Method for separating extracellular vesicles, and kit for separating extracellular vesicles

Abstract

The present invention addresses the problem of providing: a method for separating extracellular vesicles; and a kit for separating extracellular vesicles.　Provided is a separation method for separating extracellular vesicles in a sample, the separation method comprising: bringing extracellular vesicles in a sample into contact with a C-type lectin; and separating the extracellular vesicles from bonded products of the extracellular vesicles and the C-type lectin. Also provided is a kit for separating extracellular vesicles from a sample containing the extracellular vesicles, the kit including a C-type lectin.

Description

Method for isolating extracellular vesicles and kit for isolating extracellular vesicles

[0001] The present disclosure relates to a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles.

[0002] Extracellular vesicles (EVs) are spherical membrane vesicles composed of a lipid bilayer membrane approximately 30 to 1000 nm thick. They are secreted extracellularly by all cells. Extracellular vesicles contain biomolecules, such as DNA, RNA (e.g., mRNA, miRNA, ncRNA), proteins, and glycans, which act as signaling molecules and mediate various physiological functions. Detection of encapsulated miRNA in EVs has led to their use in the diagnosis of various diseases, including cancer. Furthermore, mesenchymal stem cell-derived EVs have been used in therapeutic formulations due to their anti-inflammatory, immunosuppressive, and tissue repair properties. Development of methods for using EVs as a delivery system for drugs, including nucleic acid medicines, is also underway. Accordingly, methods for identifying and isolating EVs are also being developed. However, no established method exists at this stage, and many methods have been developed, proposed, and used. Ultracentrifugation is commonly used to isolate EVs. However, this method has the problem that it cannot process a large number of samples at once and is prone to contamination with impurities. Furthermore, there are fine subtypes of extracellular vesicles, and a method for isolating only extracellular vesicles containing the desired active ingredient is required. Recently, a method for isolating extracellular vesicles using magnetic beads immobilized with Tim4, which binds to phosphatidylserine present on the surface of extracellular vesicles, has also been developed. However, even with this method, extracellular vesicles that do not contain active ingredients are also contaminated.

[0003] JP 2021-012200 A, International Publication No. 2016 / 088689 Pamphlet

[0004] An objective of the present disclosure is to provide a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles.

[0005] The present disclosure includes the following aspects. [1-1] A separation method for separating extracellular vesicles in a sample, comprising: contacting extracellular vesicles in the sample with a C-type lectin; and separating the extracellular vesicles bound to the C-type lectin from the C-type lectin. [1-2] The separation method according to [1-1], comprising: contacting extracellular vesicles in the sample with a C-type lectin in the presence of calcium ions; and separating the extracellular vesicles bound to the C-type lectin from the C-type lectin in the absence of calcium ions. [1-3] The separation method according to [1-1] or [1-2], wherein the C-type lectin is ASGR1, DCSIGN, and / or CLEC1B. [1-4] The separation method according to any one of [1-1] to [1-3], wherein the C-type lectin is ASGR1. [2-1] A kit for separating extracellular vesicles in a sample, comprising a C-type lectin. [2-2] A kit for detecting extracellular vesicles, comprising a C-type lectin and an antibody that specifically binds to extracellular vesicles. [2-3] The kit according to [2-1] or [2-2], wherein the C-type lectin is ASGR1, DCSIGN, and / or CLEC1B. [2-4] The kit according to [2-3], wherein the C-type lectin is ASGR1.

[0006] According to the present disclosure, a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles can be provided.

[0007] 1 shows silver staining (A) of extracellular vesicles separated by the TIM4 method, and Western blots using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) in Example 1. The figures show the results of Western blots using anti-CD9 antibody, anti-CD63 antibody, and anti-81 antibody for the separation of extracellular vesicles using human C-type lectin in Example 2. The figures show silver staining (A) of extracellular vesicles separated by human C-type lectin, and Western blots using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) in Example 3. The figures show the protein concentration, particle number per ml, and particle size (mean ± standard error) of extracellular vesicles separated from iPS cell culture supernatant using three types of C-type lectins (ASGR1, DCSIGN, CLEC1B), ultracentrifugation, and the TIM4 method in Example 4. Figure 5 shows silver staining (A) of extracellular vesicles isolated from iPS cell culture supernatant using C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method, as well as Western blot analysis using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) in Example 5. The right panel shows the protein concentration, particle number, particle number per ml, and particle diameter (mean ± standard error) of extracellular vesicles obtained by each separation method. Figure 6 shows silver staining (A) of extracellular vesicles isolated from HEK293T cell culture supernatant using C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method, as well as Western blot analysis using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D). The right panel shows the protein concentration, particle number, particle number per ml, and particle diameter (mean ± standard error) of extracellular vesicles obtained by each separation method. FIG. 10 is a diagram showing the results of miRNA expression analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 7. FIG. 11 is a diagram showing the results of heat map analysis of miRNA expression levels of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 8. FIG. 12 is a diagram showing the results of glycan microarray analysis of ASGR1 in Example 9.This figure shows a list of glycans used in the glycan array in Example 9. This figure shows graphs illustrating the results of flow cytometry analysis of extracellular vesicles using ASGR1-immobilized beads in Example 10. The vertical axis indicates mean fluorescence intensity. "Beads" refers to a group in which beads on which ASGR1 was not immobilized were reacted with cell culture supernatant and then reacted with an anti-CD63 antibody. "Isotype" refers to a group in which ASGR1-immobilized beads were reacted with cell culture supernatant and then reacted with a control antibody. "CD63" refers to a group in which ASGR1-immobilized beads were reacted with cell culture supernatant and then reacted with an anti-CD63 antibody. This figure shows transmission electron micrographs of extracellular vesicles isolated from HEK293T cell culture supernatant using C-type lectin (ASGR1) (M-R), ultracentrifugation (UC) (A-F), and the TIM4 method (G-L) in Example 11. The arrows indicate vesicles with lipid bilayer membranes. Scale bar: 500 nm (A, B, G, H, M, N), 100 nm (C-F, 1-L, O-R). This figure shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method (analysis of the types of proteins in extracellular vesicles separated by each separation method) in Example 12. This figure shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method (analysis of the proportion (%) of proteins associated with each cellular component in extracellular vesicles separated by each separation method) in Example 12. 1 shows the results of proteomic analysis (heat map analysis of the expression levels of exosome markers in extracellular vesicles separated by each separation method) of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 12. FIG. 1 shows graphs showing the results of flow cytometry analysis of extracellular vesicles separated from HEK293T cell culture supernatant by TIM4-immobilized beads (A) or ASGR1 immobilization (B) using buffer, a control antibody (isotype), and an exosome marker (CD9, CD63, or CD81) antibody in Example 13.10 is a graph showing the results of flow cytometry analysis using buffer, a control antibody (isotype), and an exosome marker (CD9, CD63, or CD81) antibody of extracellular vesicles separated from 201B7 iPS cell culture supernatant using TIM4-immobilized beads (A) or ASGR1-immobilized beads (B) in Example 14. In Example 15, a graph showing the results of ELISA analysis in which culture supernatants of HEK293T cells (A), 201B7 iPS cells (B), adipose-derived mesenchymal stem cells (C), or bone marrow-derived mesenchymal stem cells (D) were reacted on an ASGR1-Fc-immobilized plate, followed by detection with a CD63 antibody.

