti-CD63 antibody is an ExoBio antibody (MEM-259) and a BD antibody (H5C6)
- the anti-CD81 antibody is a GENETEX antibody (1D6).
- the following samples were used for immunoprecipitation.
- the anti-CD63 antibody and the anti-CD81 antibody Exosomes prepared from the culture supernatant of COS7 cells into which a plasmid having a FLAG tag added to the C terminus of the polynucleotide encoding CD63 or CD81 polypeptide was used.
- the CD9 antibody the serum of a healthy person was used.
- Immunoprecipitation was performed as follows. In the case of anti-CD63 or anti-CD81 antibody, 1 ⁇ g of antibody was added to 1 ⁇ g of Exosome solution dissolved in PBS solution containing 1% BSA, and reacted at 4 ° C. overnight. After adding 20 ⁇ L Protein G agarose (50% slurry), the mixture was reacted at 4 ° C. with stirring for 2 hours. After the reaction, after centrifugal washing twice with PBS containing 1% BSA, the amount of CD63 or CD81 on the Exosomes captured by the beads was evaluated by Western blotting (WB) using an HRP-labeled anti-FLAG antibody. It was.
- WB Western blotting
- anti-CD9 antibody 100 ⁇ L of PBS containing 1% BSA was added to 100 ⁇ L of serum, M280 magnetic beads on which anti-CD9 antibody was solid-phased were added in an amount corresponding to 1 ⁇ g of antibody, and reacted at 4 ° C. overnight. After the reaction, washing with PBS containing 1% BSA was performed using a magnet, and the amount of CD9 on Exosomes captured in the supernatant was evaluated by WB using anti-CD9 antibody and HRP-labeled anti-mouse IgG antibody. went. Figures 13, 14, and 15 show comparisons of the immunoprecipitation performance of Exosome for each skillful CD antibody.
- the CD9-12A12 antibody, the CD63-8A12 antibody, the CD81-6D12 antibody, and the CD81-4G6 antibody CD81-12C4 antibody exhibited stronger exosome immunoprecipitation ability than the commercially available antibody.
- Example 9 Diagnosis of disease by detecting miRNA derived from captured Exosomes
- miRNA or protein is detected according to known methods.
- the detected miRNA or protein can be analyzed according to a known method, whereby it can be determined that the patient is suffering from analysis of physiological phenomena or a specific disease.
- the miRNA in Exosomes is, for example, K. Analysis can be performed by microarray analysis or quantitative PCR with reference to Ohshima, et al, PLoS One, 2010, 5, p1-10.
- the monoclonal antibody for detecting Exosomes of the present invention can detect CD9, CD63 or CD81 on Exosomes in vivo with good sensitivity and specificity. Therefore, for example, by quantifying Exosome in combination with an antibody against a disease-specific membrane protein, it is possible to diagnose a specific disease, so application to a diagnostic agent is expected. Furthermore, the monoclonal antibody of the present invention shows that Exosomes can be purified by immunoprecipitation, and the development of diagnostic agents applying changes in miRNA and proteins within Exosomes is also expected.
- Sequence number 1 of a sequence table is Exosome membrane protein CD9 polypeptide.
- Sequence number 2 of a sequence table is Exosome membrane protein CD63 polypeptide.
- Sequence number 3 of a sequence table is Exosome membrane protein CD81 polypeptide.
