Method for screening cells producing target substance, method for producing nucleic acid, and method for producing target substance

Abstract

The present invention addresses the problem of providing: a method for screening cells having excellent screening sensitivity and screening efficiency; a method for producing a nucleic acid for obtaining a nucleic acid from cells obtained by the screening method; and a method for producing a target substance for producing a target substance using the nucleic acid. According to the present invention, provided is a method for screening target cells, which are cells that produce a target substance, comprising the following (1)-(3): (1) immobilizing vesicles having a target substance on the membrane surface within micropores; (2) introducing candidate cells obtained from a cell population including the target cells and a labeled substance that binds to a target substance into the micropores to which the vesicles are immobilized; and (3) identifying the target cell by detecting the marker bound to a target substance bound to the target substance.

Description

Technical Field

[0001] The present invention relates to a method for screening cells producing a target substance, a method for producing a nucleic acid, and a method for producing a target substance. Background Art

[0002] In recent years, for various purposes, substances that specifically bind to proteins such as cell membrane proteins (also referred to as "specific binding substances") have been sought. Examples of specific binding substances include antibodies and protein ligands.

[0003] Such specific binding substances are useful as, for example, research reagents, diagnostic drugs, therapeutic drugs, and the like.

[0004] As a technique for searching for the above-mentioned specific binding substance, for example, all or part of the protein is immobilized in a well and the binding to a candidate substance of the specific binding substance is evaluated by ELISA (Enzyme-Linked Immuno Sorbent Assay) or the like.

[0005] Furthermore, for example, as an exploration method taking into account the three-dimensional structure of cell membrane proteins, in the above-mentioned ELISA, cells expressing target cell membrane proteins on the cell membrane are used instead of all or part of the above-mentioned proteins to evaluate the binding of candidate substances for specific binding substances.

[0006] On the other hand, the above-mentioned specific binding substances such as ligands and antibodies can be secreted into the culture supernatant by culturing cells derived from humans or non-human animals or recombinant cells using genetic recombination technology.

[0007] Here, in the case of exploring cells that produce an unknown substance that specifically binds to a target protein, a producing cell population including cells that produce candidate substances that specifically bind to the substance needs to be an object of investigation that includes a total of several thousand to several hundred thousand cells (for example, an antibody-producing hybridoma cell bank).

[0008] As such an exploration method, Patent Document 1 describes a method for isolating single antibody-producing particles specific to a selected antigen, comprising the following steps: (1) preparing vesicles, preferably exosomes, displaying the selected antigen and a marker; (2) contacting the vesicles of step 1 with an antibody library; and (3) identifying and isolating single antibody-producing particles that react with the vesicles.

[0009] Patent document 2 describes a cell selection method, which is a method for selecting target cells that produce a target substance that specifically binds to a desired cell membrane protein from a second cell population, comprising the following steps: a) providing a substrate having a plurality of micropores; b) allowing a first cell that expresses the cell membrane protein on the cell surface to adhere to each of the micropores; c) after step b), introducing one or two second cells separated from the population into each of the micropores, and allowing the first cell and the second cell to coexist in the micropores; d) after step c), determining a micropore containing a first cell bound to the target substance; and e) recovering the second cell from the micropore determined in step d) as the target cell.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application No. 2006-518212

[0013] Patent Document 2: International Publication No. 2020 / 171020 Summary of the invention

[0014] Technical issues to be solved by the invention

[0015] As described above, in the case of exploring cells that produce unknown substances that specifically bind to a target protein, it is necessary to screen the target cells from a producing cell population containing thousands to hundreds of thousands of cells, requiring improved screening sensitivity (high positive rate, few false negatives) and improved screening efficiency (i.e., more efficient cell selection).

[0016] An object of the present invention is to provide a method for screening cells having excellent screening sensitivity and screening efficiency, a method for producing nucleic acid by obtaining nucleic acid from cells obtained by the screening method, and a method for producing a target substance by using the nucleic acid.