[0008] An embodiment of the present disclosure will be described in detail below. However, the scope of the present disclosure is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the present disclosure. Each aspect disclosed in this specification can be combined with any other feature disclosed in this specification. Furthermore, when multiple upper and lower limit values ​​are described for a specific parameter, any of these upper and lower limit values ​​can be combined to form a suitable numerical range. Furthermore, the lower and / or upper limit values ​​of a numerical range described in this disclosure are numerical values ​​within that numerical range and may be replaced with numerical values ​​shown in the examples. The expression "X to Y" indicating a numerical range means "X or more and Y or less." If a specific description described for one embodiment also applies to other embodiments, that description may be omitted in other embodiments. Unless otherwise specified in the embodiments and examples, methods described in standard protocol collections such as J. Sambrook, E. F. Fritsch & T. Maniatis (Eds.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified or altered methods thereof, are used. Furthermore, when commercially available reagent kits or measuring devices are used, the protocols attached thereto are used unless otherwise specified.

[0009] ==First embodiment (separation method)== The separation method according to this embodiment is a method for separating extracellular vesicles from a sample containing extracellular vesicles, using a C-type lectin. In one embodiment, the separation method is a method for separating extracellular vesicles in a sample from other components in the sample.

[0010] (Extracellular vesicles) As used herein, extracellular vesicles include vesicles that are variously classified according to their origin, the size of small membrane vesicles, etc. Specifically, as used herein, extracellular vesicles include exosomes, microvesicles, apoptotic vesicles, adiposomes, etc.

[0011] Exosomes are small membrane vesicles derived from late endosomes, composed of a lipid bilayer and bearing phosphatidylserine on their surface. The diameter of these vesicles is typically about 50 to 250 nm. Exosomes are known to contain tetraspanins such as CD9, CD63, and CD81, as well as proteins such as Alix, TSG101, Lamp-1, and Flotillin (Thery et al., "Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines," Journal of Extracellular Vesicles, 2018, Vol. 7, 1535750, https: / / doi.org / 10.1080 / 20013078.2018.1535750). It is believed that there are further subclasses of exosomes with different production mechanisms and functions. Microvesicles are small membrane vesicles derived from plasma membranes, composed of a lipid bilayer membrane and bearing phosphatidylserine on their surface. Microvesicles are typically about 100 nm to about 1000 nm in size. Microvesicles are known to contain proteins such as integrins, selectins, and CD40 ligands. Apoptotic vesicles are small membrane vesicles derived from apoptotic cells, composed of a lipid bilayer membrane and bearing phosphatidylserine on their surface. Apoptotic vesicles are typically about 50 nm to about 500 nm in size. Apoptotic vesicles are known to contain histones. Adiposomes are small membrane vesicles derived from adipocytes, composed of a lipid bilayer membrane and bearing phosphatidylserine on their surface. Adiposomes are typically about 100 nm to about 1000 nm in size. Adiposomes are known to contain MFG-E8 (milk fat globule-EGF factor 8).

[0012] Both naturally occurring extracellular vesicles and artificially synthesized extracellular vesicles are targets for separation by the separation method according to this embodiment. Furthermore, the extracellular vesicles contained in the sample may contain extracellular vesicles other than those to be separated, and the extracellular vesicles other than those to be separated may be naturally occurring extracellular vesicles or artificially synthesized extracellular vesicles.

[0013] When the extracellular vesicles are naturally derived, the cells from which the extracellular vesicles are derived are not limited to the extent that they secrete the extracellular vesicles, and may be plant cells, animal cells, or microbial cells, and are not limited by the species of origin, developmental origin, tissue or organ of origin, cellular function, etc. Plant cells may be cells derived from either seed plants or non-seed plants, and may be cells derived from any organ such as stems, leaves, flowers, or roots. Microbial cells may be cells of either prokaryotes or eukaryotes, and more specific, non-limiting examples include Escherichia coli, Staphylococcus, halophiles, acidophiles, thermophiles, yeast, fungi, and bacteria. Animal cells may be cells derived from, for example, mammals, birds, reptiles, amphibians, fish, etc., and may be endodermal, ectodermal, mesodermal, or stem cells, or may be derived from epithelial tissue, connective tissue, muscle tissue, or nervous tissue. Non-limiting examples of cells include blood cells, muscle cells, skin cells, nerve cells, glandular cells, and the like. These cells may be primary cells collected from an individual organism or tissue, or may be cultured cells. More specific, non-limiting examples of cells from which extracellular vesicles are derived include various stem cells such as mesenchymal stem cells (MSCs) (including adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells), induced pluripotent stem cells (iPS cells), and embryonic stem cells (ES cells), cell lines (HEK293 cells, CHO cells, HeLa cells, MCF-7 cells, COS cells, PC12 cells, and the like), and primary cultured cells (skin fibroblasts, endothelial cells, muscle cells, hematopoietic cells, and the like). In one embodiment, the cells from which extracellular vesicles are derived may be human-derived stem cells, cell lines established from human cells, or primary cultured human cells.

[0014] Naturally occurring extracellular vesicles also include extracellular vesicles in which the amount of sugar chains with low specificity for C-type lectins is reduced or absent, or the proportion of sugar chains with high specificity for C-type lectins is increased, compared to naturally occurring extracellular vesicles, by treating extracellular vesicles isolated from plant cells, animal cells, microbial cells, etc. with an enzyme that acts on sugar chains. Non-limiting examples of enzyme treatment include sialidase and galactosidase.

[0015] Artificially synthesized extracellular vesicles can be obtained by culturing cultured plant cells, animal cells, or microbial cells that secrete extracellular vesicles in an appropriate medium under appropriate conditions and secreting the extracellular vesicles into the medium. Artificially synthesized extracellular vesicles may be obtained, for example, by knocking out a specific gene involved in glycan synthesis in cultured plant cells, animal cells, or microbial cells, thereby reducing or eliminating the amount of glycans with low specificity for C-type lectins in the secreted extracellular vesicles, or increasing the proportion of glycans with high specificity for C-type lectins, compared to non-genetically engineered cells. Non-limiting examples of genetic engineering include knockout of core 1 glycan synthesis enzyme (C1GALT1) and knockout of Cosmc, the molecular chaperone for C1GALT1.

[0016] In one embodiment, at least a portion of the extracellular vesicles contained in a sample may be the extracellular vesicles to be separated, in which case the extracellular vesicles to be separated have sugar chains that have binding ability to C-type lectin, and the other extracellular vesicles do not have sugar chains that have binding ability to C-type lectin. Therefore, the separation method according to this embodiment includes a step of contacting extracellular vesicles in a sample with C-type lectin and a step of separating the extracellular vesicles bound to C-type lectin from the C-type lectin, and can separate extracellular vesicles that have binding ability to C-type lectin.

[0017] In one embodiment, the extracellular vesicles to be separated are at least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles. In one embodiment, the extracellular vesicles to be separated are at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles. At least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles express glycans on their surface, as described below in "C-type lectins," and are therefore easily separated from a sample containing extracellular vesicles by the separation method using C-type lectins according to this embodiment.

