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WO2025009486A1 - Procédé de détection de vésicule extracellulaire - Google Patents

Procédé de détection de vésicule extracellulaire Download PDF

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Publication number
WO2025009486A1
WO2025009486A1 PCT/JP2024/023680 JP2024023680W WO2025009486A1 WO 2025009486 A1 WO2025009486 A1 WO 2025009486A1 JP 2024023680 W JP2024023680 W JP 2024023680W WO 2025009486 A1 WO2025009486 A1 WO 2025009486A1
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Prior art keywords
extracellular vesicles
derived
blood
platelet
target
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English (en)
Japanese (ja)
Inventor
琳子 廣野
畑下 瑠依 長坂
太一 松永
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Tosoh Corp
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Tosoh Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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

Definitions

  • the present invention relates to a method for detecting target extracellular vesicles in a blood-derived sample.
  • Body fluids such as the blood of cancer patients and culture fluids of cultured tumor cells contain proteins derived from tumor cells.
  • Tumor cell-derived proteins may be localized within the cells of circulating tumor cells (or dead cell debris), may be secreted extracellularly from tumor cells and exist as free proteins in the blood, or may exist as proteins within extracellular vesicles. By detecting these proteins, information such as early detection of tumors and prediction of the patient's condition after treatment can be obtained.
  • extracellular vesicles are colloidal particles covered with a lipid bilayer membrane, and their function as a mediator of intercellular communication in the body and their relationship to diseases such as cancer and physiological phenomena have been reported in recent years, and research is being conducted to clarify their physiological functions and apply them to disease testing.
  • Patent Document 1 discloses a method for detecting proteins present in plasma and localized on the surface of extracellular vesicles released from tumor cells using the Enzyme-Linked Immunosorbent Assay (ELISA) method. This method makes it possible to distinguish between cancer patients and healthy donors.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • the objective of the present invention is to provide a method for detecting target extracellular vesicles in blood-derived samples with high sensitivity.
  • the inventors discovered that when detecting target extracellular vesicles in blood-derived samples, platelet-derived extracellular vesicles cause problems such as increased variability in measurement values and increased non-specific signals (background).
  • target extracellular vesicles in a blood-derived sample can be detected with high sensitivity by removing platelet-derived extracellular vesicles in the blood-derived sample or by suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample, thereby completing the present invention.
  • a method for detecting target extracellular vesicles in a blood-derived sample comprising: A step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or a step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample, and a step B of detecting the target extracellular vesicles in the blood-derived sample.
  • a method comprising: [2] A method for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample, comprising: Step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample A method comprising: [3] A method for pre-treating a blood-derived sample for detecting target extracellular vesicles in the blood-derived sample, comprising: Step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample A method comprising: [4] The method (specifically, the method described in any one of [1] to [3]) wherein the target extracellular vesicles are extracellular vesicles other than platelet-derived extracellular vesicles.
  • step B includes a step B1 of capturing the target extracellular vesicles on a carrier, and a step B2 of detecting the captured target extracellular vesicles.
  • step B1 is carried out using a first substance that binds to the target extracellular vesicles
  • step B2 is carried out using a second substance that binds to the target extracellular vesicles.
  • the present invention enables highly sensitive detection of target extracellular vesicles in blood-derived samples.
  • FIG. 1 shows the results of detection of PC-3-derived extracellular vesicles (Example 6).
  • FIG. 1 shows the results of detection of PC-3-derived extracellular vesicles (Example 6).
  • FIG. 1 shows the results of detection of PC-3-derived extracellular vesicles (Example 7).
  • cancer and “cancer” may be used interchangeably.
  • the method of the present invention is a method comprising a step of removing platelet-derived extracellular vesicles in a blood-derived sample or a step of suppressing the occurrence of platelet-derived extracellular vesicles in a blood-derived sample.
  • the method of the present invention may further comprise a step of detecting target extracellular vesicles in the blood-derived sample.
  • the step of removing platelet-derived extracellular vesicles in a blood-derived sample is also referred to as a "removal step”.
  • the step of suppressing the occurrence of platelet-derived extracellular vesicles in a blood-derived sample is also referred to as a "suppression step”.
  • one or both of the removal step and the suppression step may be performed.
  • the step of detecting target extracellular vesicles in a blood-derived sample is also referred to as a "detection step”. The detection step is performed after the removal step or the suppression step.
  • Target extracellular vesicles refers to extracellular vesicles to be detected and are to be distinguished from platelet-derived extracellular vesicles.
  • target extracellular vesicles refers to extracellular vesicles other than platelet-derived extracellular vesicles that may be contained in a blood-derived sample.
  • the target extracellular vesicles may be one or more specific types of extracellular vesicles selected from extracellular vesicles other than platelet-derived extracellular vesicles, or may be all extracellular vesicles other than platelet-derived extracellular vesicles.
  • Specific extracellular vesicles include extracellular vesicles derived from tumor cells.
  • the type of tumor from which the target extracellular vesicles can be derived is not particularly limited and may include malignant tumors and benign tumors.
  • Malignant tumors may be primary cancers or metastatic cancers.
  • Tumors include hematopoietic cell malignant tumors, head and neck cancer, brain tumors, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastrointestinal stromal tumors, mesothelioma, thyroid cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular tumors, bladder cancer, skin cancer, and anal cancer.
  • Hematopoietic cell malignant tumors include leukemia, lymphoma, and multiple myeloma.
  • Lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma.
  • Head and neck cancers include floor of the mouth cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, and paranasal sinus cancer.
  • Extracellular vesicles refers to lipid-covered vesicles with a diameter of 1 nm to 1 ⁇ m that are released by cells, whether actively or passively.
  • extracellular vesicles include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles (Nature Reviews Immunology, 9, 581-593 (2009)).
  • extracellular vesicles are composed of lipids and proteins with a different composition from that of the cell membrane (Bioscience, 65, 783-797 (2015)), but proteins that reflect the characteristics of the cell from which they were released are localized in the extracellular vesicles. For example, when the source of release is a tumor cell, the presence or absence of extracellular vesicles released from the cell can be used to distinguish between cancer patients and healthy donors (Publication of International Publication No. 2011-510309).
  • one aspect of the method of the present invention is a method for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample, which includes a removal step or suppression step.
  • This method is also referred to as the "detection sensitivity improving method of the present invention.”
  • the detection sensitivity improving method of the present invention may further include a detection step. Examples of improved detection sensitivity include reducing the variation in measured values and reducing non-specific signals (background).
  • the removal step or the suppression step can be carried out as a pretreatment of the blood-derived sample. That is, one aspect of the method of the present invention is a method of pretreatment of a blood-derived sample, including a removal step or a suppression step. This method is also referred to as the "pretreatment method of the present invention.” The removal step and the suppression step are also collectively referred to as the "pretreatment step.” The removal step or the suppression step can be specifically carried out as a pretreatment of the blood-derived sample when carrying out the detection step. That is, the pretreatment method of the present invention may specifically be a method of pretreatment of the blood-derived sample in order to detect target extracellular vesicles in the blood-derived sample.
  • the removal step or the suppression step can be specifically carried out as a pretreatment for improving the detection sensitivity of target extracellular vesicles in the blood-derived sample. That is, the pretreatment method of the present invention may specifically be a method of pretreatment of the blood-derived sample in order to improve the detection sensitivity of target extracellular vesicles in the blood-derived sample.
  • one aspect of the method of the present invention is a method for detecting target extracellular vesicles in a blood-derived sample, which includes a removal or suppression step and a detection step.
  • This method is also referred to as the "detection method of the present invention.”
  • the detection method of the present invention is also a method for detecting target extracellular vesicles in a blood-derived sample, which includes a step of pretreating a blood-derived sample by the pretreatment method of the present invention, and a detection step.
  • the blood-derived sample is not particularly limited as long as it can contain target extracellular vesicles.
  • blood-derived samples include whole blood, serum, plasma, and fractions thereof.
  • blood-derived samples include serum and plasma in particular.
  • Blood-derived samples such as serum and plasma may be treated with an anticoagulant such as citric acid, heparin, or EDTA before carrying out the removal or inhibition step.
  • the blood-derived sample may be obtained from a subject.
  • the subject is not particularly limited.
  • the subject may be a human or a non-human animal.
  • Non-human animals include mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, pigs, monkeys, chimpanzees, and birds.
  • the subject may particularly be a human.
  • the subject may be, for example, a subject suspected of having cancer.
  • the subject may be, for example, a subject to be tested for cancer.
  • the embodiment of the removal step is not particularly limited as long as the platelet-derived extracellular vesicles in the blood-derived sample are removed to the desired extent.
  • the removal step may be carried out, for example, using a substance that specifically binds to platelet-derived extracellular vesicles.
  • a substance that specifically binds to platelet-derived extracellular vesicles is also called a "platelet-derived extracellular vesicle-specific binding substance.”
  • “Specifically binds to platelet-derived extracellular vesicles” may mean that it binds to platelet-derived extracellular vesicles but does not substantially bind to target extracellular vesicles.
  • platelet-derived extracellular vesicle-specific binding substances include substances that bind to markers that are specifically present on the surface of platelet-derived extracellular vesicles.
  • a marker that is specifically present on the surface of platelet-derived extracellular vesicles is also called a "platelet-derived extracellular vesicle-specific marker.”
  • “Specifically present on the surface of platelet-derived extracellular vesicles” may mean that it is present on the surface of platelet-derived extracellular vesicles but is substantially not present on the surface of target extracellular vesicles.
  • platelet-derived extracellular vesicle-specific markers include proteins that are specifically present on the surface of platelet-derived extracellular vesicles.
  • proteins that are specifically present on the surface of platelet-derived extracellular vesicles include CD41, CD42a, CD42b, CD61, and CD62P.
  • proteins that are specifically present on the surface of platelet-derived extracellular vesicles include CD41.
  • substances that bind to platelet-derived extracellular vesicles-specific markers include antibodies and aptamers against platelet-derived extracellular vesicles-specific markers.
  • substances that bind to platelet-derived extracellular vesicles-specific markers include antibodies against platelet-derived extracellular vesicles-specific markers.
  • antibodies against platelet-derived extracellular vesicles-specific markers include anti-CD41 antibodies, anti-CD42a antibodies, anti-CD42b antibodies, anti-CD61 antibodies, and anti-CD62P antibodies.
  • antibodies against platelet-derived extracellular vesicles-specific markers include anti-CD41 antibodies.
  • the antibodies may be, for example, whole immunoglobulins or parts of immunoglobulins.
  • Antibodies include Fab, Fab', F(ab')2, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and diabodies.
  • Antibodies may be monoclonal or polyclonal. Antibodies may in particular be monoclonal antibodies.
