METHOD FOR PRODUCING Tim4 PROTEIN, AND USE THEREOF

Abstract

The present invention addresses the problem of providing: a method for producing a Tim4 protein having higher binding activity with extracellular vesicles; a method for improving binding activity of Tim4 protein with extracellular vesicles; a method for obtaining, removing or detecting extracellular vesicles comprising the method for producing the Tim4 protein; a Tim4 protein having improved binding activity with extracellular vesicles; and a kit for obtaining, removing or detecting extracellular vesicles comprising the Tim4 protein. The present invention provides a method for producing a Tim4 protein, the method comprising bringing a T-cell immunoglobulin and mucin domain containing molecule 4 protein, also referred to as Tim4, into contact with an acidic solution having a pH of 2 or less.

Description

Method for producing Tim4 protein and its use

[0001] The present invention relates to a method for producing Tim4 protein (T-cell immunoglobulin mucin domain-containing molecule 4 protein). The present invention further relates to a method for improving the extracellular vesicle binding activity of Tim4 protein, and a method for obtaining, removing, or detecting extracellular vesicles. The present invention also relates to a kit for obtaining, removing, or detecting Tim4 protein and extracellular vesicles.

[0002] Extracellular vesicles contain proteins and nucleic acids such as microRNA inside the particles and are known to play a role in intercellular communication. Extracellular vesicles are also secreted into body fluids such as blood, and the proteins and microRNA contained in extracellular vesicles have attracted attention as diagnostic markers for diseases. Extracellular vesicles have also attracted attention as a delivery tool for nucleic acid drugs.

[0003] As a method for obtaining, removing, or detecting extracellular vesicles, Patent Document 1 describes a method for obtaining, removing, and detecting extracellular vesicles using a Tim protein-binding carrier. Patent Document 1 describes eluting and purifying an Fc-tagged Tim4 protein with a glycine-hydrochloride buffer solution at pH 3.0.

[0004] Meanwhile, Patent Documents 2 to 4 describe purifying an antibody by treating it with acetic acid at pH 2.0, and Patent Document 5 describes purifying a protein by treating it with caprylic acid at 4.5<pH≦6.0.

[0005] Japanese Patent Application Publication No. 2021-012200 Special Publication No. 2021-534196 Special Publication No. 2021-529520 Special Publication No. 2020-530263 Japanese Patent Application Publication No. 2022-069571

[0006] Tim4 protein is known to bind to phosphatidylserine, which constitutes extracellular vesicles, and is used to isolate and detect extracellular vesicles by capturing them. Therefore, there is a need to produce Tim4 protein with higher binding activity to extracellular vesicles.

[0007] An object of the present invention is to provide a method for producing Tim4 protein having higher binding activity to extracellular vesicles.A further object of the present invention is to provide a method for improving the binding activity of Tim4 protein to extracellular vesicles.A further object of the present invention is to provide a method for obtaining, removing, or detecting extracellular vesicles using the above-mentioned method for producing Tim4 protein.A further object of the present invention is to provide a Tim4 protein with improved binding activity to extracellular vesicles.A further object of the present invention is to provide a kit for obtaining, removing, or detecting extracellular vesicles, comprising the above-mentioned Tim4 protein.

[0008] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that treating Tim4 protein with strong acid improves the extracellular vesicle-binding activity of Tim4 protein. The present invention was completed based on the above findings.

[0009] That is, the present invention provides the following: <1> A method for producing Tim4 protein, which comprises contacting T cell immunoglobulin mucin domain-containing molecule 4 protein, also known as Tim4, with an acidic solution having a pH of 2 or less. <2> The production method according to <1>, wherein the pH of the acidic solution is 1.75 or less. <3> The production method according to <1> or <2>, wherein the produced Tim4 protein has improved extracellular vesicle-binding activity compared to when the produced Tim4 protein is contacted with an acidic solution having a pH of 2.5. <4> A method for improving the extracellular vesicle-binding activity of Tim4 protein, which comprises contacting T cell immunoglobulin mucin domain-containing molecule 4 protein, also known as Tim4, with an acidic solution having a pH of 2 or less. <5> A method for obtaining, removing, or detecting extracellular vesicles, which comprises producing Tim4 protein by the method according to any one of <1> to <3>, and contacting the produced Tim4 protein with extracellular vesicles. <6> Tim4 protein produced by the method according to any one of <1> to <3>. <7> A kit for obtaining, removing or detecting extracellular vesicles, comprising a carrier to which the Tim4 protein according to <6> is bound, and an antibody that binds to the extracellular vesicles. <8> A kit for obtaining, removing or detecting extracellular vesicles, comprising the Tim4 protein according to <6>, a carrier, and an antibody that binds to the extracellular vesicles.

[0010] According to the present invention, it is possible to produce a Tim4 protein having higher binding activity to extracellular vesicles. According to the present invention, it is possible to obtain, remove, or detect extracellular vesicles with high efficiency.

[0011] Figure 1 shows the results of measuring the binding ability of Tim4 protein to extracellular vesicles. Figure 2 shows the change in the binding ability of Tim1 protein to extracellular vesicles due to acid treatment. Figure 3 shows the results of measuring the binding ability of Tim4 protein to extracellular vesicles.

[0012] The present invention will be described in detail below. In this specification, the word "to" is used to mean that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0013] The present invention relates to a method for producing Tim4 protein, which comprises contacting T cell immunoglobulin mucin domain-containing molecule 4 protein, also known as Tim4, with an acidic solution having a pH of 2 or less. The present invention further relates to a method for improving the extracellular vesicle binding activity of Tim4 protein, which comprises contacting T cell immunoglobulin mucin domain-containing molecule 4 protein, also known as Tim4, with an acidic solution having a pH of 2 or less.

[0014] In general, proteins such as antibodies tend to denature at low pH. Therefore, elution at low pH should be avoided as much as possible, and is performed only when the binding between antibody and potein A is strong enough to make elution difficult. Since the in vivo binding of Tim4 protein to PS (phosphatidylserine) on EV occurs in an environment of pH 5.5 to 7.0, it was predicted that strong acid treatment would likely denature Tim4 protein and adversely affect its binding to phosphatidylserine. Among the Tim proteins, only Tim4 protein showed improved extracellular vesicle-binding activity upon strong acid treatment; Tim1 protein was not affected (Comparative Example). It was completely unexpected that Tim4 protein could withstand strong acid treatment, and the improvement in extracellular vesicle-binding activity upon strong acid treatment was also an unexpected effect.

[0015] <Extracellular vesicles> Extracellular vesicles are small membrane vesicles with a lipid bilayer membrane secreted from cells such as cells in vivo or cultured cells. The diameter of the vesicles is usually 20 nm to 1000 nm, preferably 50 nm to 500 nm, more preferably 50 nm to 200 nm. Extracellular vesicles are known to have phosphatidylserine on their membrane surface.

[0016] Extracellular vesicles can be classified in various ways based on their origin and the size of the small membrane vesicles, as described in Nature Reviews Immunology 9, 581-593 (August 2009) and "Obesity Research" Vol. 13 No. 2 2007 Topics by Naoto Aoki et al. Specific examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic vesicles, and adiposomes.

[0017] Exosomes are small membrane vesicles composed of a lipid bilayer membrane derived from late endosomes, etc. The diameter of the vesicles is usually 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Exosomes are known to contain proteins such as tetraspanins such as CD63 and CD9, Alix, TSG101, Lamp-1, and Flotillin. Exosomes are known to have phosphatidylserine on their membrane surface.

[0018] Microvesicles are small membrane vesicles composed of a lipid bilayer membrane derived from the cell membrane. Microvesicles are usually 100 nm to 1000 nm, preferably 100 nm to 800 nm, more preferably 100 nm to 500 nm. Microvesicles are known to contain proteins such as integrins, selectins, and CD40 ligands. Microvesicles are known to have phosphatidylserine on their membrane surface.

[0019] Ectosomes are small membrane vesicles composed of a lipid bilayer membrane derived from the plasma membrane. Ectosomes are typically 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Ectosomes are known to contain CR1 and proteolytic enzymes but not CD63. Ectosomes are known to have phosphatidylserine on their membrane surface.