[0018] (Sample) In this embodiment, extracellular vesicles are separated from a sample containing extracellular vesicles using a C-type lectin. The sample is not limited to samples containing extracellular vesicles, and may be a biological sample collected from a living organism or an experimental sample. Biological samples include, for example, body fluid samples, solid samples, and semi-solid samples containing extracellular vesicles. Here, body fluids include body fluids in a broad sense, such as blood (plasma, serum, whole blood), digestive fluids such as gastric juice, bile, pancreatic juice, and intestinal juice, tissue fluids such as interstitial fluid, intercellular fluid, and interstitial fluid, body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), synovial fluid, aqueous humor (aqueous humor), lymph, amniotic fluid, saliva, sputum, urine, semen, sweat, tears, nasal discharge, vaginal fluid, and milk. Solid samples include, for example, tissue fragments and feces collected from individual organisms. Semi-solid samples include vomit and the like. Other examples of biological samples include plant or fruit extracts, cultured cell cultures, and microbial cultures. Cultures include cultures containing cells or microorganisms, and their supernatants. The microbial cultures may be, for example, yeast cultures, and more specifically, mash (moromi) produced in the sake production process, or sake products containing at least some components of mash or extracellular vesicles derived from mash.

[0019] The biological species from which the biological sample is derived is not limited as long as it contains extracellular vesicles, and may be animal, plant, or microbial cells. The biological species is the same as the biological species from which the extracellular vesicles are derived, as described in the "Extracellular Vesicles" section. If the sample is a biological sample, it may be stored according to methods known to those skilled in the art after collection from the subject until the isolation method is performed. Non-limiting examples of experimental samples include culture supernatant containing extracellular vesicles, physiological saline containing extracellular vesicles, buffer containing extracellular vesicles, etc. These extracellular vesicles may also be artificially synthesized extracellular vesicles, as described in the "Extracellular Vesicles" section above.

[0020] (C-type lectin) Lectin is generally a general term for proteins or glycoproteins present in plants, animals, microorganisms, etc. that have specific binding activity, mainly for glycans. C-type lectins are one of the structurally defined major lectin families, and in a broad sense include lectins that bind to glycans and ligands other than glycans, regardless of whether they are calcium-dependent or not. In consideration of the binding ability to the extracellular vesicles to be separated, the C-type lectin in this embodiment is preferably a lectin with calcium-dependent glycan-binding activity, and more preferably a C-type lectin receptor (transmembrane receptor). The C-type lectin receptor is not limited by its structure and may be any C-type lectin receptor classified into, for example, Group 2, Group 4, Group 5, Group 6, Group 14, etc. As long as it has binding affinity for extracellular vesicles, the C-type lectin receptor is not limited by other functions or the level of binding ability to extracellular vesicles.

[0021] The binding ability between a C-type lectin receptor and an extracellular vesicle means the binding ability between a C-type lectin receptor and a sugar chain expressed on the surface of the extracellular vesicle. The sugar chain is not limited to a range in which it is expressed on the surface of the extracellular vesicle in a form capable of binding to a C-type lectin, and is not limited by its expression level.

[0022] In one embodiment, the sugar chain is a sugar chain having αGalNAc or βGalNAc at the non-reducing end. Non-limiting examples of sugar chains having αGalNAc or βGalNAc at the non-reducing end include type A sugar chain (GalNAcα1-3Galβ1-4GlcNAcβ1-), Core6 (GlcNAcβ1-6GalNAcα1-), di-GalNAcβ (GalNAcβ1-3GalNAcβ1-), LDN (GalNAcβ1-4GlcNAcβ1-), Forssman (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-), Forssman Examples of such sugar chains include dissaccharide (GalNAcα1-3GalNAcβ1-), βGalNAc (GalNAcβ1-), Tn (GalNAcα1-), A-di (GalNAcα1-3Galβ1-), GA2 (GalNAcβ1-4Galβ1-4Glcβ1-), GM2 (GalNAcβ1-4(Siaa2-3)Galβ1-4Glcβ1-), and GD2 (GalNAcβ1-4(Siaa2-8Siaα2-3)Galβ1-4Glcβ1-Cer). In one embodiment, the sugar chain is αGal (Galα1-). In one embodiment, the sugar chain may exist as a glycoprotein, and examples of such glycoproteins include BSM (bovine submaximal mucin), asialo-BSM, etc. In another embodiment, the sugar chain may be LeX (Galβ1-4(Fucα1-3)GlcNAc), LeA (Galβ1-3(Fucα1-4)GlcNAc), a high-mannose sugar chain, an O-type sugar chain (di-sialyl T(Siaα2-3Galβ1-3(Siaα2-6)GalNAc), etc.), etc.

[0023] That is, considering the binding affinity of C-type lectins to extracellular vesicles, C-type lectin receptors are A-type glycans (GalNAcα1-3Galβ1-4GlcNAcβ1-), Core6 (GlcNAcβ1-6GalNAcα1-), Tn (GalNAcα1-), di-GalNAcβ (GalNAcβ1-3GalNAcβ1-), LDN (GalNAcβ1-4GlcNAcβ1-), Forssman (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-), βGalNAc (GalNAcβ1-), Tn (GalNAcα1-, A-di (GalNAcα1-3Galβ1-), BSM (sialylated Tn), αGal (Galα1-), BSM (Bovine It is preferable that the C-type lectin receptor is a C-type lectin receptor having binding ability to one or more of the sugar chains selected from the group consisting of A-type sugar chains (GalNAcα1-3Galβ1-4GlcNAcβ1-), Core6 (GlcNAcβ1-6GalNAcα1-), Tn (GalNAcα1-), di-GalNAcβ (GalNAcβ1-3GalNAcβ1-), L The C-type lectin receptor may be a C-type lectin receptor having binding affinity to one or more sugar chains selected from the group consisting of DN(GalNAcβ1-4GlcNAcβ1-), Forssman (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-), βGalNAc(GalNAcβ1-), Tn(GalNAcα1-, A-di(GalNAcα1-3Galβ1-), and / or one or more glycoproteins selected from the group consisting of BSM (sialylated Tn), αGal(Galα1-), BSM (Bovine submaximal mucin), and asialo-BSM. The presence or absence of binding affinity between the C-type lectin receptor and these sugar chains can be evaluated by methods well known to those skilled in the art, for example, by sugar chain arrays.Considering the binding affinity to the extracellular vesicles to be isolated, the C-type lectin receptors were ASGR1 (Asialoglycoprotein receptor 1 (UniProt No. P07306)), DCSIGN (CD209 antigen (UniProt No. Q9NNX6)), or CLEC1B (C-type lectin domain family 1 member B (UniProt No. Q9NNX6)). No. Q9P126), more preferably ASGR1. When the C-type lectin receptor is ASGR1, DCSIGN, and / or CLEC1B, the separation method of this embodiment makes it easier to separate CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles. When the C-type lectin receptor is ASGR1, it makes it easier to separate CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles with higher purity.

[0024] In terms of the types of extracellular vesicles that can be separated, the use of C-type lectin receptors has the advantage of making it easier to separate types of extracellular vesicles that are different from those obtainable by conventional methods (e.g., the TIM4 method, ultracentrifugation, etc.). Specifically, the use of C-type lectin receptors makes it easier to separate extracellular vesicles that encapsulate and / or express genes (e.g., miRNA) and / or proteins that are different from those obtained by conventional methods. In terms of the types of extracellular vesicles that can be separated, the C-type lectin receptor is preferably ASGR1, DCSIGN, and / or CLEC1B, and more preferably ASGR1. The ability to separate different types of extracellular vesicles enables analysis and use for disease diagnosis and / or treatment.