  • the removal step may be carried out by, for example, binding a platelet-derived extracellular vesicles-specific binding substance to the platelet-derived extracellular vesicles. That is, the removal step may include a step of binding a platelet-derived extracellular vesicles-specific binding substance to the platelet-derived extracellular vesicles.
  • the binding of the platelet-derived extracellular vesicles-specific binding substance to the platelet-derived extracellular vesicles can be carried out by contacting the platelet-derived extracellular vesicles-specific binding substance with the platelet-derived extracellular vesicles.
  • the binding of the platelet-derived extracellular vesicles-specific binding substance to the platelet-derived extracellular vesicles can be carried out, for example, by contacting the platelet-derived extracellular vesicles-specific binding substance with a blood-derived sample (which may contain platelet-derived extracellular vesicles).
  • the contact of the platelet-derived extracellular vesicles-specific binding substance with the platelet-derived extracellular vesicles can be carried out in a liquid medium.
  • the liquid medium include aqueous media such as water and aqueous buffer solutions, and organic solvents such as dimethyl sulfoxide.
  • the platelet-derived extracellular vesicles-specific binding substance can be brought into contact with the platelet-derived extracellular vesicles by mixing the blood-derived sample with the platelet-derived extracellular vesicles-specific binding substance in a liquid medium.
  • the platelet-derived extracellular vesicles-specific binding substance can be brought into contact with the platelet-derived extracellular vesicles by passing a blood-derived sample prepared in liquid form through the column.
  • the platelet-derived extracellular vesicles-specific binding substance may be bound to a carrier and used in the removal step. By binding the platelet-derived extracellular vesicles-specific binding substance to a carrier, the platelet-derived extracellular vesicles can be captured on the carrier via the platelet-derived extracellular vesicles-specific binding substance.
  • the order of binding between the platelet-derived extracellular vesicles-specific binding substance and the carrier is not particularly limited.
  • the platelet-derived extracellular vesicles-specific binding substance may typically be bound to the carrier before binding to the platelet-derived extracellular vesicles.
  • the platelet-derived extracellular vesicles-specific binding substance may typically be used for binding to the platelet-derived extracellular vesicles in a state in which it is bound to the carrier in advance.
  • the mode of binding between the platelet-derived extracellular vesicles-specific binding substance and the carrier is not particularly limited.
  • the platelet-derived extracellular vesicles-specific binding substance and the carrier may be bound by a covalent bond or a non-covalent bond. Examples of non-covalent bonds include hydrogen bonds and hydrophobic bonds.
  • the bond between the platelet-derived extracellular vesicles-specific binding substance and the carrier may be, in particular, a covalent bond.
  • the carrier may be a water-insoluble carrier.
  • the carrier may be hydrophilic or hydrophobic.
  • Specific examples of the carrier include polysaccharides such as agarose, dextran, chitosan, and cellulose; organic synthetic polymers such as polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, styrene-divinylbenzene copolymer, polystyrene, acrylic acid ester, methacrylic acid ester, polyethylene, polypropylene, poly4-ethylene fluoride, ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate, and polyethersulfone; ceramics such as glass, activated carbon, alumina, silica, and hydroxyapatite; and metals such as iron oxide, titanium, and gold.
  • the carrier may be in any shape, such as particles, fibers, hollow fibers, membranes, and flat plates.
  • the carrier may be
  • the platelet-derived extracellular vesicles can be removed from the blood-derived sample. That is, the removal step may include a step of separating the carrier that has captured the platelet-derived extracellular vesicles from the blood-derived sample. Separation of the carrier and the blood-derived sample can be performed, for example, by a normal solid-liquid separation means.
  • the solid-liquid separation means can be appropriately selected depending on various conditions, for example, the shape of the carrier.
  • the carrier that has captured the platelet-derived extracellular vesicles when dispersed in a liquid containing a blood-derived sample, the carrier can be separated from the blood-derived sample by subjecting the liquid to centrifugation, magnetic separation, or filtration.
  • the carrier is an antibody-immobilized magnetic particle
  • the blood-derived sample and the antibody-immobilized magnetic particle are mixed to capture the platelet-derived extracellular vesicles on the carrier, and then the antibody-immobilized magnetic particle that has captured the platelet-derived extracellular vesicles is removed using a magnet, thereby separating the carrier (i.e., the antibody-immobilized magnetic particle that has captured the platelet-derived extracellular vesicles) from the blood-derived sample. That is, the blood-derived sample can be recovered as a non-adsorbed fraction (i.e., the supernatant).
  • the blood-derived sample when a column is filled with a carrier, the blood-derived sample can be passed through the column to capture platelet-derived extracellular vesicles on the carrier, and then the blood-derived sample can be discharged from the column, thereby separating the carrier from the blood-derived sample. That is, the blood-derived sample can be recovered from the column as a non-adsorbed fraction.
  • the embodiment of the suppression step is not particularly limited as long as the generation of platelet-derived extracellular vesicles in the blood-derived sample is suppressed to a desired degree.
  • Platelet-derived extracellular vesicles can be generated from platelets contained in the blood-derived sample. Therefore, the suppression step can be achieved by removing platelets from the blood-derived sample.
  • the removal of platelets from the blood-derived sample may be performed, for example, by performing centrifugation two or more times during the preparation of a blood-derived sample (particularly, plasma).
  • the conditions of the centrifugation are not particularly limited as long as the conditions allow the separation of platelets and target extracellular vesicles.