[0020] Membrane particles are small membrane vesicles composed of a lipid bilayer membrane derived from the plasma membrane. Membrane particles are typically 50 to 80 nm in size. Membrane particles are known to contain CD133 but not CD63. Membrane particles are known to have phosphatidylserine on their membrane surface.

[0021] Exosome-like vesicles are small membrane vesicles composed of a lipid bilayer membrane derived from early endosomes. Exosome-like vesicles are typically 20 nm to 50 nm in size. Exosome-like vesicles are known to contain TNF-RI. Exosome-like vesicles are known to have phosphatidylserine on their membrane surface.

[0022] Apoptotic vesicles are small membrane vesicles composed of a lipid bilayer membrane derived from apoptotic cells. Apoptotic vesicles are usually 50 nm to 500 nm, preferably 50 nm to 300 nm, more preferably 50 nm to 200 nm. Apoptotic vesicles are known to contain histones. Apoptotic vesicles are known to have phosphatidylserine on their membrane surface.

[0023] Adiposomes are small membrane vesicles derived from fat cells and composed of a lipid bilayer membrane. Adiposomes are generally 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. Adiposomes containing MFG-E8 (milk fat globule-EGF factor 8) are known. Adiposomes having phosphatidylserine on their membrane surface are known.

[0024] <Tim4 Protein> The Tim4 (T cell immunoglobulin mucin domain-containing molecule 4) protein may be any protein capable of binding to extracellular vesicles, with animal-derived Tim4 proteins being preferred. Of these, human or mouse Tim4 proteins are preferred (hereinafter, human Tim4 proteins may be abbreviated as "human-derived Tim4 protein," and mouse Tim4 proteins may be abbreviated as "mouse-derived Tim4 protein"). More specifically, any protein may be used as long as it has at least the amino acid sequence of a phosphatidylserine-binding domain (IgV domain), and may have either the full-length amino acid sequence of the Tim4 protein or a portion of the Tim4 protein. Examples of the amino acid sequence of the phosphatidylserine-binding domain (IgV domain) include the N-terminal 22-135 amino acid region of mouse-derived Tim4 protein (RefSeq NP_848874.3) and the N-terminal 25-137 amino acid region of human-derived Tim4 protein (RefSeq NP_612388.2). Examples of the full-length amino acid sequence of Tim4 protein include the 1-343 amino acid region of the full-length sequence of mouse-derived Tim4 protein (RefSeq NP_848874.3) and the 1-378 amino acid region of the full-length sequence of human-derived Tim4 protein (RefSeq NP_612388.2). Examples of Tim4 protein fragments include those having the amino acid sequence of a phosphatidylserine-binding domain (IgV domain) and a mucin domain, such as the N-terminal 22-273 amino acid region of mouse-derived Tim4 protein (RefSeq NP_848874.3), the N-terminal 22-279 amino acid region of mouse-derived Tim4 protein (RefSeq NP_848874.3), and the N-terminal 25-315 amino acid region of human-derived Tim4 protein (RefSeq NP_612388.2). If necessary, these sequences may have a signal sequence.

[0025] Furthermore, the Tim4 protein may be a mutant in which one or more amino acids of the above-mentioned amino acid sequence have been deleted, substituted, inserted and / or added, so long as it is capable of binding to extracellular vesicles.

[0026] The Tim4 protein may be any protein having the above-mentioned properties, and may be extracted from cells (e.g., immune cells such as macrophages) or tissues of organisms that contain Tim4 protein, such as animals (e.g., mice and humans) or plants, or prepared using genetic engineering technology based on these.

[0027] When a Tim4 protein prepared by genetic recombination technology is used, it is preferable that the Tim4 protein has one or more affinity tags for ease of purification.

[0028] The affinity tag may be any that is used when preparing a protein by genetic recombination technology, and examples thereof include affinity tags such as an Fc tag, a FLAG tag, a His tag, a GST tag, an MBP tag, an HA tag, a Myc tag, a Strep(II) tag, and a PA tag.

[0029] The affinity tag is preferably fused to the C-terminus of the Tim4 protein.

[0030] Tim4 protein includes not only proteins consisting of the amino acid sequence of Tim4 protein (full-length or partial sequence) but also proteins having the amino acid sequence of Tim4 protein (full-length or partial sequence) and the amino acid sequence of the affinity tag described above. Furthermore, the affinity tag and Tim4 protein may be directly linked, or may be linked via a spacer as described in "Protein Expression Using Transdirect Insect Cell Cell-Derived Cell-Free Protein Synthesis Reagent Kit," by Ezure Toru, Suzuki Takashi, Ito Masaaki, and Shikata Masamitsu (Shimadzu Corporation, Analytical and Measurement Division), published June 9, 2008: Protein Science Society Archives, 1, e005 (2008)." Therefore, Tim4 protein also includes proteins having the amino acid sequence of Tim4 protein (full-length or partial sequence), the amino acid sequence of an affinity tag, and the amino acid sequence of a spacer.

[0031] <Method for Preparing Tim4 Protein> Tim4 protein can be produced by a general chemical method according to its amino acid sequence. For example, Tim4 protein can be obtained by a standard chemical method (chemical synthesis method) such as the fluorenylmethyloxycarbonyl method (Fmoc method) or the t-butyloxycarbonyl method (tBoc method). Alternatively, Tim4 protein can be chemically synthesized using a commercially available peptide synthesizer.

[0032] Tim4 protein can also be obtained by a well-known method using genetic engineering technology, in which a nucleic acid molecule encoding the Tim4 protein is incorporated into an expression vector such as a suitable plasmid or phage, host cells are transformed (or transduced) with the recombinant expression vector, the resulting host cells are amplified, and the protein is secreted intracellularly or extracellularly.

[0033] A method for preparing Tim4 protein using gene recombination technology will be described below.

[0034] <Expression Vector> Any expression vector for expressing Tim4 protein may be used as long as it contains a nucleic acid sequence encoding Tim4 protein (hereinafter sometimes abbreviated as "Tim coding sequence").

[0035] Among Tim coding sequences, examples of nucleic acid sequences encoding Tim4 protein include the nucleotide sequence of cDNA encoding the 1-343 amino acid region of the full-length sequence of mouse-derived Tim4 protein (RefSeq No. NM_178759.4) and the nucleotide sequence of cDNA encoding the 1-378 amino acid region of the full-length sequence of human-derived Tim4 protein (RefSeq No. NM_138379.2).

[0036] The expression vector may be a commercially available vector into which a Tim coding sequence is introduced using standard cloning techniques. Examples of such expression vectors include those obtained by incorporating, using standard cloning techniques, a cDNA encoding the N-terminal 1-273 amino acid region of mouse-derived Tim4 protein, a cDNA encoding the N-terminal 1-279 amino acid region of mouse-derived Tim4 protein, or a cDNA encoding the N-terminal 1-315 amino acid region of human-derived Tim4 protein into an expression vector such as the commercially available pCAG-Neo vector (Wako Pure Chemical Industries, Ltd.). Any vector capable of expressing and producing Tim4 protein in host cells may be used for introducing the Tim coding sequence, and it is convenient to use a commercially available vector. When the host is an animal cell, commercially available vectors for this purpose include the pCAG-Neo vector and pcDNA vector.

[0037] Any host may be used as long as it is capable of expressing the Tim4 protein, including, for example, Escherichia coli, insect cells, mammalian cells, plant cells, and yeast cells, with mammalian cells being preferred. Examples of mammalian cells include HEK293T cells, COS-7 cells, CHO-K1 cells, and CHO-S cells.

[0038] <Gene introduction into host> The expression vector is introduced into the host according to a standard method for introducing a vector into a host, as described in, for example, "Protein expression protocols selectable by purpose, Chapter 3: Protein expression protocols, ISBN 978-4-7581-0175-2, Yodosha."