[0025] C-type lectins are not limited by their preparation method and may be naturally occurring lectins or artificially synthesized lectins. Naturally occurring lectins may be isolated from biological tissues or cells of plants, animals, microorganisms, etc., according to methods well known to those skilled in the art. Artificially synthesized lectins can be prepared by expressing recombinant lectins in mammalian cells, plant cells, yeast, or Escherichia coli using methods well known to those skilled in the art. They can also be prepared using an in vitro translation system. C-type lectins may be commercially available lectins provided by reagent companies such as Acro Biosystems, R&D Systems, Sigma-Aldrich, Takara Bio, and Fujifilm Wako Pure Chemical Industries. C-type lectins also include proteins or peptides other than full-length proteins, as long as they maintain their binding ability to the target glycans. For example, they may be structures in which a portion of the protein is deleted, structures in which an arbitrary peptide is added to a full-length protein, extracellular domains, proteins or peptides containing extracellular domains, sugar-binding domains, proteins or peptides containing sugar-binding domains, etc.

[0026] The C-type lectin may be immobilized on any carrier suitable for the separation method of this embodiment. The carrier can be appropriately selected from carriers known to those skilled in the art and may be in the form of a microplate, a tube, a disk-shaped piece, particles (beads), or the like. When the carrier is in the form of particles, the carrier may be packed into a purification or separation column. The carrier may be a porous material having through-holes, such as a sponge monolith. The size of the through-holes is not limited and may be about 1 nm to about 100 μm. Using a carrier made of a material with a large surface area, such as a porous material, tends to improve the separation efficiency in the separation method. The material of the carrier can also be appropriately selected from substances known to those skilled in the art, and may include polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, glass, silicon oxide, diatom, porous glass, ground glass, alumina, silica gel, metal oxide, iron, cobalt, nickel, magnetite, chromite, etc. For example, in the case of particles (beads), the particle size can be appropriately selected by those skilled in the art depending on the intended use, and may be, for example, about 10 nm to about 100 μm, about 100 nm to about 10 μm, or about 1 μm to about 5 μm. The method for immobilizing C-type lectins to carriers can be appropriately selected from methods known to those skilled in the art, and may include, for example, binding by affinity binding, chemical binding, or physical adsorption. More specifically, for example, the Fc region of an antibody may be attached to a lectin, and the lectin may be bound to beads to which protein G has been attached by utilizing the binding force between the Fc region and protein G. Alternatively, biotinylated lectin may be immobilized on streptavidin-immobilized beads, or lectin may be immobilized on beads activated with N-hydroxysuccinimide (NHS) or epoxy groups by amine coupling.

[0027] (Separation method) In one embodiment, the separation method is a separation method for separating extracellular vesicles in a sample, comprising the steps of contacting extracellular vesicles in a sample with a C-type lectin and separating the extracellular vesicles bound to the C-type lectin from the C-type lectin.

[0028] As explained above in the "Sample" section, the sample containing extracellular vesicles may be a biological sample collected from a living organism or an experimental sample. Regardless of the sample type, the sample may be pretreated prior to the separation method according to this embodiment. Pretreatment may involve, for example, concentrating extracellular vesicles by removing unwanted substances from the sample. More specifically, substances in the sample can be selected based on their size or properties and unwanted substances can be removed by ultrafiltration, tangential flow filtration, ultracentrifugation, ion exchange separation, or the like.

[0029] Contact between extracellular vesicles and C-type lectin in a sample can be achieved by, for example, mixing the C-type lectin with a sample containing extracellular vesicles. For example, when C-type lectin is immobilized on a carrier such as a microplate, a tube, or a disk-shaped piece, mixing can be performed by dropping a liquid sample containing extracellular vesicles onto the side where the C-type lectin is exposed. C-type lectin immobilized on a particulate carrier such as beads and a sample containing extracellular vesicles can be mixed in a single solution. Alternatively, non-immobilized C-type lectin and a sample containing extracellular vesicles can be mixed in a single solution. In this way, at least a portion of the mixed C-type lectin and at least a portion of the extracellular vesicles bind to each other due to their binding properties to form a complex.

[0030] The solution used in the step of contacting extracellular vesicles in a sample with a C-type lectin is not limited as long as it can dissolve or suspend the extracellular vesicles and C-type lectin in a stable state and does not interfere with the contact and / or binding of the C-type lectin to the extracellular vesicles. Examples include water or a buffer solution with a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.). In one embodiment, the step of contacting extracellular vesicles in a sample with a C-type lectin is preferably carried out in the presence of calcium ions. The C-type lectin in this embodiment is preferably a lectin with calcium-dependent glycan-binding activity, and in this case, the presence of calcium ions improves its binding ability to extracellular vesicles. When calcium ions are present, it is preferable to use a buffer solution that does not bind to calcium to form precipitates. The source of calcium ions is not limited and may be, for example, calcium chloride, calcium hydroxide, calcium bicarbonate, calcium iodide, calcium bromide, or calcium acetate. Calcium chloride, calcium bicarbonate, or calcium iodide is preferred, and calcium chloride or calcium bicarbonate is more preferred. The calcium ion concentration can be adjusted by adding one or more of these ionic compounds before or at the time of contacting extracellular vesicles in a sample with a C-type lectin. The final concentration is about 0.5 to about 100 mM, preferably about 0.5 to about 10 mM, more preferably about 0.5 to about 5.0 mM, and even more preferably about 0.5 to about 2.0 mM. When the calcium ion concentration is about 0.5 to about 2.0 mM, it may be about 1.0 mM. The step of contacting extracellular vesicles in a sample with C-type lectin can be carried out by incubating at about 10°C or lower, preferably at about 1 to about 6°C, more preferably at about 4°C, for about 0.5 to about 24 hours, preferably about 3 to about 20 hours, more preferably about 6 to 12 hours.

[0031] In one embodiment, when a non-immobilized C-type lectin and a sample containing extracellular vesicles are mixed in a single solution, a carrier such as beads that specifically binds to the C-type lectin may be introduced into the complex formed by binding the C-type lectin to the extracellular vesicles, thereby forming a complex of the C-type lectin, the carrier, and the extracellular vesicles.

[0032] The separation of extracellular vesicles bound to the C-type lectin from the C-type lectin can be carried out by incubating the complex of the C-type lectin and the extracellular vesicles in an elution solution, which separates at least a portion of the extracellular vesicles bound to the C-type lectin from the C-type lectin.

[0033] The solution used in this step is not limited as long as it can dissolve or suspend extracellular vesicles and C-type lectin in a stable state and does not interfere with the separation of the C-type lectin from the extracellular vesicles, and examples thereof include water or a buffer solution (e.g., Tris buffer, Hepes buffer, etc.) with a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6. In one embodiment, the eluate is preferably free of calcium ions. The C-type lectin according to this embodiment is preferably a lectin with calcium-dependent glycan-binding activity, and the absence of calcium ions facilitates the separation of extracellular vesicles from the C-type lectin. In one embodiment, the eluate may contain a glycan recognized by the C-type lectin. Use of such an eluate tends to improve the efficiency of separation of extracellular vesicles from the C-type lectin. Use of an eluate that is free of calcium and contains a glycan recognized by the C-type lectin further facilitates the separation of extracellular vesicles from the C-type lectin. Such sugar chains may be any sugar chains that are recognized by the C-type lectin used, depending on the type of C-type lectin used. For example, when the C-type lectin is ASGR1, DCSIGN, or CLEC1B, the sugar chain is preferably a sugar chain having αGalNAc or βGalNAc, αMan, αFuc, or di-sialyl T (Siaα2-3Galβ1-3(Siaα2-6)GalNAc) at the non-reducing end. Examples of αGalNAc and βGalNAc are as described above in "C-type lectins."