  • the conditions of the centrifugation may be, for example, the conditions of normal centrifugation used in the preparation of plasma.
  • Platelet-derived extracellular vesicles can be generated when a blood-derived sample containing platelets is frozen. Therefore, the suppression step may be performed, for example, by not freezing the blood-derived sample.
  • the method of the present invention includes a suppression step, and the suppression step is performed by not freezing the blood-derived sample” may mean that the method of the present invention does not include a step of freezing the blood-derived sample.
  • the embodiment of the detection step is not particularly limited as long as it can detect target extracellular vesicles to the desired degree. Detection of target extracellular vesicles may be qualitative detection (e.g., detection of the presence or absence of target extracellular vesicles) or quantitative detection (e.g., detection of the amount of target extracellular vesicles present). Detection of target extracellular vesicles may particularly be quantitative detection.
  • the method of detecting target extracellular vesicles can be appropriately selected depending on various conditions such as the desired detection sensitivity. "Measurement of target extracellular vesicles" and “detection of target extracellular vesicles" may be used interchangeably.
  • the detection step is performed after the removal step or the suppression step.
  • the detection step may be carried out, for example, using a substance that binds to the target extracellular vesicles.
  • a substance that binds to the target extracellular vesicles is also referred to as a "target extracellular vesicle binding substance.”
  • target extracellular vesicle-specific binding substances include substances that bind to markers present on the surface of target extracellular vesicles. Markers present on the surface of target extracellular vesicles are also referred to as "target extracellular vesicle markers.”
  • the target extracellular vesicle binding substance may preferably be a substance that specifically binds to the target extracellular vesicles.
  • a substance that specifically binds to the target extracellular vesicles is also referred to as a "target extracellular vesicle specific binding substance.”
  • “Specifically binds to target extracellular vesicles” may mean that the substance binds to the target extracellular vesicles but does not substantially bind to platelet-derived extracellular vesicles.
  • Examples of target extracellular vesicle-specific binding substances include substances that bind to markers specifically present on the surface of target extracellular vesicles.
  • target extracellular vesicle specific markers Markers specifically present on the surface of target extracellular vesicles are also referred to as "target extracellular vesicle specific markers.” “Specifically present on the surface of target extracellular vesicles” may mean that it is present on the surface of target extracellular vesicles but is substantially absent on the surface of platelet-derived extracellular vesicles.
  • Target extracellular vesicle markers include proteins and lipids present on the surface of target extracellular vesicles.
  • Proteins present on the surface of target extracellular vesicles include tetraspanins such as TM4SF1, CD9, CD63, and CD81; antigen presentation-related proteins such as Major Histocompatibility Complex (MHC) I and MHCII; adhesion molecules such as integrins, cadherins, InterCellular Adhesion Molecule 1 (ICAM-1), and Epithelial Cell Adhesion Molecule (EpCAM); cytokines or growth factors such as EGFR (Epidermal Growth Factor Receptor) vIII and TGF (Transforming Growth Factor)- ⁇ ; receptors for cytokines or growth factors; and enzymes.
  • Lipids present on the surface of target extracellular vesicles include phosphatidylserine (PS) and sphingomyelin.
  • Substances that bind to target extracellular vesicle markers include antibodies and aptamers (peptide aptamers and nucleic acid aptamers) against the target extracellular vesicle markers.
  • the antibody may be, for example, an entire immunoglobulin or a part of an immunoglobulin.
  • antibodies include Fab, Fab', F(ab')2, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and diabodies.
  • Substances that bind to target extracellular vesicle markers include, in particular, antibodies against the target extracellular vesicle markers.
  • the detection step may be, in particular, performed by immunoassay (i.e., an assay using antibodies).
  • Substances that bind to target extracellular vesicle markers include, in particular, antibodies against target extracellular vesicle-specific markers. Examples of antibodies against target extracellular vesicle-specific markers include anti-TM4SF1 antibodies and anti-CD81 antibodies.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be, in particular, a monoclonal antibody.
  • the antibody or aptamer may be appropriately modified with a labeling substance.
  • the labeling substance examples include fluorescent substances such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Alexa Fluor (trade name); enzymes such as luciferase, alkaline phosphatase, and horseradish peroxidase; and radioactive substances.
  • the binding mode between the antibody or aptamer and the labeling substance is not particularly limited.
  • the antibody or aptamer may or may not be directly bound to the labeling substance.
  • the antibody may be directly bound to the labeling substance by chemical bonding or the like, or may be indirectly bound to the labeling substance by binding to a secondary antibody (labeled secondary antibody) bound to the labeling substance.
  • Examples of immunological measurement methods include EIA (Enzyme Immunoassay) and Western blotting.
  • EIA method include the ELISA (Enzyme-Linked Immunosorbent Assay) method, the CLEIA (Chemiluminescent Enzyme Immunoassay) method, and the FEIA (Fluorescence Enzyme Immunoassay) method.
  • a detection substrate may be used as appropriate.
  • the detection substrate include colorimetric substrates, fluorescent substrates, and luminescent substrates.
  • Examples of the detection substrate include, in particular, luminescent substrates.
  • the luminescent substrate include substrates that generate luminescence through an enzyme reaction such as horseradish peroxidase.
  • an enzyme corresponding to the luminescent substrate is used as a labeling substance, and luminescence can be generated by reacting with the luminescent substrate.