[0039] <Culturing the host> The host into which the gene has been introduced is cultured according to a standard method for culturing the host. The culture conditions vary depending on the host into which the gene has been introduced, but may be according to a standard method for each host. For example, in the case of animal cells, the culture conditions are usually 5 to 10%, preferably 5 to 8% CO ２ The cells are cultured under the above conditions, typically at 36° C. to 38° C., preferably 36.5° C. to 37.5° C., for 1 to 10 days, preferably 3 to 4 days. Since the Tim4 protein does not contain a transmembrane domain or an intracellular domain, it is expressed and secreted into the culture supernatant.

[0040] <Purification of Tim4 Protein> The resulting culture medium of the gene-transfected host is centrifuged (usually at 200 to 400×g for 3 to 10 minutes, preferably at 300×g for 3 to 6 minutes) to recover the culture supernatant, and if necessary, (i) the recovered culture supernatant may be centrifuged typically at 1000 to 2000×g for 20 to 60 minutes, preferably at 1200×g for 20 to 40 minutes, and / or (ii) filtered to separate impurities and obtain a culture supernatant filtrate.

[0041] Furthermore, if necessary, the obtained culture supernatant filtrate may be concentrated, typically 5- to 20-fold, and preferably 8- to 12-fold, by a standard method such as ultrafiltration to obtain a concentrated culture supernatant filtrate. Next, when the Tim4 protein has an affinity tag, the Tim4 protein (a fusion protein of the Tim4 protein and an affinity tag) may be purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or a concentrate of the obtained culture supernatant filtrate according to a standard method for purifying a protein using the affinity tag for each affinity tag (e.g., a method using a carrier onto which a substance having affinity for the affinity tag is immobilized), as described in "Protein Expression Protocols Selectable for Each Purpose, Chapter 3: Protein Expression Protocols, Section 6: Protein Purification, Purification by Tags, ISBN 978-4-7581-0175-2, Yodosha" or the like. When the Tim4 protein does not have an affinity tag, the Tim4 protein can be purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or a concentrate of the obtained culture supernatant filtrate by various types of chromatography according to standard protein purification methods such as those described in "Protein Expression Protocols Selected for Each Purpose, Chapter 3: Protein Expression Protocols, Section 6: Protein Purification, Purification by Chromatography, ISBN 978-4-7581-0175-2, Yodosha." Purification may also be performed by appropriately combining the above purification methods.

[0042] In the present invention, Tim4 may be contacted with an acidic solution having a pH of 2 or less in the above-mentioned purification, or may be contacted with an acidic solution having a pH of 2 or less in a step different from the above-mentioned purification. The pH of the acidic solution is preferably 1.75 or less, more preferably 1.6, such as 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0. Examples of acidic solutions include KCl solution, glycine hydrochloride solution, acetic acid, and hydrochloric acid. Of the above, KCl solution or glycine hydrochloride solution is preferred.

[0043] When a KCl solution is used, the concentration is preferably 0.1 mol / L to 1.0 mol / L, more preferably 0.1 mol / L to 0.5 mol / L, and even more preferably 0.2 mol / L to 0.4 mol / L. When a glycine hydrochloride solution is used, the concentration is preferably 0.02 mol / L to 0.5 mol / L, more preferably 0.03 mol / L to 0.3 mol / L, and even more preferably 0.05 mol / L to 0.2 mol / L.

[0044] It is preferable that the extracellular vesicle binding activity of Tim4 protein produced by contact with an acidic solution of pH 2 or less is improved compared to when it is contacted with an acidic solution of pH 2.5.

[0045] According to the present invention, there is provided a Tim4 protein produced by the above-described method for producing a Tim4 protein according to the present invention.

[0046] <Tim4 Carrier> A Tim4 protein-bound carrier (hereinafter sometimes abbreviated as "Tim carrier") is a carrier in which Tim4 protein is bound. Any insoluble carrier used in conventional immunoassays can be used as the carrier, including, for example, organic materials such as 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, and ethylene-maleic anhydride copolymer; inorganic materials such as glass, silicon oxide, diatomaceous earth, porous glass, ground glass, alumina, silica gel, and metal oxides; magnetic materials such as iron, cobalt, nickel, magnetite, and chromite; and materials prepared from alloys of these magnetic materials. These carriers can also be used in a variety of forms, such as microplates, tubes, disk-shaped pieces, and particles (beads). When used in the acquisition method and removal method of the present invention described below, particles (beads) are preferably used, and the particle size is not particularly limited, but typically 10 nm to 100 μm, preferably 100 nm to 10 μm, depending on the purpose and application. Furthermore, when used in the detection method of the present invention described below, particles (beads) or microplates are preferred, and the particle size is not particularly limited, but typically 10 nm to 100 μm, preferably 100 nm to 10 μm, depending on the purpose and application. Furthermore, the number and size of the wells in the microplate are not particularly limited, but typically 12 to 1536 wells, preferably 96 to 384 wells, depending on the purpose and application.

[0047] <Method of binding Tim4 protein to a carrier> The method of binding Tim4 protein to a carrier may be any known method for binding a protein to a carrier, for example, a method of binding by affinity binding; a method of binding by chemical binding (for example, the method described in Japanese Patent No. 3269554 and WO2012 / 039395); a method of binding by physical adsorption (for example, the method described in Japanese Patent Publication No. 5-41946), etc., but the method of binding by affinity binding and the method of binding by physical adsorption are preferred. Specific methods for binding Tim4 protein to a carrier and post-treatment of the Tim4 carrier are described in paragraphs 0055 to 0096 of Japanese Patent Laid-Open No. 2021-012200, and the contents of Japanese Patent Laid-Open No. 2021-012200 are all incorporated by reference herein.

[0048] By using the Tim4 carrier, extracellular vesicles can be efficiently obtained, and extracellular vesicles in a sample can be obtained with high purity. It is also possible to efficiently remove extracellular vesicles from a sample. Furthermore, it is also possible to detect extracellular vesicles in a sample with high sensitivity.

[0049] <Method for Obtaining Extracellular Vesicles> The present invention provides a method for obtaining extracellular vesicles, comprising: producing Tim4 protein by the method for producing Tim4 protein of the present invention; and contacting the produced Tim4 protein with extracellular vesicles. The method for obtaining extracellular vesicles of the present invention (hereinafter sometimes abbreviated as "obtaining method of the present invention") preferably comprises the following steps: (1) a step of forming a complex between Tim4 protein bound to a carrier and extracellular vesicles in a sample (hereinafter also referred to as "complex formation step"), (2) a step of separating the complex from the sample (hereinafter also referred to as "complex separation step"), and (3) a step of separating the extracellular vesicles from the complex and obtaining the extracellular vesicles (hereinafter also referred to as "obtaining step").

[0050] <About the Complex Formation Step> The complex formation step is a step of forming a complex between the Tim4 protein, the carrier, and the extracellular vesicles in the sample.

[0051] <Sample> The sample may be either a liquid that contains or may potentially contain extracellular vesicles. The sample may be derived from a living organism or a solution such as a culture medium or buffer in which extracellular vesicles are dissolved or suspended. Specific examples of samples include body fluids such as blood, saliva, urine, milk, amniotic fluid, and ascites, as well as cell culture supernatants.

[0052] The solution containing (dissolving or suspending) extracellular vesicles may be any solution that stably dissolves or suspends the extracellular vesicles and does not interfere with the binding of the complex between the Tim4 protein bound to the carrier and the extracellular vesicles in the sample, and examples thereof include water and buffer solutions (e.g., TBS, HBS, etc.) that have a buffering action at pH 7.0 to 8.0, preferably 7.2 to 7.6. The buffer concentration in these buffer solutions is appropriately selected from the range of usually 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is appropriately selected from the range of usually 100 to 200 mM, preferably 140 to 160 mM.

[0053] <Amount of Sample> In the complex formation step, the amount of sample to be contacted with 1 μg of Tim4 protein is generally 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.1 to 1.0 ml.

[0054] <Temperature> In the complex formation step, the temperature at which Tim4 protein is brought into contact with extracellular vesicles in the sample is generally 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0055] <Time> In the complex formation step, the contact time between the Tim4 protein and the sample is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0056] The complex formation process can be divided into two cases: (1-A) using a pre-prepared Tim4 carrier (i.e., using a carrier bound to Tim4 protein) and (1-B) using Tim4 protein and a carrier separately.