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Abstract
Description
<1> Exosomeを検出又は捕捉することができる、配列番号1に示されるアミノ酸配列のアミノ酸番号113~195を認識するモノクローナル抗体もしくはその抗体断片、配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体もしくはその抗体断片、配列番号3に示されるアミノ酸配列のアミノ酸番号36~54を認識するモノクローナル抗体もしくはその抗体断片、及び配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体もしくはその抗体断片、からなる群より選ばれる、Exosome検出又は捕捉用モノクローナル抗体に関する。
<2> 受領番号FERM ABP-11519として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD9-12A12抗体)もしくはその断片であって配列番号1に示されるアミノ酸配列のアミノ酸番号113~195を認識するモノクローナル抗体断片、
受領番号FERM ABP-11520として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD63-8A12抗体)もしくはその断片であって配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体断片、
受領番号FERM ABP-11521として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD63-13C8抗体)もしくはその断片であって配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体断片、
受領番号NITE ABP-1480として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-4G6抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体断片、
受領番号NITE ABP-1481として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-6D12抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体断片、及び
受領番号NITE ABP-1482として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-12C4抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号36~54を認識するモノクローナル抗体断片
からなる群より選ばれる、Exosome検出又は捕捉用モノクローナル抗体。
<3> CD9-12A12抗体もしくはその抗体断片、CD63-8A12抗体もしくはその抗体断片、及びCD63-13C8抗体もしくはその抗体断片、からなる群より選ばれる同一又は二種の、抗体もしくはその抗体断片を組み合わせてなる、モノクローナル抗体もしくはその抗体断片のセット。
<4> 固相抗体がCD9-12A12抗体又はその抗体断片、標識抗体がCD9-12A12抗体又はその抗体断片であるセット、
固相抗体がCD9-12A12抗体又はその抗体断片、標識抗体がCD63-13C8抗体又はその抗体断片であるセット、
固相抗体がCD63-8A12抗体又はその抗体断片、標識抗体がCD9-12A12抗体又はその抗体断片であるセット、及び
固相抗体がCD63-8A12抗体又はその抗体断片、標識抗体がCD63-13C8抗体又はその抗体断片であるセット、
からなる群より選ばれる、前記<3>記載のセット。
<5> Exosome検出用である、前記<3>又は<4>記載のセット。
<6> CD9-12A12抗体もしくはその抗体断片、CD63-8A12抗体もしくはその抗体断片、及びCD63-13C8抗体もしくはその抗体断片からなる群より選ばれる抗体又はその抗体断片と、疾患特異的膜タンパク質抗体又はその抗体断片とを組み合わせてなる、疾患特異的なExosome検出用モノクローナル抗体のセット。
<7> 前記<3>~<5>いずれか記載のセットのモノクローナル抗体又はその抗体断片を含む、癌又は免疫系疾患の診断キット。
<8> 前記<6>記載のセットのモノクローナル抗体又はその抗体断片を含む、癌又は免疫系疾患の診断キット。
<9> 前記<6>記載のセットを含有する、癌又は免疫系疾患の診断キット。
<10> 被検者由来の生体試料と、前記<1>又は<2>記載のモノクローナル抗体とを接触させてExosomeを捕捉して、該ExosomeのmiRNAを検出する方法。
<11> 被検者由来の生体試料と、前記<3>~<5>いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する方法。
<12> 被検者が癌又は免疫系疾患を発症しているか否かを判定するための方法であって、