  • the target extracellular vesicles may be detected manually or automatically using a measuring device.
  • measuring devices include enzyme immunoassay devices such as the AIA-900 (Tosoh) and chemiluminescent enzyme immunoassay devices such as the AIA-CL2400 (Tosoh).
  • the target extracellular vesicles may be collected and the collected target extracellular vesicles may be detected. That is, the detection step may include, for example, a step of collecting the target extracellular vesicles on a carrier and a step of detecting the collected target extracellular vesicles.
  • the method for collecting the target extracellular vesicles include ultracentrifugation, sedimentation, size exclusion chromatography, affinity method, and polymer precipitation method.
  • the method for collecting the target extracellular vesicles include, in particular, affinity method.
  • the collection of the target extracellular vesicles may be specifically carried out by capturing the target extracellular vesicles on a carrier.
  • the detection step may specifically include, for example, a step of capturing the target extracellular vesicles on a carrier and a step of detecting the captured target extracellular vesicles.
  • the target extracellular vesicles can be captured on a carrier by an affinity method.
  • the target extracellular vesicles can be captured on a carrier via a target extracellular vesicle-binding substance by an affinity method.
  • the capture of target extracellular vesicles on a carrier can be carried out in the same manner as the capture of platelet-derived extracellular vesicles on a carrier described above, except that a target extracellular vesicles-binding substance is used instead of a platelet-derived extracellular vesicles-specific binding substance.
  • the carrier is as described above in the description of the capture of platelet-derived extracellular vesicles on a carrier.
  • Specific examples of the carrier include magnetic particles and microplates.
  • the carrier may be, for example, magnetic particles.
  • the step of capturing the target extracellular vesicles on the carrier may be carried out, for example, using a first substance that binds to the target extracellular vesicles.
  • the step of detecting the captured target extracellular vesicles may be carried out, for example, using a second substance that binds to the target extracellular vesicles.
  • Both the first substance and the second substance are target extracellular vesicles-binding substances.
  • Both the first substance and the second substance may be, for example, an antibody against a target extracellular vesicle marker. That is, the first substance may be an antibody (also called a first antibody) against a first marker present in the target extracellular vesicles.
  • the second substance may be an antibody (also referred to as a second antibody) against a second marker present in the target extracellular vesicles.
  • the first antibody and the second antibody may be, for example, antibodies against different target extracellular vesicle markers.
  • a sandwich EIA method such as a sandwich CLEIA method can be performed using the first antibody and the second antibody.
  • One or both of the first substance and the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker).
  • one or both of the first marker and the second marker may be a target extracellular vesicle-specific marker.
  • the first substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker), and further, the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker).
  • the first marker may be a target extracellular vesicle-specific marker
  • the second marker may be a target extracellular vesicle-specific marker.
  • the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker), and the first substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker).
  • the second marker may be a target extracellular vesicle-specific marker
  • the first marker may be a target extracellular vesicle-specific marker.
  • the first marker may be, for example, a target extracellular vesicle-specific marker such as TM4SF1 or CD81, among others.
  • the second marker may be, for example, CD9 or CD81, among others.
  • the use of the detection results of target extracellular vesicles is not particularly limited.
  • the detection results of the target extracellular vesicles can be used to test for cancer.
  • the detection results of the target extracellular vesicles can be used to test for cancer in the subject.
  • Tests for cancer in a subject include testing whether the subject has cancer, testing the presence or absence or degree of the possibility that the subject has cancer, and testing the progression (e.g., stage) of cancer if the subject has cancer. "Cancer testing” may be read as "cancer diagnosis.”
  • kit of the present invention is a kit for pretreatment of a blood-derived sample.
  • This kit is also referred to as the "pretreatment kit of the present invention.”
  • the pretreatment of the blood-derived sample is as described above in the method of the present invention.
  • kit of the present invention is a kit for detecting target extracellular vesicles in a blood-derived sample.
  • This kit is also referred to as the "detection kit of the present invention.” Detection of target extracellular vesicles in a blood-derived sample is as described above in the method of the present invention.
  • the kit of the present invention may, for example, include a reagent for use in the method of the present invention.
  • the kit of the present invention may, for example, include laboratory equipment for use in the method of the present invention.
  • the kit of the present invention may, for example, include instructions for the kit of the present invention.
  • the kit of the present invention may include, for example, a platelet-derived extracellular vesicles-specific binding substance.
  • the kit of the present invention may include, for example, a carrier for binding to the platelet-derived extracellular vesicles-specific binding substance.
  • the kit of the present invention may include, for example, a pre-bound platelet-derived extracellular vesicles-specific binding substance and a carrier.
  • the kit of the present invention may, for example, include a target extracellular vesicle binding substance.
  • the kit of the present invention may, for example, include a first substance and/or a second substance.
  • the kit of the present invention may, for example, include a carrier for binding to the first substance.
  • the kit of the present invention may, for example, include a first substance pre-bound and a carrier.
  • the kit of the present invention may, for example, include a labeling substance for labeling the target extracellular vesicle binding substance (e.g., the second substance).
  • the kit of the present invention may, for example, include a target extracellular vesicle binding substance pre-bound (e.g., the second substance) and a labeling substance.
  • the kit of the present invention may, for example, include a detection substrate.
  • the kit of the present invention can be used, for example, to carry out the method of the present invention.