[0017] Means for solving technical problems

[0018] Representative embodiments of the present invention are shown below, but the present invention is not limited to these.

[0019] <1> A method for screening target cells that produce a target substance, comprising the following (1) to (3):

[0020] (1) Immobilizing vesicles with target substances on the membrane surface into micropores;

[0021] (2) introducing candidate cells obtained from a cell population containing the target cells and a substance that binds to the target substance and is labeled into the micropores in which the vesicles are immobilized; and

[0022] (3) The target cell is identified by detecting the marker bound to the target substance bound to the target substance.

[0023] <2> The screening method according to <1>, wherein

[0024] The above-mentioned target substance is a membrane protein.

[0025] <3> The screening method according to <1> or <2>, wherein:

[0026] The above-mentioned target substance is an antibody.

[0027] <4> The screening method according to any one of <1> to <3>, wherein

[0028] The target cells are spleen cells or B cells derived from lymphoid tissue.

[0029] <5> The screening method according to any one of <1> to <3>, wherein

[0030] The above-mentioned target cells are CHO cells or hybridoma cells.

[0031] <6> The screening method according to any one of <1> to <5>, wherein

[0032] In the above (1), the vesicles are fixed in the micropores using at least one selected from the group consisting of Tim family proteins, annexin V, anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies.

[0033] <7> The screening method according to <6>, wherein

[0034] In the above (1), the vesicles are fixed in the micropores using Tim family proteins.

[0035] <8> The screening method according to any one of <1> to <7>, wherein

[0036] In the above (2), the candidate cell introduced into the microwell is a single cell to be separated.

[0037] <9> The screening method according to any one of <1> to <8>, wherein

[0038] The above-mentioned vesicles are extracellular vesicles.

[0039] <10> The screening method according to any one of <1> to <9>, wherein

[0040] The diameter of the above vesicles is 30 to 1000 nm.

[0041] <11> The screening method according to any one of <1> to <10>, wherein

[0042] The detection in (3) above is fluorescence detection.

[0043] <12> The screening method according to <11>, wherein

[0044] Identification of target cells based on the above-mentioned fluorescence detection is performed by excitation at one wavelength and fluorescence measurement at one wavelength.

[0045] <13> The screening method according to <11> or <12>, wherein

[0046] In the above (3), the target cells are identified by detecting the wells where fluorescence is confirmed at the side walls of the microwells.

[0047] <14> The screening method according to any one of <1> to <13>, further comprising the following (4):

[0048] (4) Recovering the target cells identified in the above (3).

[0049] <15> A method for producing a nucleic acid, comprising the step of obtaining a nucleic acid encoding a target substance from the target cell obtained by the screening method according to any one of <1> to <14>.

[0050] <16> A method for producing a target substance, comprising producing the target substance using the nucleic acid obtained by the method for producing a nucleic acid according to <15>.

[0051] Effects of the Invention

[0052] According to the present invention, there are provided a screening method for cells having excellent screening sensitivity and screening efficiency, a method for producing nucleic acid by obtaining nucleic acid from cells obtained by the screening method, and a method for producing a target substance by using the nucleic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a graph showing the analysis results of the particle size distribution in Example 1.

[0054] Figure 2 This is a graph showing the values ​​of fluorescence intensity in Example 2.

[0055] Figure 3 This is a diagram showing an example of the imaging result in Example 3.

[0056] Figure 4 This is a diagram showing an example of the imaging results of negative cells in Example 3.

[0057] Figure 5 This is a diagram showing an example of the imaging results of positive cells observed by Cell-ELISA in Example 4.

[0058] Figure 6 This is a diagram showing an example of the imaging results of negative cells observed by Cell-ELISA in Example 4.

[0059] Figure 7 This is a graph showing the absorbance measurement results in Example 5.