[0034] In one embodiment, the separation method according to this embodiment may include a step of washing the complex to remove undesired substances before the step of separating the extracellular vesicles bound to the C-type lectin from the C-type lectin in the eluate. Washing is preferably performed about once to about three times, and may be performed twice. The washing solution is not limited as long as it can stably dissolve or suspend the extracellular vesicles and the C-type lectin and does not interfere with the separation of the C-type lectin from the extracellular vesicles, and examples thereof include water or a buffer solution having a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.). For example, when the C-type lectin is immobilized on a carrier such as a microplate, a tube, or a disk-shaped piece, the surface of the carrier on which the complex of the C-type lectin and the extracellular vesicles is present can be washed by dropping a washing solution on it. For example, when C-type lectin is immobilized on beads, the complex of bead-immobilized C-type lectin and extracellular vesicles can be washed by adding a washing solution to the complex, agglutinating or precipitating the complex using the beads, and then removing the supernatant. For example, when the beads are magnetic beads, the complex containing the beads can be agglutinated or precipitated by magnetic force.

[0035] The separation method of this embodiment can separate extracellular vesicles that have binding affinity to C-type lectins. In one embodiment, when ASGR1, DCSIGN, or CLEC1B is used as the C-type lectin, a population of extracellular vesicles containing at least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles can be separated. In one embodiment, when ASGR1, DCSIGN, or CLEC1B is used as the C-type lectin, a population of extracellular vesicles containing at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles can be separated. In one embodiment, the extracellular vesicles separated by the separation method of this embodiment may have an average particle diameter of about 50 nm to about 250 nm, about 100 nm to about 250 nm, about 150 nm to about 250 nm, or about 200 nm to about 250 nm.

[0036] For example, when the C-type lectin is immobilized on a carrier, the extracellular vesicles to be separated from the C-type lectin can be isolated from the sample by obtaining only the solution containing the free extracellular vesicles.

[0037] The separation method according to this embodiment can be used in a method for purifying extracellular vesicles by further isolating the extracellular vesicles separated from other components in a sample by the separation method as described above. The separated extracellular vesicles can also be used in a method for detecting extracellular vesicles by labeling them with a label (e.g., a specific antibody) that binds to a marker specific to the extracellular vesicles.

[0038] In one embodiment, the population of extracellular vesicles separated by the separation method contains at least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles at a higher density than the sample containing extracellular vesicles before being subjected to the separation method. In one embodiment, the population of extracellular vesicles separated by the separation method contains at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles at a higher density than the sample containing extracellular vesicles before being subjected to the separation method. In one embodiment, the separation method according to this embodiment can enrich for at least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles, or at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles.

[0039] Furthermore, the separation method according to this embodiment can be used to diagnose symptoms, diseases, etc. of a subject based on analysis of bioinformation molecules contained in the extracellular vesicles, using extracellular vesicles purified by a method utilizing this method. That is, in one embodiment, a diagnostic method includes the separation method according to this embodiment, and preferably includes a step of analyzing information molecules in the separated extracellular vesicles. Examples of symptoms or diseases of a subject include, but are not limited to, cancer, neurodegenerative diseases, psychiatric disorders, and chronic diseases (lifestyle-related diseases).

[0040] Second Embodiment (Kit) A kit according to this embodiment is a kit for separating extracellular vesicles from a sample containing extracellular vesicles, comprising a C-type lectin. In one embodiment, the kit is a kit for separating extracellular vesicles in a sample from other components in the sample.

[0041] In this embodiment, examples and preferred examples of "extracellular vesicles," "C-type lectins," and "samples" are as described in the first embodiment.

[0042] The kit according to this embodiment can be used to carry out any of the separation methods described in the first embodiment.

[0043] In one embodiment, the kit may contain, in addition to the C-type lectin, one or more reagents necessary for each step of the separation method described in the first embodiment, i.e., the "step of contacting extracellular vesicles in a sample with the C-type lectin" and / or the "step of separating extracellular vesicles bound to the C-type lectin from the C-type lectin." The reagents are, for example, a buffer solution used in these steps, an ionic compound containing calcium ions, a sugar chain used in the elution solution, and / or a washing solution. In the kit, the C-type lectin may be immobilized on a carrier as described in the first embodiment; however, if the C-type lectin is not immobilized, the kit may contain a carrier for immobilizing the C-type lectin and one or more reagents for immobilizing the C-type lectin on the carrier.

[0044] In one embodiment, the kit can be used as a kit for purifying extracellular vesicles by further isolating extracellular vesicles separated from other components in a sample by a separation method. Alternatively, the kit can be used as a kit for detecting extracellular vesicles by labeling the separated extracellular vesicles with a label (e.g., a specific antibody) that binds to a marker specific to the extracellular vesicles. In this case, the kit may include a means for labeling the marker specific to the extracellular vesicles. Such a means may be an antibody that specifically binds to a marker specifically expressed on extracellular vesicles. The antibody is preferably an anti-CD9 antibody, an anti-CD63 antibody, and / or an anti-CD81 antibody. In one embodiment, the kit includes a C-type lectin and an antibody that specifically binds to extracellular vesicles. The antibody that specifically binds to a marker specifically expressed on extracellular vesicles may be modified for detection. The modification can be appropriately selected from techniques known to those skilled in the art, and may be, for example, biotin, a fluorescent substance, an enzyme, or the like.

[0045] The kit for detecting extracellular vesicles may be, for example, a kit for EIA (Enzyme Immunoassay) including chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmunoassay), Western blotting, latex agglutination, immunochromatography, sandwich method, flow cytometry, or the like.

[0046] The kit according to this embodiment and various kits using the same contain the C-type lectin described above, and further include one or more of the reagents described above, a means for labeling a marker specific to extracellular vesicles, positive / negative controls, and / or instructions for use of the kit.

[0047] The kit for isolating extracellular vesicles in a sample according to this embodiment, and various kits using the kit, can be used as a kit for diagnosing a symptom or disease in a subject based on the analysis of bioinformation molecules contained in the extracellular vesicles using the extracellular vesicles purified by the kit. Furthermore, the kit for detecting extracellular vesicles can be used to quantify the amount of extracellular vesicles in a sample, and the subject's symptom or disease can be diagnosed based on the results. That is, the kit for detecting extracellular vesicles according to this embodiment can be used as a kit for diagnosing a symptom or disease in a subject based on the results of the detection or quantification. Examples of the symptom or disease in a subject include, but are not limited to, cancer, neurodegenerative disease, mental illness, and chronic disease (lifestyle-related disease).

[0048] The present invention will be explained in more detail below by showing examples, but interpretation of the present disclosure is not limited to these examples.

[0049] Throughout the following examples, the antibodies used against CD9, CD63, and CD81 were Exosome CD9 Antibody (Ts9) (Invitrogen, 10626D), Anti-CD63 Human (Mouse) Unlabeled, 8A12 (Cosmo Bio Co., Ltd., SHI-EXO-MO2), and Anti-CD81 Human (Mouse) Unlabeled, 12C4 (Cosmo Bio Co., Ltd.: SHI-EXO-MO3). Unless otherwise specified, each analysis was performed according to the method described in the previous examples.