  • Example 1 Preparation of antibody-immobilized magnetic particles (1) 600 ⁇ L of 0.01 M MES (2-morpholinoethanesulfonic acid) buffer solution (pH 6.0) (hereinafter referred to as "buffer A”) was added to 400 ⁇ L of a magnetic particle solution having carboxy groups on the surface (Magnosphere MS300/Carboxyl, 1.0% (w/v) slurry, manufactured by JSR Life Sciences Corporation).
  • buffer A 0.01 M MES (2-morpholinoethanesulfonic acid) buffer solution having carboxy groups on the surface
  • Buffer A containing 1 mg/mL anti-human TM4SF1 antibody (R&D Systems)
  • Buffer A containing 0.5 mg/mL anti-human CD41 antibody (BioLegend)
  • Buffer solution A containing 1 mg/mL mouse IgG (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
  • Example 2 Preparation of biotinylated modified antibody (1) 200 ⁇ L of a solution containing any of the antibodies shown in [d] and [e] below was added to a filtration tube (Dojin Chemical Laboratory Co., Ltd.) and centrifuged at 8,000 ⁇ g for 10 minutes at room temperature to immobilize the antibody on the filter.
  • [d] 1 mg/mL anti-human CD9 antibody (manufactured by Frontier Research Institute)
  • [e] 1 mg/mL anti-human CD81 antibody manufactured by Frontier Research Institute, Inc.
  • Example 3 Detection of background in plasma measurement
  • PC-3-derived extracellular vesicles are used as target extracellular vesicles.
  • TM4SF1 a member of the four-transmembrane protein (Transmembrane 4 superfamily, tetraspanin), a protein that is highly expressed in many tumor cells including PC-3 and extracellular vesicles derived from tumor cells) was selected as a marker protein for detecting PC-3-derived extracellular vesicles.
  • TM4SF1 is not expressed in extracellular vesicles derived from blood cells contained in plasma.
  • plasma is used as a specimen to be used as a sample. In this example, detection of background in plasma measurement was performed.
  • background refers to the measurement value (signal) of a specimen that does not contain target extracellular vesicles, and if a large amount of impurities are contained, the noise signal increases, resulting in a high background and making it impossible to perform accurate measurement.
  • Plasma Samples (1-1) 5 mL of blood was collected from one healthy subject who had given informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation). The blood collection tube was centrifuged at 1,500 ⁇ g for 20 minutes at 4° C. to separate blood cells, and the supernatant was collected. The supernatant was centrifuged at 1,500 ⁇ g for 20 minutes at 4° C. to separate remaining blood cells, and the supernatant was collected to obtain healthy subject plasma.
  • EDTA-2K blood collection tube VP-DK050K, manufactured by Terumo Corporation
  • BSA buffer bovine serum albumin
  • 10 ⁇ L of the anti-TM4SF1 antibody-immobilized magnetic particles prepared in Example 1 were mixed in a 2 mL tube and stirred by inversion for 3 minutes.
  • the 2 mL tube was brought close to a magnet and left for 1 minute, the solution was removed, and then 1 mL of BSA buffer was added and stirred by inversion for 10 minutes or more.
  • the 2 mL tube was brought close to a magnet and left for 1 minute, the solution was removed, and then 50 ⁇ L of BSA buffer was added to prepare an anti-TM4SF1 antibody-immobilized magnetic particle suspension.
  • washing buffer PBS containing 0.05% (v/v) Tween 20 (trade name)
  • Example 3 (1-1) Plasma samples were obtained in the same manner as in Example 3 (1), except that the blood collection tubes were each centrifuged at 1,500 ⁇ g for 10 minutes at 4°C to separate blood cells, and the supernatant was collected to obtain plasma from healthy subjects.
  • Comparative Example 2 Detection of background in plasma measurement when pretreatment was performed using mouse IgG-immobilized magnetic particles (1) Preparation of plasma sample Plasma samples were prepared in the same manner as in Comparative Example 1 (1).
  • Example 3(2) Measurement of Background Background in plasma measurement was detected in the same manner as in Example 3(2), except that the plasma sample in Example 3(2) was the pretreated plasma sample obtained in (2-3).
  • Example 4 Detection of background in plasma measurement when pretreatment was performed using anti-CD41 antibody-immobilized magnetic particles
  • background in plasma measurement was detected in the same manner as in Comparative Example 2, except that anti-CD41 antibody-immobilized magnetic particles were used.
  • Example 3 and 4 and Comparative Examples 1 and 2 are shown in Table 1.
  • Example 3 almost all blood cells were removed from the plasma by two centrifugations, while in Comparative Example 1, a large number of blood cells remained in the plasma due to milder conditions and one centrifugation.
  • the remaining blood cells are thought to release extracellular vesicles through freezing and thawing.
  • platelets are known to be activated by physical stimuli such as freezing and thawing, and release extracellular vesicles as a result of activation.
  • platelet-derived extracellular vesicles which are particularly abundant among blood cell-derived extracellular vesicles, were removed by pretreatment using magnetic particles immobilized with antibodies against CD41 expressed on platelets, and it was confirmed whether the background would be reduced.
  • a pretreatment was performed in which a plasma sample was reacted with magnetic particles immobilized with mouse IgG protein as an antibody control, and then background measurement was performed (Comparative Example 2).
  • the amount of luminescence was equivalent to that of Comparative Example 1 in which no pretreatment was performed (1 million), and therefore no reduction in the background due to nonspecific adsorption to the antibody-immobilized magnetic particles was observed.