[0060] Figure 8 This is a graph showing the absorbance measurement results in Example 6.

[0061] Fig. 9 This is a graph showing the value of the S / B ratio in Example 6.

[0062] Fig.10 This is a graph showing the results of measuring antibody activity in Example 7. DETAILED DESCRIPTION

[0063] (Cell Screening Method)

[0064] The screening method of the present invention is a method for screening cells that produce a target substance, that is, target cells, and includes the following (1) to (3).

[0065] (1) Immobilizing vesicles with target substances on the membrane surface into micropores;

[0066] (2) introducing candidate cells obtained from a cell population containing the target cells and a substance that binds to the target substance and is labeled into the micropores in which the vesicles are immobilized; and

[0067] (3) The target cell is identified by detecting the marker bound to the target substance bound to the target substance.

[0068] Hereinafter, a vesicle having a target substance bound to the target substance on the membrane surface is also referred to as “EV” for short.

[0069] According to the present invention, a screening method having excellent screening sensitivity and screening efficiency is provided.

[0070] Here, for example, the use of vesicles expressing selected antigens and markers is described in Patent Document 1. The expression levels of antigens and markers that can be expressed on vesicles are limited, and it is difficult to identify cells producing target substances with high sensitivity.

[0071] Furthermore, the method described in Patent Document 1 is a method using a flow cytometer. Here, the method using a flow cytometer also has the following problems: it is impossible to obtain secretory antibody-producing cells, the criterion (threshold) for determining whether or not a positive result is vague, it is difficult to reliably recover a single cell, the cell is severely damaged, and false positives due to nonspecific fluorescence are generated.

[0072] In the method described in Patent Document 2, it is necessary to place two cells, a first cell expressing a cell membrane protein such as a CHO (Hamster Ovary) cell, and a second cell producing a target substance such as a B cell, into one microwell. Therefore, it is necessary to increase the size of the microwell itself, resulting in a decrease in the number of holes on the substrate.

[0073] Furthermore, wells that do not include both cells expressing cell membrane proteins and cells producing target substances cannot be used for analysis, thereby reducing the number of wells that can be analyzed.

[0074] As described above, in the method described in Patent Document 2, the number of wells that can be used for analysis is limited, and it is difficult to identify cells that efficiently produce a target substance.

[0075] On the other hand, in the screening method according to the present invention, various well-known markers having excellent detection capabilities can be used as markers to be detected.

[0076] Furthermore, compared to the method using a flow cytometer, the screening method of the present invention can obtain secretory antibody-producing cells, etc., and since it can determine whether a test result is positive based on the detected shape, the criterion for determining whether a test result is positive or not is clear, it is easy to recover a cell, there is little damage to the cell, and it has the advantages of being less likely to produce false positives caused by nonspecific fluorescence.

[0077] Furthermore, in the screening method according to the present invention, it is only necessary to introduce candidate cells into the microwells on which EVs are immobilized, so the microwells themselves can be made smaller, thereby increasing the number of wells present on the substrate.

[0078] In addition, in the screening method involved in the present invention, since EVs can be easily fixed to microwells, any microwell into which candidate cells are introduced can be used for analysis. That is, compared with the method described in Patent Document 2, which requires both the first cell and the second cell to be placed in one microwell, the number of wells that can be analyzed increases.

[0079] In summary, it can be said that the screening method of the present invention is more excellent in screening sensitivity and screening efficiency than the prior art.

[0080] The screening method of the present invention is described in detail below. In the present invention, mol / l is also described as M. "mmol / l" and "μmol / l" are also described as "mM" and "μM".

[0081] Furthermore, in the present invention, a combination of preferred embodiments is a more preferred embodiment.

[0082] <Cells producing target substances (target cells)>

[0083] The target cells are cells that produce the target substance.

[0084] [Target substance]

[0085] Examples of the target substance include polypeptides, cyclic peptides, proteins, and the like that selectively bind to the target substance.