[0050] Example 1: Separation of Extracellular Vesicles by the TIM4 Method 1 μg of galactose-binding lectin rLSLN (a recombinant form of the N-terminal domain of Laetiporus sulphureus-derived lectin) was mixed with 10 μl of Dynabeads M280 Streptavidin (Veritas) and allowed to bind at 4°C for 30 minutes to prepare rLSLN lectin-immobilized beads. 50 ml of 201B7 iPS cell culture supernatant was concentrated to 1 ml using a 100 kDa cut Amicon Ultra (Merck). The concentrate was then mixed with the rLSLN-immobilized beads and allowed to react overnight at 4°C. After washing four times with 1 ml of PBS, the beads were eluted with 0.2 M lactose solution. To use as a positive control, extracellular vesicles were isolated using the MagCapture™ Exosome Isolation Kit PS (TIM4 method, Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The resulting extracellular vesicles were subjected to protein quantification using a micro BCA assay (PIERCE, Inc.), and particle size and particle count were measured using a NanoSight (Japan Quantum Design Co., Ltd.). Furthermore, 7.4 × 10 of each sample was analyzed. ７ After electrophoresis, the samples were transferred to a PVDF membrane and Western blotted with antibodies against CD9, CD63, and CD81. The results of the Western blot are shown in Figure 1. Extracellular vesicles isolated by the TIM4 method were positive for CD9, CD63, and CD81. Therefore, a population of extracellular vesicles containing at least CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles was isolated. On the other hand, extracellular vesicles isolated by the rLSLN-immobilized beads were negative for CD9, CD63, and CD81, and CD9-positive, CD63-positive, or CD81-positive extracellular vesicles could not be isolated.

[0051] Example 2: Separation of extracellular vesicles using human C-type lectin The nucleotide sequence encoding the extracellular domain of human C-type lectin (Table 1, SEQ ID NOS: 1 to 23) was ligated into the XhoI / EcoRV restriction enzyme sites of the pSecTag / FRT / V5-His TOPO vector (ThermoFisher). An expression vector was then constructed by ligating the human IgG1 Fc region into the AgeI / PmeI restriction enzyme sites. The constructed expression vector was transfected into HEK293T cells using Lipofectamine LTX Reagent with PLUS Reagent (ThermoFisher), and the cells were incubated for 24 hours at 4°C. ２ The cells were cultured in an incubator, and the culture supernatant was collected. Human C-type lectin-Fc fusion proteins were purified from the collected culture supernatant using rProtein A Sepharose Fast Flow (GE Healthcare Japan). The purity of the purified proteins was confirmed by electrophoresis and Western blotting, and the protein amount was quantified using the Pierce (registered trademark) BCA Protein Assay Kit (ThermoFisher). A total of 23 types of human C-type lectin-Fc fusion proteins were produced. TIM4 and the other 23 types of human C-type lectins used in this example are shown in Figure 2.

[0052] C-type lectin-immobilized beads were prepared by binding 12 μg of C-type lectin-Fc fusion protein to 50 μl of Dynabeads Protein G (Veritas) at 4°C for 30 minutes. 50 ml of culture supernatant from 201B7 iPS cells cultured in mTeSR PLUS (Veritas) was collected and concentrated to 1 ml using a 100 kDa cut Amicon Ultra (Merck). CaCl ２ The mixture was added to a final concentration of 1 mM, mixed with the C-type lectin-immobilized beads, and incubated overnight at 4°C while mixing. After spinning down in a tabletop centrifuge, the beads were placed on a magnetic stand and the culture supernatant was removed. Then, 1 ml of washing solution (10 mM Tris, 137 mM NaCl, pH 7.4, 1 mM CaCl ２, 0.02% Tween 20) was added, vortexed, and then reacted with a magnetic stand, and the supernatant was removed. This washing procedure was repeated twice, and the supernatant was then removed. The sample was then eluted by incubation with 50 μl of elution solution (10 mM Tris, 137 mM NaCl, pH 7.4, with 1 mM EDTA) at room temperature for 10 minutes. This elution procedure was repeated twice to prepare 100 μl of a solution containing extracellular vesicles.

[0053] For use as a positive control, extracellular vesicles were isolated using the MagCapture™ Exosome Isolation Kit PS (TIM4 method, Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions.

[0054] The obtained extracellular vesicles were subjected to protein quantification using a micro BCA assay (PIERCE), and particle size and particle number were measured using a NanoSight (Japan Quantum Design Co., Ltd.). 36 μl of each sample was electrophoresed, transferred to a PVDF membrane, and Western blotted with antibodies against CD9, CD63, and CD81.

[0055] The results are shown in Figure 2. As shown in the "Stats: Mean + / - Standard Error" column in Figure 2, the TIM4 method was able to separate extracellular vesicles of 200 nm or less that were reactive with antibodies against CD9, CD63, and CD81. Furthermore, many C-type lectin-immobilized beads were able to separate extracellular vesicles of approximately 100 nm to approximately 250 nm. Among these, ASGR1, DCSIGN, and CLEC1B were able to separate a population of extracellular vesicles containing extracellular vesicles reactive with antibodies against tetraspanins CD9, CD63, and CD81. Western blot bands were evaluated as follows: - No antibody reactivity detected: 1 - Antibody reactivity detected: 2 - Strong antibody reactivity detected: 3 As shown in Figure 2, extracellular vesicles separated by TIM4 were reactive with anti-CD9 and anti-CD63 antibodies, and strong reactivity with anti-CD81 antibodies was observed. Extracellular vesicles isolated by DCSIGN were reactive with anti-CD9 and anti-CD63 antibodies, and showed strong reactivity with anti-CD81 antibodies. Extracellular vesicles isolated by ASGR1 were reactive with anti-CD9 and anti-CD63 antibodies, and showed strong reactivity with anti-CD81 antibodies. CLEC1B was reactive with anti-CD9 and anti-CD63 antibodies, and showed strong reactivity with anti-CD81 antibodies. No reactivity was detected with anti-CD9, anti-CD63, or anti-CD81 antibodies with lectins other than those mentioned above.

[0056] [Example 3] Marker expression in extracellular vesicles separated using human C-type lectins The extracellular vesicles prepared in Example 2 were electrophoresed at 0.15 μg / well each and Western blotted with antibodies against CD9, CD63, and CD81. As a result, as shown in Figure 3, the population of extracellular vesicles separated using beads immobilized with three types of C-type lectins (ASGR1, DCSIGN, CLEC1B) contained CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles.

[0057] Example 4 Comparison of Extracellular Vesicles Separated by Different Separation Methods Furthermore, the separation of extracellular vesicles was investigated using beads immobilized with three types of C-type lectins (ASGR1, DCSIGN, CLEC1B), and the results were compared with those of extracellular vesicles separated by ultracentrifugation and the TIM4 method. C-type lectin-immobilized beads were prepared using the same method as in Example 2, and extracellular vesicles were separated using the C-type lectin-immobilized beads and the TIM4 method. For ultracentrifugation (UC), 20 ml of iPS cell culture supernatant was filtered through a 0.22 μm filter, placed in an ultracentrifuge tube, and centrifuged at 100,000 × g for 70 minutes at 4°C in an ultracentrifuge (Beckman Optima MAX-XP). After removing the supernatant, a small amount of PBS was added to the ultracentrifuge tube, and the tube was vortexed to recover the eluate containing extracellular vesicles. As shown in Figure 4, by using beads immobilized with three types of C-type lectins (ASGR1, DCSIGN, CLEC1B), extracellular vesicles with particle diameters (average value) of approximately 100 nm to approximately 250 nm could be separated.