工程(I):被検者由来の生体試料と、前記<3>~<5>いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程、及び;
工程(II):前記工程(I)で測定されたシグナル強度と、対照者におけるシグナル強度とを対比して、前記被検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に、前記被検者が癌又は免疫系疾患を発症していると判定する工程
を含む、癌又は免疫系疾患の判定方法。
<13> 抗癌剤又は抗免疫系疾患薬の薬効評価方法であって、
工程(A):抗癌剤又は抗免疫系疾患薬の投与前及び投与後の被検者由来の生体試料と、前記<3>~<5>いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程、及び;
工程(B):前記抗癌剤又は抗免疫系疾患薬の投与後の被検者由来の生体試料における該複合体由来のシグナル強度が、前記抗癌剤又は抗免疫系疾患薬の投与前の被検者由来の生体試料における該複合体由来のシグナル強度より弱いと認められる場合に、抗癌剤又は抗免疫系疾患薬が薬効を示している可能性が高いと判定する工程
を含む、抗癌剤又は抗免疫系疾患薬の薬効評価方法。
(抗原の調製)
本発明のモノクローナル抗体は、抗原を下記のように設計して調製したものであることから、抗原に対する感度及び特異性に優れる。
本発明のExosome検出用モノクローナル抗体(以下、単に、本発明のモノクローナル抗体ともいう)は、特に限定されるものではなく、公知の方法、例えば、K.Watanabe et al.,Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis,J.Clin.Invest.114(2004),898-907に記載した方法に従って調製することができる。
FERM ABP-11519(産生されるモノクローナル抗体がCD9-12A12抗体、表示CD9:12A12、受領日2011年11月8日)
FERM ABP-11520(産生されるモノクローナル抗体がCD63-8A12抗体、表示CD63:8A12、受領日2011年11月8日)
FERM ABP-11521(産生されるモノクローナル抗体がCD63-13C8抗体、表示CD63:13C8、受領日2011年11月8日)。
NITE ABP-1480(産生されるモノクローナル抗体がCD81-4G6抗体、表示CD81-4G6、受領日2012年12月12日)
NITE ABP-1481(産生されるモノクローナル抗体がCD81-6D12抗体、表示CD81-6D12、受領日2012年12月12日)
NITE ABP-1482(産生されるモノクローナル抗体がCD81-12C4抗体、表示CD81-12C4、受領日2012年12月12日)
また、本発明は、前記本発明のモノクローナル抗体又はその抗体断片を少なくとも1種含むモノクローナル抗体のセットを提供する。かかるセットを用いることにより、試料中のExosomeの捕捉が感度良く行なえるため、例えば、サンドイッチELISA法による定量精度が向上する。
態様1:本発明のモノクローナル抗体又はその抗体断片を2種以上含むセット
態様2:本発明のモノクローナル抗体又はその抗体断片と疾患特異的膜タンパク質抗体又はその抗体断片を含むセット
(抗体の2次評価法)
CD9又はCD63を強制発現させたVLPs(Virus-Like Particles)に任意の試験抗体を作用させて、試験抗体のCD9又はCD63への結合性を評価することができる。
Exosomeを捕捉する際に、本発明のモノクローナル抗体が好適に用いられる。例えば、本発明のモノクローナル抗体をビオチン化してExosomeと反応させた後、ストレプトアビジン固相磁性ビーズを用いることにより単離が可能となる。
Exosomeは、種々の細胞、例えば免疫系の細胞や各種癌細胞から分泌されることから、Exosome由来のmiRNAを検出することができれば、それを解析することにより生理現象や各種疾患の判定が可能になる。
本発明のモノクローナル抗体のセットを用いて免疫測定を行う。免疫測定法としては、酵素免疫測定法(EIA)、酵素イムノメトリックアッセイ法(ELISA)、蛍光免疫測定法(FIA)、放射線免疫測定法(RIA)、発光免疫測定法、イムノブロット法、ウェスタンブロット法等が挙げられ、簡便に感度よく抗体を検出し得ることから、ELISA法が好ましい。
また、本発明は、癌又は免疫系疾患の診断方法(発症予測方法)を提供する。本発明の態様1のセットは、生体内に分布するExosomeを良好な感度及び特異性で検出することが可能である。態様2のセットは、疾患特異的なExosomeを良好な感度で検出することが可能である。Exosomeは、種々の細胞、例えば免疫系の細胞や各種癌細胞から分泌されることから、癌又は免疫系疾患を発症している場合には、生体内のExosome量が多くなっていると考えられる。また、癌細胞などは、正常細胞と比べ細胞表面に変化が起き、CD9やCD63などの膜タンパク質の発現量が亢進する可能性もある。従って、前記Exosomeに由来するシグナル量を指標とすることにより癌又は免疫系疾患の診断(発症予測)が可能となり、さらに、態様2の場合は癌又は免疫系疾患の特定を行うことも可能であり、前記方法は、診断方法(発症関連因子の測定方法又は判定方法)としても使用可能である。