[0057] (1-A) is a method in which a Tim4 carrier is brought into contact with extracellular vesicles in a sample to form a complex between the Tim4 protein bound to the carrier and the extracellular vesicles in the sample.

[0058] (1-B) is a method in which Tim4 protein and a carrier are used separately. For example, when Tim4 protein and a carrier are bound by affinity binding, the following (1-Bi), (1-B-ii), or (1-B-iii) may be used.

[0059] (1-Bi) Tim4 protein, a carrier, and extracellular vesicles in a sample are simultaneously contacted to form a complex.

[0060] (1-B-ii) Tim4 protein is contacted with extracellular vesicles in a sample to form a complex between Tim4 protein and extracellular vesicles, and then the complex is contacted with a carrier to form a complex.

[0061] (1-B-iii) After contacting the carrier with extracellular vesicles in the sample, Tim4 protein is further contacted to form a complex.

[0062] <Complex separation step> The complex separation step in the acquisition method is a step in which, after the complex formation step, the complex between the Tim4 protein bound to the carrier (Tim4 carrier) obtained and the extracellular vesicles in the sample is separated from the sample, and the separated complex is obtained.

[0063] The complex separation step in the acquisition method of the present invention may be any method that can separate the complex from the sample and obtain the complex, and examples thereof include the following methods.

[0064] (1) When a magnetic carrier is used as the carrier, the container in which the complex formation step has been carried out is placed on a magnetic stand if necessary, and the complex is collected on the wall of the tube using magnetic force, and the supernatant sample is removed to separate them. (2) When the carrier is in the form of beads, the container in which the complex formation step has been carried out is centrifuged to collect the complex as a precipitate, and the supernatant sample is then removed to separate them. (3) When a plate or the like in which the carrier is not in the form of beads is used, the sample is removed alone to separate them. (4) A method in which the complex and the sample are separated by filtration. After separating the complex and the sample in this manner, the separated complex can be obtained (recovered) by a method known per se.

[0065] <Washing Procedure> After the complex formation step and the complex separation step, the resulting complex may be washed with a washing solution, if necessary (hereinafter also referred to as the "washing procedure"). This washing procedure allows for the removal of contaminants in the sample, such as cell-derived components attached to the carrier surface. As a washing method, any washing method commonly used in this field can be used, except for the use of the washing solution described above. The washing solution used in the washing procedure may be any solution that does not affect the binding of the extracellular vesicles or viruses of the present invention, the Tim4 protein of the present invention, and the carrier of the present invention in the complex. For example, a buffer solution (e.g., TBS or HBS) containing calcium ions typically at 0.5 to 100 mM, preferably typically 1 to 10 mM, more preferably typically 2 to 5 mM, and having a buffering action at pH 7.0 to 8.0, preferably 7.2 to 7.6, that does not precipitate calcium, may be used. The buffer concentration in these buffer solutions is generally selected from the range of 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is generally selected from the range of 100 to 200 mM, preferably 140 to 160 mM. The solutions may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., in amounts that do not interfere with the binding of the extracellular vesicles, Tim4 protein, and carrier in the complex.

[0066] For example, the case where magnetic particles are used as carriers will be described. First, a washing solution is added to a container containing the complex obtained in the complex separation step, and the container is stirred. Then, the container is placed on a magnetic stand, and the complex is collected on the tube wall using magnetic force, and the solution in the container is discarded. These washing procedures may be repeated several times as necessary.

[0067] <Regarding the Obtaining Step> The obtaining step is a step of performing a complex formation step, a complex separation step, and optionally a washing step (washing operation), followed by separating the extracellular vesicles from the resulting complex between the Tim4 protein bound to the carrier and the extracellular vesicles in the sample, thereby obtaining the extracellular vesicles. This allows for the obtaining of highly pure extracellular vesicles.

[0068] Specific examples of the obtaining step include the following (2-A) and (2-B): (2-A) A method using a protein denaturant (2-B) A method of reducing calcium ion concentration

[0069] <(2-A): Method using a protein denaturant> (2-A) is a method in which, after performing a complex formation step, a complex separation step, and optionally a washing operation, the resulting complex is treated with a protein denaturant to denature the Tim4 protein in the complex, and extracellular vesicles are separated from the complex. This method allows for the production of highly pure extracellular vesicles.

[0070] -Protein Denaturant- The protein denaturant used in (2-A) may be any compound generally used in this field as a compound for denaturing proteins, and examples thereof include anionic surfactants such as SDS (sodium dodecyl sulfate) and N-lauroyl sarcosine; zwitterionic surfactants such as CHAPS (3-(3-cholamidopropyl) tetrahydrofuran-1-propanesulfonate) and Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); Brij 35 (manufactured by Takara Bio Inc.), dodecyl-β-D-maltoside, Nonidet (registered trademark) P-40, octyl-β-D-glucoside, and Triton Examples of suitable surfactants include nonionic surfactants such as X-100 (polyoxyethylene (10) octylphenyl ether) and Tween 20 (polyoxyethylene (20) sorbitan monolaurate); and chaotropic agents such as urea, formamide, and guanidine. Anionic surfactants are preferred, with SDS being particularly preferred.

[0071] In (2-A), the action of these protein denaturants on the complex is generally carried out by contacting a solution containing the protein denaturant (hereinafter sometimes abbreviated as "protein denaturant-containing solution") with a complex of Tim4 protein bound to a carrier and extracellular vesicles in a sample, and then allowing the protein denaturant-containing solution to act on the complex. Contact between the protein denaturant-containing solution and the complex can be achieved, for example, by suspending the complex in the solution (when the carrier is beads, etc.), immersing the complex in the solution (when the carrier is a disk-shaped piece or tube, etc.), or adding the solution to the complex (carrier) (when the carrier is a microplate or tube, etc.).

[0072] - Solution containing protein denaturant - In (2-A), examples of the solution containing the protein denaturant include purified water and a buffer solution capable of dissolving the protein denaturant. Examples of the buffer solution include buffer solutions (e.g., Tris, HEPES, etc.) that typically have a buffering effect at a pH of 6 to 9, preferably 7 to 8. The concentration of the buffer in these buffer solutions is typically selected from the range of 5 to 100 mM, preferably 10 to 50 mM.

[0073] -Protein Denaturant-Containing Solution- In (2-A), the pH of the protein denaturant-containing solution is typically 6.0 to 9.0, preferably 7.0 to 8.0. The concentration of the protein denaturant in the protein denaturant-containing solution varies depending on the type of protein denaturant, but may be within the range of concentrations generally used in this field. For example, in the case of SDS, the concentration is typically 0.1 to 10%, preferably 0.3 to 4%, and more preferably 0.5 to 2%. The protein denaturant-containing solution may also contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc. In (2-A), the protein denaturant-containing solution is typically used in an amount of 10 μL to 500 μL, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL per 1 mg of Tim4 carrier.

[0074] -Contact (reaction) conditions- In (2-A), the temperature and time for which the protein denaturant is allowed to act (contact) on the complex are generally 4.0 to 37°C, preferably 10 to 30°C, and more preferably 20 to 30°C, and the time is generally 5.0 to 60 seconds, preferably 10 to 30 seconds, and more preferably 10 to 20 seconds.

[0075] <(2-B): Method of reducing calcium ion concentration> (2-B) is a method in which, after performing a complex formation step, a complex separation step, and optionally a washing operation, the concentration of calcium ions bound to the resulting complex and the calcium ions in the solution containing the complex are reduced to separate extracellular vesicles from the complex. This method allows for obtaining highly purified, intact extracellular vesicles.

[0076] It is known that calcium ions mediate the binding between Tim4 protein and phosphatidylserine (Immunity 2007 December; 27(6): 941-951). After the complex formation step, complex separation step, and optional washing step, the resulting complex must be allowed to coexist with calcium ions in order to maintain the binding between Tim4 protein in the complex and extracellular vesicles in the sample. For example, when the complex is allowed to coexist in solution, 0.5 mM or more of calcium ions are required in the complex solution. Here, when the complex is allowed to coexist in solution, this also includes the carrier in a pellet state after the complex formation step, complex separation step, and optional washing step. Note that even if the complex is dried after the complex formation step, complex separation step, and optional washing step, calcium ions will not be eluted from the complex unless the recovery step is performed, and the binding between the carrier and extracellular vesicles or viruses will be maintained.