[0058] [Example 5] Separation of iPS cell-derived extracellular vesicles by ASGR1 Among the three C-type lectins (ASGR1, DCSIGN, and CLEC1B), ASGR1 was further investigated. In the same manner as in Example 2, a solution (100 μl) containing extracellular vesicles separated from the culture supernatant (20 ml) of 201B7 iPS cells using ASGR1-immobilized beads was added to the same number of particles (4.5 × 10 ７The beads were electrophoresed (number of cells / well) and Western blotted with antibodies against CD9, CD63, and CD81. The results are shown in Figure 5. As a result, extracellular vesicles separated using ASGR1-immobilized beads had a particle diameter of approximately 220 nm, and the reactivity of antibodies against CD9, CD63, and CD81 was highest compared to extracellular vesicles separated by the TIM4 method and ultracentrifugation. Therefore, when ASGR1 was used, a greater number of intact extracellular vesicles positive for markers (CD9, CD63, CD81) and with a particle diameter (average) of approximately 220 nm were separated from the same number of extracellular vesicles compared to ultracentrifugation and the TIM4 method. This demonstrates that the use of ASGR1 enables the separation of CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles with a higher purity than the ultracentrifugation and TIM4 methods.

[0059] Example 6 Separation of Extracellular Vesicles Derived from HEK293T Cells Using ASGR1 HEK293T cells were cultured in RPMI1640, and 20 ml of the resulting culture supernatant was collected. Extracellular vesicles (100 μl of solution) were separated using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation. As a result, as with the ultracentrifugation and TIM4 methods, extracellular vesicles with a particle diameter of approximately 200 nm could be separated using ASGR1-immobilized beads. Furthermore, the same number of particles (4.5 × 10 ７ The beads were electrophoresed (number of cells / well) and Western blotted with antibodies against CD9, CD63, and CD81. The results are shown in Figure 6. As a result, the extracellular vesicles separated using ASGR1-immobilized beads had a particle diameter (average) of approximately 200 nm, and the reactivity of antibodies against CD9, CD63, and CD81 was highest compared to extracellular vesicles separated by the TIM4 method and ultracentrifugation. Therefore, when ASGR1 was used, a greater number of extracellular vesicles positive for markers (CD9, CD63, CD81) and with a particle diameter (average) of approximately 200 nm were separated compared to the ultracentrifugation and TIM4 methods for the same particle number of extracellular vesicles. This indicates that the use of ASGR1 enables the separation of CD9-positive, CD63-positive, and / or CD81-positive extracellular vesicles with a higher purity than the ultracentrifugation and TIM4 methods.

[0060] [Example 7] Analysis of miRNA expression in extracellular vesicles separated by different separation methods. 1 x 10 miRNAs were separated by ultracentrifugation (UC), TIM4 method, or ASGR1. ９ RNA was purified from each extracellular vesicle fraction, and miRNA expression analysis was performed using a 3D-Gene DNA chip (Toray). The results are shown in Figure 7. 1,143 miRNAs were detected in extracellular vesicles purified by ultracentrifugation (UC), 1,139 miRNAs in extracellular vesicles purified by the TIM4 method, and 1,273 miRNAs in extracellular vesicles purified by ASGR1, with the highest number of miRNAs detected in extracellular vesicles purified by ASGR1. Analysis of the similarity of the detected miRNAs revealed that the similarity of miRNAs in extracellular vesicles purified by ASGR1 was 76% with that by ultracentrifugation and 74% with that by the TIM4 method, indicating that while extracellular vesicles containing the same type of miRNA were isolated, some miRNAs were detected only in extracellular vesicles purified by ASGR1. This result indicates that by using C-type lectin (ASGR1), a population of extracellular vesicles different from those obtainable by ultracentrifugation or the TIM4 method can be obtained.

[0061] [Example 8] Heat map analysis of miRNA levels in extracellular vesicles isolated by different isolation methods The levels of miRNA in extracellular vesicles purified by three methods, ultracentrifugation (UC), TIM4, and ASGR1, were analyzed using Heatmapper (http: / / www.heatmapper.ca / ) and displayed as a heat map. The results are shown in Figure 8. It was found that extracellular vesicles purified using ASGR1 contained more miRNA than extracellular vesicles purified by other methods.

[0062] Example 9 Analysis of Glycoaffinity of ASGR1 by Glycomicroarray ASGR1 was mixed with Cy3-conjugated AffiniPure Goat Anti-Human IgG and Fcγ Fragment Specific antibody (Jackson ImmunoResearch) and analyzed by glycan microarray (Tateno et al., Glycobiology, Vol. 18, Issue 10, Pages 789-798, 2008). As shown in Figure 9, ASGR1 was found to exhibit high reactivity with αGal, Asialo-BSM (Bovine submaximal mucin), A-di (GalNAcα1-3Galβ1-PAA), Core6 (GlcNAcβ1-6GalNAcα1-), Tn (GalNAcα1-), di-GalNAcβ (GalNAcβ1-3GalNAcβ1-), BSM (Bovine submaximal mucin), LDN (GalNAcβ1-4GlcNAcβ1-), Forssman disaccharide (GalNAcα1-3GalNAcβ1-), and βGalNAc (GalNAcβ1-), and to exhibit high affinity for glycans having αGalNAc at the non-reducing end. A list of the glycans used in the glycan array is shown in FIG.

[0063] Example 10 Flow Cytometry Analysis of Extracellular Vesicles Using ASGR1-Immobilized Beads 24 μg of the ASGR1-Fc fusion protein prepared in Example 2 was mixed with 50 μl of Dynabeads Protein G (Veritas) and allowed to bind at 4°C for 30 minutes to prepare ASGR1-immobilized beads. 10 ml of culture supernatant from 201B7 iPS cells cultured in mTeSR PLUS (Veritas) was collected and concentrated to 1 ml using a 100 kDa cut Amicon Ultra (Merck). CaCl ２was added to a final concentration of 1 mM, mixed with ASGR1-immobilized beads, and incubated overnight at 4°C with mixing. After washing, the cells were reacted with 10 μg / ml AF647-Human anti-CD63 (CD63 antibody, BD Biosciences) or a control antibody (isotype antibody) and analyzed by flow cytometry. As shown in Figure 11, the CD63 antibody ("CD63" in the figure) exhibited significantly higher fluorescence intensity than the control antibody ("Isotype" in the figure). It was found that extracellular vesicles could be analyzed by flow cytometry using ASGR1-immobilized beads. On the other hand, when the culture supernatant was reacted with Protein G beads ("Beads" in the figure) without immobilized ASGR1-Fc and detected with AF647-Human anti-CD63, no reactivity was observed.

[0064] Example 11 Separation of HEK293T Cell-Derived Extracellular Vesicles Using ASGR1 Extracellular vesicles were separated from the culture supernatant of HEK293T cells using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation in the same manner as in Example 6. The results of observation of the separated extracellular vesicles under a transmission electron microscope are shown in Figure 12. The extracellular vesicles separated using ASGR1-immobilized beads were vesicles with lipid bilayer membranes and had particle diameters of approximately 200 to 300 nm.

[0065] Example 12: Proteomic Analysis of Extracellular Vesicles Separated by Different Separation Methods Using the same method as in Example 6, extracellular vesicles were isolated from the culture supernatant of HEK293T cells using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation. A volume equivalent to 1.5 μg of total protein was collected from the extracellular vesicle suspension and subjected to acetone precipitation. The precipitate was dissolved in solubilization buffer. The lysate was hydrolyzed with trypsin to obtain a peptide mixture. The MS / MS data obtained by liquid chromatography-tandem mass spectrometry (LC-MS / MS) of the peptide mixture was collated with a human amino acid sequence database. Both peptide identification and peptide detection intensity information were integrated, and the peptide detection intensity was integrated for each protein (protein metric value) (Figure 13). The data obtained as described above was analyzed using FunRich software (http: / / www.funrich.org / ) to determine the proportion of proteins associated with each cellular component (Figure 14).