  • a pretreatment was performed in which anti-CD41 antibody-immobilized magnetic particles were reacted with the plasma sample, and then background measurement was performed (Example 4).
  • the amount of luminescence was reduced to 380,000 compared to Comparative Example 1 and Comparative Example 2. Therefore, it was found that the background was reduced by specifically binding and removing platelet-derived extracellular vesicles contained in the plasma to the anti-CD41 antibody-immobilized magnetic particles.
  • PC-3 cells which are human prostate cancer cells, were selected as diseased cells to be used in the following examples.
  • PC-3 cells were suspended in Ham's F-12K (Fujifilm Wako Pure Chemical Industries, Ltd.) medium containing 15% (v/v) FBS (fetal bovine serum) to a concentration of 1 x 105 cells/mL, and then seeded in five 15 cm dishes at 30 mL each and cultured at 37°C in a 5% CO2 environment .
  • Ham's F-12K Flujifilm Wako Pure Chemical Industries, Ltd.
  • FBS fetal bovine serum
  • PC-3 cells obtained in (3) were cultured for an additional 3 days, and the entire culture supernatant (approximately 125 mL) was then collected.
  • the cells were centrifuged at 300 ⁇ g for 10 minutes at room temperature to remove floating cells, and 120 mL of the supernatant was collected.
  • the supernatant obtained in (4) was diluted to a uniform volume and dispensed into ultracentrifuge tubes, centrifuged at 300,000 x g for 70 minutes at 4°C, and 120 mL of the supernatant was removed.
  • the remaining precipitate was suspended in 4 mL of PBS, and the resulting suspension was centrifuged at 259,000 x g for 70 minutes at 4°C, after which 4 mL of the supernatant was removed. 1 mL of the remaining precipitate was used as the PC-3-derived extracellular vesicle solution.
  • Plasma samples containing PC-3-derived extracellular vesicles at six concentrations shown below in [f] to [k] were prepared by adding 100 ⁇ L of a solution obtained by diluting the PC-3-derived extracellular vesicles obtained in Example 5 with PBS to 250 ⁇ L of plasma from a healthy subject, and the plasma samples were stored frozen at ⁇ 80° C.
  • PC-3-derived extracellular vesicles were detected in the same manner as in Example 3(2), except that the plasma sample obtained in (1) was used as the plasma sample in Example 3(2).
  • Example 6 Detection of PC-3-Derived Extracellular Vesicles in Plasma of Healthy Subjects from Which Platelet-Derived Extracellular Vesicles Have Been Removed (1) Preparation of Plasma Samples Plasma samples were prepared in the same manner as in Comparative Example 3(1).
  • PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Example 3(2), except that the plasma sample was the pretreated plasma sample obtained in (2).
  • Comparative Example 3 (1-2) the healthy subject's plasma was the healthy subject's plasma obtained in (1-1), and a plasma sample was prepared in the same manner as in Comparative Example 3 (1-2), except that the plasma was not frozen and stored at -80°C, and the plasma was immediately subjected to step (2) after preparation.
  • Example 3 (1-3) the healthy subject's plasma was the healthy subject's plasma obtained in (1-1), and a plasma sample not containing PC-3-derived extracellular vesicles was prepared in the same manner as in Comparative Example 3 (1-3), except that the healthy subject's plasma was not frozen and stored at -80°C, and the process was immediately carried out after preparation in step (2).
  • PC-3-derived extracellular vesicles were detected in the same manner as in Example 3(2), except that the plasma sample obtained in (1) was used as the plasma sample in Example 3(2).
  • Comparative Example 3, Example 6, and Reference Example 1 are shown in Table 2 and Figures 1 to 3.
  • the "variation coefficient” is the standard deviation of the luminescence amount divided by the average luminescence amount, and the lower this value is, the smaller the variation in the measured values between samples is, and the higher the measurement accuracy is.
  • the “threshold value” is the value obtained by adding twice the standard deviation of the luminescence amount in a plasma sample that does not contain cell-derived extracellular vesicles to the average luminescence amount.
  • the "lower detection limit concentration” refers to the lowest concentration of added PC-3-derived extracellular vesicles at which the luminescence amount is higher than the threshold value, and the lower the luminescence amount and standard deviation in a plasma sample that does not contain PC-3 cell-derived extracellular vesicles is, the lower the lower detection limit concentration is, and the higher the detection sensitivity is.
  • Example 6 In Comparative Example 3, Example 6, and Reference Example 1, the amount of luminescence increased depending on the concentration of PC-3-derived extracellular vesicles added, confirming that PC-3-derived extracellular vesicles can be detected in plasma.
  • Comparative Example 3 when focusing on the average luminescence intensity of plasma samples not containing PC-3 cell-derived extracellular vesicles, Comparative Example 3 was significantly higher than Example 6 and Reference Example 1. This result, like the result of Comparative Example 1, is considered to be due to an increase in background luminescence intensity caused by platelet-derived extracellular vesicles released by freezing and thawing from platelets remaining in the plasma. Furthermore, in Comparative Example 3, the noise signal amount differed between samples, resulting in a high coefficient of variation (0.50), and therefore a high detection limit concentration (1.0 x 109 particles/mL).
  • the freeze-thawed plasma was pretreated with anti-CD41 antibody-immobilized magnetic particles (Example 6), which reduced the background luminescence and reduced the coefficient of variation (0.23) and the detection limit concentration (2.5 x 108 particles/mL).