[0077] In (2-B), the concentration of calcium ions carried over from calcium ions bound to the complex of the present invention is reduced below the concentration (effective concentration) required to maintain the binding between Tim4 protein and extracellular vesicles, thereby enabling separation of extracellular vesicles from the complex. Specifically, the calcium ion concentration in the solution containing the complex is typically less than 0.5 mM, preferably less than 0.4 mM, and more preferably less than 0.2 mM.

[0078] Methods for lowering the (effective) concentration of calcium ions include, for example, the following (2-Bi) and (2-B-ii).

[0079] (2-B-i) A method using a calcium ion chelating agent. (2-B-ii) A method using a solution containing no calcium ions.

[0080] (2-B-i): Method using a calcium ion chelating agent (2-B-i) is a method in which, after performing a complex formation step, a complex separation step, and optionally a washing operation, a calcium ion chelating agent is allowed to act on calcium ions bound to the resulting complex and calcium ions carried over from the solution containing the complex, and then the (effective) concentration of calcium ions bound to the complex and calcium ions carried over from the solution containing the complex is reduced (to chelate the calcium ions), thereby separating extracellular vesicles from the complex. This method allows for the production of highly purified, intact extracellular vesicles.

[0081] -Calcium ion chelating agent- Any compound capable of chelating calcium ions may be used as the calcium ion chelating agent, and examples thereof include EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), GLDA (L-glutamic acid diacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), GEDTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), HIDA (2-hydroxyethyliminodi(acetic acid)), DHEG (N,N-bis(2-hydroxyethyl)glycine), CyDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid), Examples of suitable calcium ion chelating agents include EDTA, GEDTA, and CyDTA, with EDTA, GEDTA, and CyDTA being preferred. To allow these calcium ion chelating agents to act on the calcium ions bound to the complex of the present invention, a solution containing the calcium ion chelating agent (hereinafter sometimes abbreviated as "calcium ion chelating agent-containing solution") is generally brought into contact with the pellet-shaped complex, and the calcium ions bound to the complex are reacted with the calcium ion chelating agent in the calcium ion chelating agent-containing solution. The contact of the calcium ion chelating agent-containing solution with the complex can be achieved, for example, by suspending the complex in the solution (when the carrier is beads, etc.), immersing the complex in the solution (when the carrier is a disk-shaped piece or tube, etc.), or adding the solution to the complex (carrier) (when the carrier is a microplate or tube, etc.).

[0082] -Calcium ion chelating agent-containing solution- In (2-Bi), the solution containing the calcium ion chelating agent may be any solution capable of dissolving the calcium ion chelating agent, such as purified water or a buffer solution. Preferred buffer solutions are those that have a buffering effect at a pH of typically 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.). The buffer concentration in these buffer solutions is typically selected from the range of 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is typically selected from the range of 100 to 200 mM, preferably 140 to 160 mM. The calcium ion chelating agent-containing solution may also contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

[0083] In (2-Bi), the concentration of the calcium ion chelating agent in the calcium ion chelating agent-containing solution is usually 0.5 to 500 mM, preferably 0.5 to 100 mM, more preferably 0.5 to 50 mM.

[0084] In (2-Bi), the pH of the calcium ion chelating agent-containing solution is usually 6.0 to 9.0, preferably 7.0 to 8.0, more preferably 7.2 to 7.6.

[0085] In (2-Bi), the amount of the calcium ion chelating agent-containing solution to be mixed with the reaction solution may be an amount that makes the calcium ion concentration in the reaction solution less than the effective concentration and allows extracellular vesicles or viruses to be separated from the complex of the present invention.

[0086] -Contact (reaction) conditions- In (2-Bi), the temperature and time for reacting (contacting) the complex with the calcium ion chelating agent are generally 4.0 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, and the time is generally 5 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds.

[0087] (2-B-ii): Method using a solution containing no calcium ions - (2-B-ii) is a method in which a complex formation step, a complex separation step, and optionally a washing operation are performed, and then the resulting complex is brought into contact with a solution containing no calcium ions, thereby reducing (diluting) the (effective) concentration of calcium ions bound to the complex, thereby separating extracellular vesicles from the complex.

[0088] The contact of the complex with a solution not containing calcium ions can be carried out, for example, by suspending the complex in the solution (when the carrier is beads, etc.), immersing the complex in the solution (when the carrier is a disk-shaped piece or a tube, etc.), or adding the solution to the complex (carrier) (when the carrier is a microplate or a tube, etc.).

[0089] -Calcium ion-free solution- In (2-B-ii), the calcium ion-free solution to be added to the obtained complex may be any solution that does not denature the extracellular vesicles, such as purified water or a buffer solution. Preferred buffer solutions are those that have a buffering effect at a pH of typically 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.). The buffer concentration in these buffer solutions is typically selected from the range of 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is typically selected from the range of 100 to 200 mM, preferably 140 to 160 mM. The calcium ion-free solvent may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

[0090] In (2-B-ii), the amount of the solution not containing calcium ions to be added to the obtained complex may be any amount that can make the calcium ion concentration below the effective concentration.

[0091] Furthermore, if the complex obtained after the complex formation step, complex separation step, and optional washing operation is present in a solution containing calcium ions (e.g., a reaction solution after the complex formation step, or a calcium ion-containing washing solution), the calcium ion-containing solution may be replaced with a solution not containing calcium ions and / or diluted with a solution not containing calcium ions so that the final calcium ion concentration is less than the above-mentioned effective concentration.

[0092] The solutions that have been brought into contact with and reacted with the complex (protein denaturant-containing solution, calcium ion chelator-containing solution, calcium ion-free solution) contain the carrier and extracellular vesicles that have separated (released) from the carrier (complex). Therefore, by removing the carrier from the above solution and recovering only the solution, a solution containing extracellular vesicles can be obtained.

[0093] By using the Tim4 carrier, extracellular vesicles can be efficiently removed from a sample, and a sample with few contaminants can be obtained.

[0094] The removal method of the present invention can accurately and efficiently remove extracellular vesicles from a sample that cannot be completely removed by conventional methods such as ultracentrifugation and polymer precipitation treatment.

[0095] <Method for Removing Extracellular Vesicles from a Sample> The present invention provides a method for removing extracellular vesicles, comprising: producing Tim4 protein by the method for producing Tim4 protein of the present invention; and contacting the produced Tim4 protein with extracellular vesicles. The method for removing extracellular vesicles of the present invention (hereinafter sometimes abbreviated as "the removal method of the present invention") is characterized by comprising the following steps: (1) a step of forming a complex between Tim4 protein bound to a carrier and extracellular vesicles in a sample (hereinafter also referred to as "complex formation step"), and (2) a step of separating the complex from the sample (hereinafter also referred to as "complex separation step").

[0096] <Complex Formation Step> The complex formation step in the removal method of the present invention is the same as the complex formation step in the acquisition method of the present invention, and various preferred conditions are also the same.

[0097] <Complex Separation Step> The complex separation step in the removal method of the present invention is a step of separating the resulting complex of the Tim4 protein bound to the carrier (Tim4 carrier) and extracellular vesicles in the sample from the sample after the complex formation step, thereby obtaining the separated sample. This allows for the production of a sample from which extracellular vesicles or viruses have been removed.

[0098] The complex separation step in the removal method of the present invention may be any method that can remove the complex of the present invention from the sample, and examples thereof include the same methods as those used in the complex separation step in the above-mentioned acquisition method of the present invention. After the complex and the sample are separated in this manner, the separated sample can be obtained (recovered) by a method known per se.

[0099] This makes it possible to accurately and efficiently remove extracellular vesicles from samples that cannot be completely removed by conventional methods such as ultracentrifugation and polymer precipitation treatment.