[0086] More specifically, peptide hormones, cytokines, antibodies, artificial polypeptides, artificial cyclic peptides and the like are mentioned.

[0087] Among these, the target substance is preferably an antibody or a ligand, and more preferably an antibody.

[0088] In the present invention, the term "antibody" can be replaced by "immunoglobulin." The antibody in the present invention also includes a functional fragment of the antibody.

[0089] Here, the "functional fragment of an antibody" refers to a partial fragment of an antibody (ie, immunoglobulin) that retains at least one function against an antigen.

[0090] Examples of the partial fragments include F(ab')2, Fab, Fv, disulfide bond Fv, single-chain antibody (scFv, VH-VL), VH, VHH and polymers thereof, and fusions of these with the heavy chain CH3 region.

[0091] The antibody may be a multispecific antibody. Examples of multispecific antibodies include diabodies (eg, International Publication No. 1993 / 011161), which are one type of bispecific antibodies.

[0092] The class (isotype) of the antibody in the present invention is not particularly limited, and may be, for example, any of IgG, IgM, IgA, IgD, and IgE.

[0093] Furthermore, the subclass of the antibody is not particularly limited. For example, if it is IgG, it may be any subclass such as IgG1, IgG2, IgG3, or IgG4.

[0094] Furthermore, the antibody may be any of a fully human antibody, a humanized antibody, and a chimeric antibody.

[0095] [Target cells and cell populations containing target cells]

[0096] The target cells are not particularly limited as long as they are cells expected to produce a desired target substance.

[0097] The cell population including the target cell may be a cell population that has a possibility of including the target cell, and may not actually include the target cell.

[0098] - Antibody-producing cells -

[0099] When the target substance is an antibody, the target cell is an antibody-producing cell.

[0100] Examples of antibody-producing cells include spleen cells, B cells or plasma cells derived from lymphoid tissue or blood, hybridoma cells, and CHO cells.

[0101] As spleen cells, B cells or plasma cells derived from lymphatic tissue or blood, cells collected from healthy humans or animals, humans or animals suffering from cancer, humans or animals suffering from known or unknown infectious diseases, humans or animals suffering from autoimmune diseases, humans or animals vaccinated, etc. can be used. For example, the collected cell population can be used as a cell population containing the above-mentioned target cells.

[0102] Among these, an embodiment in which the target cell is a spleen cell or a B cell derived from a lymphoid tissue is one of the preferred embodiments of the present invention.

[0103] In order to more effectively identify the target cells relative to the target substance, cell concentration can be performed. For example, activated B cells or plasma cells obtained from the bone marrow, spleen, lymphoid tissue or blood cells of a non-human animal immunized with the target substance can be concentrated and used as a cell population containing the above-mentioned target cells.

[0104] Furthermore, for example, B cells (particularly, activated B cells) or plasma cells derived from cells obtained from human lymphatic tissue or blood can be concentrated and used as a cell population containing target cells.

[0105] The concentration of activated B cells or plasma cells can be carried out, for example, using CD antigens on the cell surface as an index. For example, it is possible to use antibody magnetic beads for specific CD antigens for concentration. For example, activated B cells or plasma cells can be concentrated by using one or more negative selection methods of antibody magnetic beads for CD2, CD3, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD34, CD40, CD43, CD45R, CD49b, CD56, CD61, CD79a, CD90.2, CD138, CD235a, etc., or activated B cells or plasma cells can be concentrated by using one or more positive selection methods of antibody magnetic beads for CD19, CD20, CD25, CD27, CD38, CD78, CD138, CD319, etc.

[0106] The degree of concentration is not particularly limited, but for example, B cells or plasma cells can be concentrated 50 times or more from a lymphocyte population of approximately 10,000,000 lymphoid tissues.