  • anti-CD41 antibody-immobilized magnetic particles By removing platelet-derived extracellular vesicles from plasma by pretreatment with anti-CD41 antibody-immobilized magnetic particles, it became possible to detect the target PC-3-derived extracellular vesicles with higher accuracy and sensitivity.
  • the concentration of disease cell-derived extracellular vesicles contained in the plasma of a patient with a disease, including cancer is generally estimated to be 10 8 particles/mL or less
  • the measurement method for detecting the disease cell-derived extracellular vesicles in the plasma must have a detection limit concentration of 10 8 particles/mL or more. Therefore, the method for measuring plasma in a state containing platelet-derived extracellular vesicles as shown in Comparative Example 3 has a detection limit concentration that is significantly higher than the concentration of the disease cell-derived extracellular vesicles, making it difficult to apply to detection of disease cell-derived extracellular vesicles from a plasma sample of a patient with a disease.
  • Example 6 and Reference Example 1 it is suggested that the method for measuring plasma including a step of removing platelet-derived extracellular vesicles or suppressing the generation of platelet-derived extracellular vesicles can be applied to a plasma sample of a patient with a disease.
  • Example 7 Detection of PC-3-derived extracellular vesicles in healthy subject plasma from which platelet-derived extracellular vesicles have been removed PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Example 6 (3), except that 0.5 ⁇ g/mL biotin-modified anti-CD81 antibody was used instead of 0.5 ⁇ g/mL biotin-modified anti-CD9 antibody.
  • Comparative Example 4, Example 7, and Reference Example 2 are shown in Table 3 and Figures 4 to 6.
  • the "variation coefficient” is the standard deviation of the amount of luminescence divided by the average amount of luminescence, and the lower this value is, the smaller the variation in the measured values between samples, and the higher the measurement accuracy.
  • the “threshold” is the value obtained by adding twice the standard deviation of the amount of luminescence in plasma samples that do not contain cell-derived extracellular vesicles to the average amount of luminescence.
  • the “lower detection limit concentration” refers to the lowest concentration of PC-3-derived extracellular vesicles added at which the amount of luminescence is higher than the threshold, and the lower the lower detection limit concentration, the higher the detection sensitivity. Therefore, the detection sensitivity decreases as the amount of luminescence and standard deviation increase in plasma samples that do not contain PC-3 cell-derived extracellular vesicles.
  • Example 7 In Comparative Example 4, Example 7, and Reference Example 2, the amount of luminescence increased depending on the concentration of added PC-3-derived extracellular vesicles, confirming that PC-3-derived extracellular vesicles can be detected in plasma. In addition, in all results, the average amount of luminescence in plasma samples that did not contain PC-3 cell-derived extracellular vesicles was equally low. This is presumably because, while Comparative Example 3, Example 6, and Reference Example 1 used CD9 as a detection marker, which is also expressed in platelets, Comparative Example 4, Example 7, and Reference Example 2 used CD81 as a detection marker, which is not expressed in platelets, suppressing the generation of noise signals.
  • Example 7 When paying attention to the lower detection limit concentration, Example 7 had a lower value compared to Comparative Example 4 and Reference Example 2. This is presumably because, compared to Comparative Example 4 and Reference Example 2, the total amount of extracellular vesicles contained in the plasma was reduced by the process of removing platelet-derived extracellular vesicles in Example 7, and the reactivity between the target extracellular vesicles (PC-3-derived extracellular vesicles) and the anti-TM4SF1 antibody-immobilized magnetic particles was improved, which increased the measurement stability and reduced the variability in the measurement values between samples.
  • the detection method of the present invention can detect target extracellular vesicles from plasma with high sensitivity and high accuracy, suggesting that the method is also applicable to the detection of disease cell-derived extracellular vesicles in plasma samples from disease patients.

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé de détection, avec une sensibilité élevée, d'une vésicule extracellulaire cible dans un échantillon dérivé du sang. Le problème est résolu par l'élimination d'une vésicule extracellulaire dérivée de plaquettes dans un échantillon dérivé du sang ou la suppression de la génération de la vésicule extracellulaire dérivée de plaquettes dans l'échantillon dérivé du sang, avant la détection d'une vésicule extracellulaire cible dans l'échantillon dérivé du sang.
PCT/JP2024/023680 2023-07-05 2024-06-28 Procédé de détection de vésicule extracellulaire Pending WO2025009486A1 (fr)

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JP2018179957A (ja) * 2017-04-17 2018-11-15 日本光電工業株式会社 脂質二重膜粒子またはその断片の検出方法
US20190049438A1 (en) * 2016-02-01 2019-02-14 The Board Of Trustees Of The Leland Stanford Junior University Exosome-Total-Isolation-Chip (ExoTIC) Device for Isolation of Exosome-Based Biomarkers
WO2023004082A1 (fr) * 2021-07-21 2023-01-26 Mercy Bioanalytics, Inc. Compositions et méthodes pour la détection du cancer de la prostate
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US20170146542A1 (en) * 2015-06-09 2017-05-25 The Board Of Regents Of The University Of Texas System Diagnostic test for early stage cancer
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JP2018179957A (ja) * 2017-04-17 2018-11-15 日本光電工業株式会社 脂質二重膜粒子またはその断片の検出方法
WO2023004082A1 (fr) * 2021-07-21 2023-01-26 Mercy Bioanalytics, Inc. Compositions et méthodes pour la détection du cancer de la prostate
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