[0100] Furthermore, by repeating the above-described complex formation step and complex separation step multiple times, extracellular vesicles can be removed from a sample more efficiently. When repeating the above-described complex formation step and complex separation step, a new carrier can be used, or a used carrier can be reused. To reuse the carrier, the complex removed in the complex separation step of the removal method of the present invention can be treated in the same manner as in the acquisition step of the acquisition method of the present invention ((2-B) Method of Reducing Calcium Ion Concentration) using a solution containing a calcium ion chelating agent or a solution not containing calcium ions. This allows the Tim4 carrier and extracellular vesicles to be separated from the complex. In other words, since the Tim4 carrier can be reused in the removal method of the present invention, reusing the Tim4 carrier allows for accurate and efficient removal of extracellular vesicles or viruses from a sample.

[0101] <Method for detecting extracellular vesicles in a sample> The present invention provides a method for detecting extracellular vesicles, comprising: producing Tim4 protein by the method for producing Tim4 protein of the present invention; and contacting the produced Tim4 protein with extracellular vesicles. The method for detecting extracellular vesicles of the present invention (hereinafter also referred to as the "detection method of the present invention") is characterized by comprising the following steps: (1) a step of forming a complex between Tim4 protein bound to a carrier and extracellular vesicles in a sample (complex formation step); and (2) a step of detecting the complex (detection step).

[0102] <Complex Formation Step> The complex formation step in the detection method of the present invention is a step of forming a complex between the Tim4 carrier and extracellular vesicles in the sample.

[0103] -Sample- The sample is the same as the sample in the acquisition method of the present invention.

[0104] - Amount of sample - When beads are used as the carrier, the amount of sample in the complex formation step of the detection method of the present invention is usually 0.1 to 1000 ml, preferably 0.1 to 500 ml, and more preferably 0.1 to 100 ml per 1 mg of Tim4 carrier. When a microplate is used as the carrier, the amount of sample is usually 50 μL to 300 μL, preferably 100 μL to 200 μL per well.

[0105] - Temperature - In the complex formation step of the detection method of the present invention, the temperature at which the Tim4 carrier is brought into contact with extracellular vesicles in the sample is generally 2 to 37°C, preferably 2 to 30°C.

[0106] -Time- In the complex formation step of the detection method of the present invention, the contact time between Tim4 protein and the sample is generally 0.5 to 24 hours, preferably 1 to 20 hours, and more preferably 1 to 12 hours.

[0107] <Detection step in the detection method of the present invention> The detection step in the detection method of the present invention is a step of detecting a complex between the Tim4 carrier and extracellular vesicles in the sample after a complex formation step, if necessary, a complex separation step in the sample, and if necessary, a further washing operation.

[0108] - Complex separation step - The complex separation step in the detection method of the present invention is a step of removing the sample from the complex between the Tim4 protein bound to the carrier (Tim4 carrier) and extracellular vesicles in the sample, if necessary, after the complex formation step.

[0109] The complex separation step in the detection method of the present invention may be any method that can separate the complex from the sample, in other words, so-called B / F separation, and a B / F separation method used in this field can be used. Such a method is the same as the complex separation step in the acquisition method of the present invention.

[0110] <Washing Procedure> After the complex formation step, a complex separation step may be carried out if necessary, and the obtained complex may further be washed with a washing solution containing calcium ions (hereinafter also referred to as "washing procedure"). The washing procedure can remove contaminants in the sample, such as cell-derived components attached to the carrier surface. Various conditions, such as the washing method, are the same as those for the washing procedure in the acquisition method of the present invention.

[0111] <Detection Step> The detection step in the detection method of the present invention may be any method capable of detecting the presence and / or amount of a complex, and any method known per se for immunological measurement can be used. The detection step of the present invention is not particularly limited except for the use of a Tim4 carrier prepared by the method described above. Such immunological measurement methods include, for example, measurement methods that utilize agglutination reactions, such as the reverse passive agglutination reaction (Tokyo Kagaku Dojin, Continued Biochemical Experimental Lectures 5, Immunobiochemical Research Methods, pp. 36-37; Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, pp. 844-845, etc.), optical measurement methods that apply agglutination reactions, such as the nephelometry method (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, pp. 851-853, etc.) and immunoturbidimetry (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, pp. 853-854, etc.), radioimmunoassay (RIA) (Tokyo Kagaku Dojin, Continued Biochemical Experimental Lectures 5, Immunobiochemical Research Methods, pp. 57-61; Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, pp. 856-862, etc.), and immunoradiometric assay (IRMA) (Kane ...). Examples of immunoassay methods include those known per se, such as enzyme immunoassay (EIA) (Tokyo 10 Kagaku Dojin, Continued Biochemistry Experiment Course 5, Immunobiochemistry Research Methods, p. 62-65; Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, p. 862-865; JP 56-106154 A, JP 58-23796 A, etc.), solid-phase enzyme-linked immunosorbent assay (ELISA) (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, p. 1145-1149 A, etc.), fluorescent / luminescent immunoassay (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Guide, 30th Edition, p. 865-867 A, etc.), and flow cytometry, and among these, enzyme-linked immunosorbent assay (ELISA) or flow cytometry is preferred.

[0112] When the detection step of the present invention is performed by enzyme-linked immunosorbent assay (ELISA) or flow cytometry, it can be performed according to a method known per se, as long as the complex is the target of detection. For example, a method using a labeled primary antibody in which the primary antibody is labeled with a label such as an anti-extracellular vesicle antibody that binds to extracellular vesicles, or a method using the above primary antibody and a labeled secondary antibody that binds to the above primary antibody, can be used. The labeled primary antibody and labeled secondary antibody can be any antibody used in ELISA or flow cytometry, such as fluorescently labeled antibodies labeled with fluorescent substances such as Cy3, Cy5, FITC, rhodamine, and PE, enzyme-labeled antibodies labeled with enzymes such as peroxidase and alkaline phosphatase, magnetic bead-labeled antibodies, and infrared-labeled antibodies. The fluorescence of these labeled antibodies can be measured by a method known per se corresponding to the labeling method (label) of the labeled antibody.

[0113] -Dilution of Labeled Antibody- In the detection method of the present invention, the dilution of the labeled primary antibody, or the primary and secondary antibodies, to be reacted with the complex may be any solution that does not interfere with the binding of the complex of Tim4 protein and extracellular vesicles in the sample to the antibody. Examples include water and buffer solutions (e.g., TBS, HBS, etc.) that have a buffering action at pH 7.0 to 8.0, preferably 7.2 to 7.6. The buffer concentration in these buffer solutions is typically selected from the range of 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is typically selected from the range of 100 to 200 mM, preferably 140 to 160 mM. This solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., in amounts that do not interfere with the binding of the complex of Tim4 carrier and extracellular vesicles in the sample. The surfactant concentration in the solution containing extracellular vesicles is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%. The calcium ion concentration in the diluent is usually 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM.

[0114] In the detection method of the present invention, the dilution ratio of the labeled primary antibody, or the primary antibody and labeled secondary antibody, to be reacted with the complex varies depending on the activity and concentration of the antibody, but is usually 10-fold to 1,000,000-fold, preferably 1,000-fold to 100,000-fold.

[0115] In the detection method of the present invention, the amount of the labeled primary antibody, or the diluted solution of the primary antibody and labeled secondary antibody solution, to be reacted with the complex is usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL per 1 mg of carrier when the carrier is beads, and is usually 50 μL to 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to 100 μL per well when the carrier is a microplate.

[0116] -Reaction (Contact) Temperature- In the detection method of the present invention, the reaction temperature between the complex and the labeled primary antibody, or between the primary antibody and the labeled secondary antibody, is generally 2 to 37°C, preferably 11 to 337°C, and more preferably 20 to 30°C.

[0117] -Reaction (contact) time- In the detection method of the present invention, the reaction time between the complex and the labeled primary antibody, or between the primary antibody and the labeled secondary antibody, is usually 0.5 to 12 hours, preferably 1 to 4 hours, and more preferably 1 to 2 hours.