工程(I):被検者由来の生体試料と、前記本発明のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程、及び;
工程(II):前記工程(I)で測定されたシグナル強度と、対照者におけるシグナル強度とを対比して、前記被検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に、前記被検者が癌又は免疫系疾患を発症していると判定する工程を含む、癌又は免疫系疾患の判定方法を含む。
本発明の別の態様では、癌又は免疫系疾患の診断を行うためのキットが提供される。
また、本発明の別の態様では、抗癌剤又は抗免疫系疾患薬の薬効評価方法が提供される。
工程(A):抗癌剤又は抗免疫系疾患薬の投与前及び投与後の被検者由来の生体試料と、本発明のモノクローナル抗体のセットに含まれるモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程
工程(B):前記抗癌剤又は抗免疫系疾患薬の投与後の被検者由来の生体試料における該複合体由来のシグナル強度が、前記抗癌剤又は抗免疫系疾患薬の投与前の被検者由来の生体試料における該複合体由来のシグナル強度より弱いと認められる場合に、抗癌剤又は抗免疫系疾患薬が薬効を示している可能性が高いと判定する工程
を含む。
本発明の別の態様では、抗癌剤又は抗免疫系疾患薬の薬効評価を行うためのキットが提供される。
(抗原の調製)
CD9及びCD63タンパクの部分ペプチド、即ち、CD9ポリペプチドのArg36-Asn50、CD63ポリペプチドのVal38-Pro54の各アミノ末端にシステイン残基を付加した2種類のペプチドをシグマアルドリッチ社にて合成した。これらをマレイミド化したKLH〔Keyhole Limpet Hemocyanin、Imject(登録商標)Maleimide Activated mcKLH、サーモサイエンティフィック社製〕を用いて、ペプチドのSH基を介してハプテン抗原を調製した。図2Aにテトラスパニンファミリー(CD9、CD63)の構造、図2Bに抗原を模式的に示した。また、図3Aにペプチドの配列を示した。
CD9及びCD63のハプテン抗原、Fc融合タンパク質を、初回免疫には完全アジュバントと、2回目以降は不完全アジュバントと等量混和することにより、免疫原としての乳剤を調製した。
抗血清とハイブリドーマ上清の抗体価の評価(1次評価)は下記に記述するELISA法にて行った。即ち、ヤギ抗マウスIgG抗体を固相した96穴マイクロプレートに、抗血清又はハイブリドーマ上清を添加し、さらにビオチン化CD9、CD63タンパク質とHRP標識ストレプトアビジンを混合攪拌後、室温で2時間あるいは4℃で終夜反応させた。反応後、洗浄液(0.01% Tween20と0.1% ProClin 150を含む生理食塩水)にて3回洗浄を行い、100μL TMB試薬を加え、攪拌後15~20分間静置し、1N 硫酸溶液50μLを添加して反応を停止させた。450nmにおける吸光度をARVO MX(Perkin Elmer社製)にて測定し、抗血清やハイブリドーマを未添加の場合に得られるシグナルの3倍を超えるシグナル強度を示す場合を陽性と判断した。
インビトロジェン社のMembranePro機能性タンパク質発現キットを用い、CD9又はCD63とI型膜タンパク質TEM7とを共発現させたVLPs(virus-like particles)を調製した。具体的には、CD9又はCD63とTEM7遺伝子をpEF V5-His TA Vector Kitに組み込んだプラスミドベクターを、293FT細胞(インビトロジェン社製)にLipofectamine 2000にてトランスフェクトした。トランスフェクト後の培養上清にPrecipitation Mix試薬を添加し、VLPsを沈殿させた。VLPsにおけるTEM7及び各CD抗原の発現はWestern blotting(WB)により確認した(結果示さず)。
血中Exosomeの測定が可能となるサンドイッチELISA法における抗体の組合せを探索するため、前記実施例1で得られた全てのモノクローナル抗体に関して固相抗体及び標識抗体を調製して、血中Exosome測定用のサンドイッチELISAを行った。
固相抗体:CD9-12A12抗体、標識抗体:CD9-12A12抗体
固相抗体:CD63-8A12抗体、標識抗体:CD63-13C8抗体
したがって、上記の実験結果から、以下の組み合わせも、同様に良い組み合わせであることが十分に予測される。
固相抗体:CD9-12A12抗体、標識抗体:CD63-13C8抗体、
固相抗体:CD63-8A12抗体、標識抗体:CD9-12A12抗体
実施例3で選択した最も良い抗体の組合せについて、さらに感度を上げるため、抗体に直接標識を行うことにより調製した標識抗体を用いてELISA法を構築した。
健常者、乳癌患者、及び大腸癌患者各10名の血清中Exosome量を、固相抗体:CD9-12A12抗体、標識抗体:CD9-12A12抗体の組合せ、固相抗体:CD63-8A12抗体、標識抗体:CD63-13C8抗体の組合せを用いて、実施例4と同様にしてExosome ELISAにて測定した。結果を図9に示す。なお、群間比較はt検定により行なった。