[0066] Furthermore, the expression intensity of exosome markers described in the MISEV (Minimum Information for Extracellular Vesicle Research) guidelines (MISEV2023) was analyzed for the peptide mixture obtained as described above, and the results were displayed as a heat map using the R program ( FIG. 15 ).

[0067] As shown in Figure 13, 1,630 types of proteins were commonly detected by all three separation methods. The total number of proteins detected by ASGR1-immobilized beads was 1,789, the total number of proteins detected by the TIM4 method was 2,102, and the total number of proteins detected by ultracentrifugation was 2,109. In addition, 59 types of proteins were detected only by ASGR1-immobilized beads, 48 ​​types of proteins were detected only by the TIM4 method, and 25 types of proteins were detected only by ultracentrifugation. The results of this example demonstrate that separation using ASGR1 can separate extracellular vesicles containing different types of proteins than conventional methods.

[0068] As shown in Figure 14, exosome-associated proteins were detected in 49.80% of extracellular vesicles isolated using ASGR1-immobilized beads, 47.98% by the TIM4 method, and 47.34% by ultracentrifugation. The results of this example demonstrate that exosomes can be isolated more selectively (with higher purity) by using ASGR1 for separation than by conventional methods.

[0069] As shown in Figure 15, it was shown that the extracellular vesicles isolated using ASGR1-immobilized beads contained the largest amount of exosome markers compared to those isolated using the TIM4 method and the ultracentrifugation method.

[0070] Example 13 Flow Cytometry Analysis of Extracellular Vesicles Derived from HEK293T Cells Using Exosome Marker Antibodies The culture supernatant obtained by culturing HEK293T cells was incubated overnight at 4°C with ASGR1-immobilized beads or TIM4-immobilized beads (MagCapture™ Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, Ltd.). The beads were then diluted with 1 mM CaCl ２ The cells were suspended in Wash Buffer (Tris-HCl; NaCl; pH 7.4) supplemented with 10 μg / ml of a labeled isotype control antibody (clone number: A-1, MC2B-TF2, Cosmo Bio Co., Ltd.) or an exosome marker (CD9, CD63, or CD81) antibody (clone number: 12A12, SHI-EXO-M01, -M02, and -M03 (manufacturer: CAC), Cosmo Bio Co., Ltd.). Flow cytometry data was analyzed using CytoFLEX (Beckman Coulter, Inc.) and is shown in FIG. 16.

[0071] 16, the detection patterns of exosomes positive for the exosome markers CD9, CD63, and CD81 were different between the ASGR1-immobilized bead group and the TIM4-immobilized bead group. The results of this example showed that the expression patterns of each exosome marker in exosomes obtained from HEK293T cells were different when separated using ASGR1-immobilized beads and when separated using the TIM4 method, i.e., different exosome groups were separated.

[0072] Example 14 Flow Cytometry Analysis of iPS Cell-Derived Extracellular Vesicles Using Exosome Marker Antibodies Culture supernatant of 201B7 iPS cells (human iPS cell line) was incubated with ASGR1-immobilized beads or TIM4-immobilized beads (MagCapture™ Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, Ltd.) overnight at 4°C. The beads were then incubated with 1 mM CaCl ２ The cells were suspended in Wash Buffer (Tris-HCl; NaCl; pH 7.4) supplemented with 10 μg / ml of a labeled isotype control antibody (clone number: A-1, MC2B-TF2, Cosmo Bio Co., Ltd.) or an exosome marker (CD9, CD63, or CD81) antibody (clone number: 12A12, SHI-EXO-M01, -M02, and -M03 (manufacturer: CAC), Cosmo Bio Co., Ltd.). Flow cytometry data was analyzed using CytoFLEX (Beckman Coulter, Inc.) and is shown in FIG. 17.

[0073] 17, the detection patterns of exosomes positive for the exosome markers CD9, CD63, and CD81 were different between the ASGR1-immobilized bead group and the TIM4-immobilized bead group. The results of this example showed that the expression patterns of each exosome marker in exosomes obtained from iPS cells were different when separated using ASGR1-immobilized beads and when separated using the TIM4 method, i.e., different exosome groups were separated.

[0074] Example 15 ELISA Analysis of Extracellular Vesicles Derived from Different Cells Using CD63 Antibody HEK293T cells were cultured in RPMI1640, iPS cells in mTeSR1 (STEMCELL Technologies), and adipose-derived mesenchymal stem cells (ADSC, Lot No. 2118, Life Technologies) and bone marrow-derived mesenchymal stem cells (Yub622, RIKEN CELL BANK) in MesenPRO RS™ Medium (Catalog No. 12746-012, GIBCO) (supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin).

[0075] 100 μl of 2 μg / ml biotinylated ASGR1-Fc was added to an avidin-immobilized plate (BS-X7603, Sumitomo Bakelite Co., Ltd.) and immobilized by incubating at room temperature for 1 hour. After washing, 100 μl / well of culture supernatant from HEK293T cells, 201B7 iPS cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells was added and incubated at room temperature for 2 hours. The culture supernatant was diluted in three stages (2-fold (0.5), 4-fold (0.25), and 8-fold (0.125)) as shown on the horizontal axis of Figures 18A-D. The culture supernatant was washed away, and peroxidase-labeled anti-CD63 antibody (SHI-EXO-M02 (Cosmo Bio Co., Ltd.) labeled with peroxidase (LK11, Dojindo Laboratories)) (0.5 μg / ml, 100 μl) was added and incubated at room temperature for 2 hours. Finally, 100 μl / well of TMB solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after 30 minutes of reaction at room temperature, the color reaction was stopped with 1N HCl, and measurements were made at OD 450 / 620 nm.

[0076] As shown in Figure 18, the presence of the exosome marker CD63 was detected in the culture supernatant of all cells. This indicates that CD63-positive exosomes were isolated from the culture supernatant of all cells by ASGR1. Note that in HEK293T cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells, CD63-positive exosomes were not dependent on the dilution of the culture supernatant. This indicates that the supernatants of these cells contained CD63-positive exosomes at a high concentration that saturated the ASGR1 immobilized on the plate.

[0077] The method for separating extracellular vesicles and the kit for separating extracellular vesicles according to the present disclosure can separate extracellular vesicles from a sample containing extracellular vesicles, and therefore can be suitably used in diagnosis, treatment, etc. using extracellular vesicles, and has industrial applicability.

Claims

1. A method for separating extracellular vesicles in a sample, comprising: contacting extracellular vesicles in the sample with a C-type lectin; and separating extracellular vesicles bound to the C-type lectin from the C-type lectin.

2. The method of claim 1, comprising: contacting extracellular vesicles in a sample with a C-type lectin in the presence of calcium ions; and separating the extracellular vesicles bound to the C-type lectin from the C-type lectin in the absence of calcium ions.

3. The isolation method according to claim 1 or 2, wherein the C-type lectin is ASGR1, DCSIGN, and / or CLEC1B.

4. The isolation method according to claim 1 or 2, wherein the C-type lectin is ASGR1.

5. A kit for isolating extracellular vesicles from a sample containing extracellular vesicles, comprising a C-type lectin.

6. A kit for detecting extracellular vesicles, comprising a C-type lectin and an antibody that specifically binds to extracellular vesicles.

7. The kit according to claim 5 or 6, wherein the C-type lectin is ASGR1, DCSIGN, and / or CLEC1B.

8. The kit according to claim 5 or 6, wherein the C-type lectin is ASGR1.

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