[0118] In the detection operation of the detection method of the present invention, when a labeled primary antibody is used, the unreacted labeled primary antibody can be removed by a washing operation after reacting the complex with the labeled primary antibody. Furthermore, when a primary antibody and a labeled secondary antibody are used, the unreacted primary antibody and labeled secondary antibody can be removed by a washing operation after reacting the complex with the primary antibody or after reacting the complex of the complex and the primary antibody with the labeled secondary antibody. As the washing method, a washing method commonly used in this field can be used. The washing solution used in the washing procedure may be any solution that contains calcium ions, typically at 0.5 to 100 mM, preferably typically at 1 to 10 mM, and more preferably typically at 2 to 5 mM, and does not affect the binding of the complex between the extracellular vesicles, Tim4 protein, and carrier. Examples include buffer solutions (e.g., TBS and HBS) that contain calcium ions, typically at 0.5 to 100 mM, preferably typically at 1 to 10 mM, and more preferably typically at 2 to 5 mM, and have a buffering action at pH 7.0 to 8.0, preferably 7.2 to 7.6, and do not cause calcium precipitation. The buffer concentration in these buffer solutions is appropriately selected from the range of typically 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is appropriately selected from the range of typically 100 to 200 mM, preferably 140 to 160 mM. This solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as the amount does not interfere with the binding of the extracellular vesicles, Tim4 protein, and carrier in the complex. Examples of surfactants include Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.). The concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%. When the carrier is beads, the amount of washing solution used per 1 mg of carrier is typically 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL. When the carrier is a microplate, the amount of washing solution used per well is typically 100 μL to 300 μL, preferably 200 μL to 300 μL, and the washing solution is removed after addition. These washing procedures may be repeated multiple times as necessary.

[0119] -Detection- When the detection step in the detection method of the present invention is colorimetric detection, examples of the label for the labeled primary antibody or labeled secondary antibody include peroxidase, alkaline phosphatase, etc. These labels may be detected according to a method known per se depending on the label.

[0120] The color-developing substrate solution for color detection may be any color-developing substrate solution commonly used in this field. For example, when peroxidase is used as the label, examples include a TMB (tetramethylbenzidine) solution or an OPD (orthophenylenediamine) solution, and preferably a TMB solution.

[0121] The amount of colorimetric substrate used in colorimetric detection is usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL per 1 mg of carrier when the carrier is a bead, and usually 50 μL to 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to 100 μL per well when the carrier is a microplate.

[0122] When the detection step in the detection method of the present invention is color development detection, the reaction time with the color development substrate solution is usually 5 to 60 minutes, preferably 10 to 40 minutes.

[0123] When the detection step in the detection method of the present invention is color development detection, the reaction temperature with the color development substrate solution is usually 2°C to 37°C, preferably 20°C to 30°C.

[0124] When the detection step in the detection method of the present invention is color development detection, a strong acid such as 1 mol / L hydrochloric acid or 1 mol / L sulfuric acid is usually added as a reaction stop solution in an amount equal to the amount of the color development substrate solution to stop the color development reaction.

[0125] When the detection step in the detection method of the present invention is fluorescence detection and a fluorescent substance is used as the label for the labeled primary antibody or labeled secondary antibody, a fluorescence measurement solution is added to the complex between the complex and the labeled primary antibody, or to the complex between the complex and the primary antibody and the labeled secondary antibody, and fluorescence is measured. The fluorescence measurement solution may be a fluorescence measurement solution (hereinafter sometimes abbreviated as "measurement solution") that is typically used in this field. The measurement solution used in the fluorescence measurement solution may be any solution that contains calcium ions typically at 0.5 to 100 mM, preferably typically at 1 to 10 mM, and more preferably typically at 2 to 5 mM, and does not affect the binding between the extracellular vesicles, Tim4 protein, and carrier in the complex. For example, a buffer solution (e.g., TBS or HBS) that contains calcium ions typically at 0.5 to 100 mM, preferably typically at 1 to 10 mM, and more preferably typically at 2 to 5 mM, and that does not affect the binding between the extracellular vesicles, Tim4 protein, and carrier in the complex and that does not precipitate calcium, may be used. The buffer concentration in these buffer solutions is typically selected from the range of 5 to 50 mM, preferably 10 to 30 mM, and the NaCl concentration is typically selected from the range of 100 to 200 mM, preferably 140 to 160 mM. This solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins such as BSA, and the like, as long as the amount does not interfere with the binding of the extracellular vesicles, Tim4 protein, and carrier in the complex. Examples of surfactants include Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.), and the concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%. When the detection method of the present invention is an ELISA method and the carrier of the present invention is a microplate, the amount of measurement solution used per well is typically 50 μL to 300 μL, preferably 50 μL to 200 μL. When the detection method according to the present invention is a flow cytometry method and the carrier according to the present invention is beads, the amount is usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, more preferably 0.1 mL to 100 mL per mg of the carrier according to the present invention.

[0126] <Kit of the present invention> A first aspect of the kit of the present invention is a kit for obtaining, removing, or detecting extracellular vesicles, comprising: a carrier to which the Tim4 protein of the present invention is bound; and an antibody that binds to extracellular vesicles.

[0127] A second aspect of the kit of the present invention is a kit for obtaining, removing, or detecting extracellular vesicles, comprising: the Tim4 protein of the present invention; a carrier; and an antibody that binds to extracellular vesicles.

[0128] The carrier to which the Tim4 protein of the present invention is bound, the Tim4 protein of the present invention, the carrier, and the antibody that binds to the extracellular vesicles are as described herein.

[0129] The reagents contained in the kit may include reagents commonly used in this field, such as buffers, sensitizers, surfactants, preservatives (e.g., sodium azide, salicylic acid, benzoic acid, etc.), stabilizers (e.g., albumin, globulin, water-soluble gelatin, surfactants, sugars, etc.), activators, agents for preventing the effects of coexisting substances, and other substances commonly used in this field, which do not interfere with the stability of coexisting reagents or with the reaction between the Tim4 protein of the present invention and the carrier, or the reaction or binding between the Tim4 protein bound to the carrier and extracellular vesicles in the sample. The concentration ranges of these reagents may be appropriately selected from those commonly used to exert the effects of each reagent.

[0130] The kit of the present invention may contain instructions for the acquisition method of the present invention, the removal method of the present invention, and the detection method of the present invention. The above-mentioned "instructions" refer to an instruction manual, attached document, pamphlet (leaflet), etc. of the kit in which the characteristics, principles, operating procedures, determination procedures, etc. of the above-mentioned methods are substantially described in text or diagrams.

[0131] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In this specification, mM represents mmol / L, and M represents mol / L.

[0132] Example 1: Evaluation of the effect of acid treatment on the EV-binding ability of Tim4 protein Tim4 protein fused with an Fc tag was prepared and the effect on EV-binding ability was evaluated. Amino acid sequence of Fc-tagged Tim4 protein (SEQ ID NO: 1) MSKGLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTLPCHYLSWSQSRNSMCWGKGSCPNSK CNAELLRTDGTRIISRKSTKYTLLGKVQFGEVSLTISNTNRGDSGVYCCRIEVPGWFNDVKKNV RLELRRATTTKKPTTTTRPTTTPYVTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTTTC PSTTPGSFSQETTKGSAFTTESETLPASNHSQRSMMTISTDIAVLRPTGSNPGILPSTSQLTTQ KTTLTTSESLQKTTKSHQINSRQTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0133]

[0134] <Preparation of culture supernatant> To transfect the coding sequence of Tim4 protein fused with an Fc tag having the amino acid sequence shown in SEQ ID NO: 1, CHO cells expressing Tim4 protein were constructed using the pCAG-Neo vector and cultured for 12 days from Day 0 to Day 11 according to the following procedure. Day 0: 0.3 × 10 CHO cells for Tim4 protein expression were placed in a 1 L Erlenmeyer flask. ６The cells were seeded into 240 mL of BalanCD CHO Growth A (Fujifilm Wako Pure Chemical Corporation) at a concentration of 100 cells / mL, and culture was initiated at 37 ° C. Day 3: 4% BalanCD CHO Feed 4 solution (Fujifilm Wako Pure Chemical Corporation) was added to the above medium. Day 4: The same procedure as Day 3 was performed. Day 5: After adding the feed as on Day 4, the glucose concentration was measured using a Cedex Bio (Roche), and a glucose solution was added to a final concentration of 10 g / mL. Day 6: The same procedure as on Day 5 was performed, and the culture temperature was changed to 32 ° C. Day 7 and Day 10: The culture temperature was maintained at 32 ° C, and the same procedure as on Day 5 was performed. Day 11: The culture medium was collected and centrifuged at 5000 g for 30 minutes, and the culture supernatant was collected.