上記抗CD9、抗CD63抗体と疾患特異的膜タンパク質に対する抗体を組み合わせ、癌に関連したExosomeの定量の可能性を調べた。具体的には、癌に関連した膜タンパク質抗体としてEpCAM抗体を選択し、AbCAM社製の抗体(クローンAUA1)を用いた。
(抗原の調製)
さらに抗CD81抗体を作製するため、CD81の小ループペプチド、即ち、CD81ポリペプチドのArg36-Ala54のアミノ末端にシステイン残基を付加したペプチドをシグマアルドリッチ社にて合成した。これをマレイミド化したKLH〔Keyhole Limpet Hemocyanin、Imject(登録商標)Maleimide Activated mcKLH、サーモサイエンティフィック社製〕を用いて、ペプチドのSH基を介してハプテン抗原を調製した。図2Aにテトラスパニンファミリー(CD9、CD63及びCD81)の構造、図2Bに抗原を模式的に示した。また、図12Aにペプチドの配列を示した。
CD81のハプテン抗原、Fc融合タンパク質を、初回免疫には完全アジュバントと、2回目以降は不完全アジュバントと等量混和することにより、免疫原としての乳剤を調製した。
抗血清とハイブリドーマ上清の抗体価の評価(1次評価)は下記に記述するELISA法にて行った。即ち、ヤギ抗マウスIgG抗体を固相した96穴マイクロプレートに、抗血清又はハイブリドーマ上清を添加し、さらにビオチン化CD9、CD63タンパク質とHRP標識ストレプトアビジンを混合攪拌後、室温で2時間あるいは4℃で終夜反応させた。反応後、洗浄液(0.01% Tween20と0.1% ProClin 150を含む生理食塩水)にて3回洗浄を行い、100μL TMB試薬を加え、攪拌後15~20分間静置し、1N 硫酸溶液50μLを添加して反応を停止させた。450nmにおける吸光度をARVO MX(Perkin Elmer社製)にて測定し、抗血清やハイブリドーマを未添加の場合に得られるシグナルの3倍を超えるシグナル強度を示す場合を陽性と判断した。
実施例1及び7で得られた抗CD9、抗CD63及び抗CD81モノクローナル抗体を使用したExosomeの免疫沈降による精製の可能性を調べた。なお、比較に用いた市販抗体としては、それぞれ任意の3つの市販抗体より免疫沈降能が良い抗体を予め選択して用いた。具体的には、抗CD9抗体はAbnova社の抗体(IVA50)、抗CD63抗体はExoBio社の抗体(MEM-259)及びBD社の抗体(H5C6)、抗CD81抗体はGENETEX社の抗体(1D6)を用いた。
抗CD63あるいは抗CD81抗体の場合は、1%BSAを含むPBS溶液に溶解した1μgのExosome溶液に、抗体1μgを加え、終夜4℃にて反応させた。20μLのProteinGアガロース(50% slurry)を添加した後、4℃にて2時間攪拌しながら反応させた。反応後、1%BSAを含むPBSにて2回遠心洗浄を行った後、ビーズに捕捉されたExosome上のCD63あるいはCD81量をHRP標識抗FLAG抗体用いたウエスタンブロッティング(WB)にて評価を行った。抗CD9抗体に関しては、血清100μLに1%BSAを含むPBSを100μL添加し、抗CD9抗体を固相したM280磁性ビーズを抗体1μg分となる量を添加し、4℃にて終夜反応させた。反応後、1%BSAを含むPBSにて、磁石を用いて洗浄を行い、上清に捕捉されたExosome上のCD9量を抗CD9抗体とHRP標識抗マウスIgG抗体を用いたWBにて評価を行った。各巧CD抗体のExosomeの免疫沈降の性能を比較した図を図13、14、15に示す。
実施例8の方法を参照にして捕捉されたExosomeから、miRNA又はタンパク質を公知の方法に従って検出する。検出したmiRNA又はタンパク質については、公知の方法に従って解析することが可能であり、それにより、生理現象の解析や特定の疾患に罹患していると判定することができる。
配列表の配列番号2は、Exosome膜タンパク質CD63ポリペプチドである。
配列表の配列番号3は、Exosome膜タンパク質CD81ポリペプチドである。
Claims (13)
- Exosomeを検出又は捕捉することができる、配列番号1に示されるアミノ酸配列のアミノ酸番号113~195を認識するモノクローナル抗体もしくはその抗体断片、配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体もしくはその抗体断片、配列番号3に示されるアミノ酸配列のアミノ酸番号36~54を認識するモノクローナル抗体もしくはその抗体断片、及び配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体もしくはその抗体断片、からなる群より選ばれる、Exosome検出又は捕捉用モノクローナル抗体。
- 受領番号FERM ABP-11519として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD9-12A12抗体)もしくはその断片であって配列番号1に示されるアミノ酸配列のアミノ酸番号113~195を認識するモノクローナル抗体断片、