[0135] <Purification of Tim4 Protein> The collected culture supernatant was treated as follows to purify Tim4 protein. The culture supernatant was filtered through a 0.22 μm filter (Corning) to obtain a 0.22 μm filtered supernatant sample. Hereinafter, 1 CV (column volume) = 400 μL. To 10 mL of the 0.22 μm filtered supernatant sample, 800 μL of 50% slurry KanCapA 3G (KANEKA) washed with 20 CV of D-PBS(-) (Fujifilm Wako Pure Chemical Corporation) was added, and the mixture was allowed to react overnight at 4°C on a rotator. The reaction solution was transferred to poly-Prep Chromatography Columns (BIORAD) and washed with 20 CV of wash buffer (1) (20 mM sodium phosphate, 1 M NaCl, 10 mM EDTA) and 5 CV of wash buffer (2) (D-PBS(-)). Subsequently, elution was performed with 10 CV of elution buffer (0.3 M KCl at pH 1.0, 1.25, 1.50, 1.75, 2.0, and 2.5), followed by adjustment to pH 7.2 with neutralization buffer (0.8 M CAPS-NaOH at pH 11). Subsequently, buffer exchange with D-PBS(-) was performed using a dialysis membrane (Fujifilm Wako Pure Chemical Corporation). The external solution was diluted 100-fold or more, twice, and each exchange lasted for at least 2 hours.

[0136] <Quantification of Tim4 Protein> The purified Tim4 protein was quantified as follows. Protein quantification was performed using a protein assay reagent (Fujifilm Wako Pure Chemical Corporation) according to the kit's protocol. Reagent A and reagent B were mixed at a ratio of 50:1 to prepare a sample buffer. BSA solution (Fujifilm Wako Pure Chemical Corporation) was used as a standard solution to prepare solutions at 0, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL. The purified Tim4 protein was diluted 20-fold with D-PBS(-) to prepare the measurement sample. The standard solution and measurement sample were added to a 96-well plate (ThermoFisher) at 25 μL / well, followed by the addition of the sample buffer at 200 μL / well and incubation at 37°C for 30 minutes. Thereafter, absorbance at 560 nm was measured using Spark (Tecan), and Tim4 protein was quantified using a calibration curve.

[0137] <Measurement of binding ability of Tim4 protein to extracellular vesicles> 100 μL / well of purified Tim4 protein (0.2 μg / mL, 50 mM MOPS buffer (pH 6.25)) was added to each well of a 96-well plate (ThermoFisher) and allowed to react overnight at 4°C. After the reaction, 100 μL / well of 10 mg / mL (TBS) BlockAce (KAC) was added to each well of the 96-well plate. The plate was shaken at room temperature for 1 hour, and then each well was washed with a washing buffer (0.05% Tween 20 + 2 mM CaCl ２ After washing, 100 μL / well of COLO201-derived exosomes (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted to 20 ng / ml was added to each well. The dilution was performed using dilution buffer (0.05% Tween 20 + 2 mM CaCl ２+ 1.5% BSA / TBS). After shaking at room temperature for 2 hours, each well was washed three times with the above washing buffer. Next, anti-CD63 monoclonal antibody (3-13), biotin conjugated (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted to 250 ng / ml with the above dilution buffer and added at 100 μL / well. After shaking at room temperature for 1 hour, each well was washed three times with the above washing buffer. Next, Poly-HRP Streptavidin (ThermoFisher) was diluted to 20 ng / ml with the above dilution buffer and added at 100 μL / well. After shaking at room temperature for 2 hours, each well was washed five times with the above washing buffer. TMB solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at 100 μL / well. After standing at room temperature for 30 minutes, 1 M (mol / L) HCl was added to each well at 100 μL / well. The absorbance at 450 nm was measured using a Spark (Tecan).

[0138] The results are shown in Table 1 and Figure 1. As the pH of the elution buffer was decreased toward the strongly acidic side, the absorbance increased and reached a plateau at pH 1.75. Therefore, it was found that contacting Tim4 protein with a strong acid unexpectedly improved its extracellular vesicle-binding ability.

[0139]

[0140] <Preparation of Tim1 Protein Solution> Tim1 protein (adipogen) was dissolved in D-PBS(-) to a concentration of 1 mg / mL.

[0141] <Changes in binding ability of Tim1 protein to extracellular vesicles due to acid treatment> Tim1 protein (0.2 μg / mL, 50 mM MOPS buffer (pH 6.25)) was added to each well of a 96-well plate at 100 μL / well and incubated overnight at 4°C. After removing the protein solution, 100 μL / well of 0.3 M KCl solutions at pH 1.0, 1.75, and 2.5 were added and incubated for 1 hour. After the reaction, each well was washed with a washing buffer (0.05% Tween 20 + 2 mM CaCl ２The wells were washed three times with PBS (TBS). 100 μL / well of 10 mg / mL (TBS) BlockAce (KAC) was added to each well of the 96-well plate. After shaking at room temperature for 1 hour, each well was washed three times with wash buffer. COLO201-derived exosomes diluted to 20 ng / ml were added to each well after washing at 100 μL / well. Dilution was performed with dilution buffer. After shaking at room temperature for 2 hours, each well was washed three times with the wash buffer. Next, anti-CD63 monoclonal antibody (3-13), biotin-conjugated, was diluted to 250 ng / ml with the dilution buffer and added at 100 μL / well. After shaking at room temperature for 1 hour, each well was washed three times with the wash buffer. Next, Poly-HRP Streptavidin was diluted to 20 ng / ml with the dilution buffer and added at 100 μL / well. After shaking at room temperature for 2 hours, each well was washed five times with the washing buffer. TMB solution was added at 100 μL / well. After leaving the plate at room temperature for 30 minutes, 1 M (mol / L) HCl was added to each well at 100 μL / well. The absorbance at 450 nm was measured using a Spark.

[0142] The results are shown in Table 2 and Figure 2. The absorbance of Tim1 protein decreased as the pH of the acid treatment decreased. Therefore, it was found that the improvement in extracellular vesicle binding activity by strong acid treatment is not a property common to Tim proteins, but is a property unique to Tim4 protein.

[0143]

[0144] <Measurement of binding ability of Tim4 protein to extracellular vesicles> The same method as in Example 1 was used, except that 0.1 M glycine-HCl buffers at pH 1.0, 1.25, 1.50, 1.75, 2.0, and 2.5 were used as the elution buffer instead of 0.3 M KCl at pH 1.0, 1.25, 1.50, 1.75, 2.0, and 2.5. The results are shown in Table 3 and Figure 3.

[0145]

Claims

1. A method for producing Tim4 protein, which comprises contacting T-cell immunoglobulin mucin domain-containing molecule 4 protein, also referred to as Tim4, with an acidic solution having a pH of 2 or less.

2. The method of claim 1, wherein the pH of the acidic solution is 1.75 or less.

3. The method of claim 1, wherein the extracellular vesicle binding activity of the Tim4 protein produced is improved compared to when the protein is contacted with an acidic solution at pH 2.

5.

4. A method for improving the extracellular vesicle binding activity of Tim4 protein, which comprises contacting T-cell immunoglobulin mucin domain-containing molecule 4 protein, also known as Tim4, with an acidic solution having a pH of 2 or less.

5. A method for obtaining, removing, or detecting extracellular vesicles, comprising: producing Tim4 protein by the method according to any one of claims 1 to 3; and contacting the produced Tim4 protein with extracellular vesicles.

6. Tim4 protein produced by the method of any one of claims 1 to 3.

7. A kit for obtaining, removing or detecting extracellular vesicles, comprising a carrier to which the Tim4 protein of claim 6 is bound, and an antibody that binds to extracellular vesicles.

8. A kit for obtaining, removing, or detecting extracellular vesicles, comprising the Tim4 protein according to claim 6, a carrier, and an antibody that binds to extracellular vesicles.

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