受領番号FERM ABP-11520として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD63-8A12抗体)もしくはその断片であって配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体断片、
受領番号FERM ABP-11521として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD63-13C8抗体)もしくはその断片であって配列番号2に示されるアミノ酸配列のアミノ酸番号104~202を認識するモノクローナル抗体断片、
受領番号NITE ABP-1480として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-4G6抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体断片、
受領番号NITE ABP-1481として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-6D12抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号113~201を認識するモノクローナル抗体断片、及び
受領番号NITE ABP-1482として寄託されているハイブリドーマにより産生されるモノクローナル抗体(CD81-12C4抗体)もしくはその断片であって配列番号3に示されるアミノ酸配列のアミノ酸番号36~54を認識するモノクローナル抗体断片
からなる群より選ばれる、Exosome検出又は捕捉用モノクローナル抗体。 - CD9-12A12抗体もしくはその抗体断片、CD63-8A12抗体もしくはその抗体断片、及びCD63-13C8抗体もしくはその抗体断片、からなる群より選ばれる同一又は二種の、抗体もしくはその抗体断片を組み合わせてなる、モノクローナル抗体もしくはその抗体断片のセット。
- 固相抗体がCD9-12A12抗体又はその抗体断片、標識抗体がCD9-12A12抗体又はその抗体断片であるセット、
固相抗体がCD9-12A12抗体又はその抗体断片、標識抗体がCD63-13C8抗体又はその抗体断片であるセット、
固相抗体がCD63-8A12抗体又はその抗体断片、標識抗体がCD9-12A12抗体又はその抗体断片であるセット、及び
固相抗体がCD63-8A12抗体又はその抗体断片、標識抗体がCD63-13C8抗体又はその抗体断片であるセット、
からなる群より選ばれる、請求項3記載のセット。 - Exosome検出用である、請求項3又は4記載のセット。
- CD9-12A12抗体もしくはその抗体断片、CD63-8A12抗体もしくはその抗体断片、及びCD63-13C8抗体もしくはその抗体断片からなる群より選ばれる抗体又はその抗体断片と、疾患特異的膜タンパク質抗体又はその抗体断片とを組み合わせてなる、疾患特異的なExosome検出用モノクローナル抗体のセット。
- 請求項3~5いずれか記載のセットのモノクローナル抗体又はその抗体断片を含む、癌又は免疫系疾患の診断キット。
- 請求項6記載のセットのモノクローナル抗体又はその抗体断片を含む、癌又は免疫系疾患の診断キット。
- 請求項6記載のセットを含有する、癌又は免疫系疾患の診断キット。
- 被検者由来の生体試料と、請求項1又は2記載のモノクローナル抗体とを接触させてExosomeを捕捉して、該ExosomeのmiRNAを検出する方法。
- 被検者由来の生体試料と、請求項3~5いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する方法。
- 被検者が癌又は免疫系疾患を発症しているか否かを判定するための方法であって、
工程(I):被検者由来の生体試料と、請求項3~5いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程、及び;
工程(II):前記工程(I)で測定されたシグナル強度と、対照者におけるシグナル強度とを対比して、前記被検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に、前記被検者が癌又は免疫系疾患を発症していると判定する工程
を含む、癌又は免疫系疾患の判定方法。 - 抗癌剤又は抗免疫系疾患薬の薬効評価方法であって、
工程(A):抗癌剤又は抗免疫系疾患薬の投与前及び投与後の被検者由来の生体試料と、請求項3~5いずれか記載のセットのモノクローナル抗体とを接触させてExosome複合体を形成させて、該複合体由来のシグナル強度を測定する工程、及び;
工程(B):前記抗癌剤又は抗免疫系疾患薬の投与後の被検者由来の生体試料における該複合体由来のシグナル強度が、前記抗癌剤又は抗免疫系疾患薬の投与前の被検者由来の生体試料における該複合体由来のシグナル強度より弱いと認められる場合に、抗癌剤又は抗免疫系疾患薬が薬効を示している可能性が高いと判定する工程
を含む、抗癌剤又は抗免疫系疾患薬の薬効評価方法。
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