[0107] There are various methods for immunizing non-human animals with target substances, as shown in Hutchings CJ, Koglin M, Olson WC, Marshall FH, "Opportunities for therapeutic antibodies directed at G-protein-coupled receptors", Nat Rev Drug Discov. 16(9), 2017.

[0108] For example, a method of synthesizing a partial peptide or partial protein exposed to the cell surface and using it as an antigen for immunization can be cited. Also, a method of dissolving and purifying the target substance from the cell using a surfactant and using it as an antigen for immunization can be cited. Also, a method of directly immunizing the cells themselves that highly express the target substance can be cited. Also, a method of immunizing a substance presenting the target substance on an artificial double-layer membrane or virus-like nanoparticle as an antigen can be cited. Also, a method of using the target substance used in (1) of the present invention in vesicles on the membrane surface as an antigen for immunization can be cited. In addition, a method of immunizing a protein expression vector in which a cDNA sequence encoding the target substance is embedded (DNA immunization) can be cited.

[0109] Among these, DNA immunization is preferred because it is likely to produce more specific and high-affinity antibodies.

[0110] An embodiment in which the target cell is a CHO cell or a hybridoma cell is also one of the preferred embodiments of the present invention.

[0111] CHO cells are preferably recombinant cells into which an antibody gene has been introduced.

[0112] Recombinant cells are produced, for example, by the following procedure.

[0113] A cDNA library is prepared from B cells or plasma cells derived from lymphoid tissue or blood cells of immune animals. Genes of antibodies or antibody fragments are selectively amplified from the cDNA library. In order to be able to express various forms of antibody molecules such as complete antibodies, functional antibody fragments, single-chain antibodies or multi-specific antibodies, the amplified genes are changed to prepare an antibody gene library. The gene library is integrated into vectors such as pcDNA, pEF / FRt / V5-DEST, Mammalian PowerExpress System (mammalian cell expression system). Then, the vector is introduced into CHO cells to obtain recombinant cells.

[0114] A cell population containing the recombinant cells can be used as a cell population containing the above-mentioned target cells.

[0115] Furthermore, among the above-mentioned recombinant cells, a cell population containing recombinant cells that survive using the drug-resistant gene retained in the vector and that stably express the gene can be used as a cell population containing the above-mentioned target cells.

[0116] Furthermore, in the present invention, the target cell may be a recombinant cell obtained by the same method using COS cells, HEK293T cells, NS0 cells, etc. instead of CHO cells in the above method.

[0117] The target cell may be a hybridoma cell.

[0118] For example, immune cells are collected from non-human animals immunized with a target substance and fused with myeloma cells to obtain a cell population containing hybridoma cells. The cell population containing the hybridoma cells can be used as the cell population containing the target cells.

[0119] About cell fusion, the selection of hybridoma cells, cloning, known methods can be used. For example, cell fusion can be carried out by a method using polyethylene glycol or a method in which a voltage is applied to a mixed solution of immune cells and myeloma cells. In addition, the selection of hybridoma cells can be carried out by using the culture of HAT selection medium. The cell colony after this selection can also be used as a cell colony comprising the above-mentioned target cells.

[0120] The target cell can be a cell immortalized by a method other than the hybridoma method. For example, in the case of B cells derived from human lymphoid tissue or blood, it can be a cell immortalized by infection with Epstein-Barr virus (Epstein-Barr virus). It is also possible to use a cell colony containing these cells as a cell colony containing the above-mentioned target cell.

[0121] - Other cells -

[0122] When the target cells are cells other than antibody-producing cells, examples of the target cells include various cells derived from non-human or human tissues, such as blood cells, nerve cells, vascular endothelial cells, vascular smooth muscle cells, immune cells, fat cells, skeletal muscle cells, lymphocytes, skin cells, etc.

[0123] For example, cells isolated from tissues of non-human animals by separation, treatment with proteolytic enzymes, etc., followed by filtration with a 30 to 100 μm mesh can be used as the cell population containing the target cells.

[0124] For example, a cell population separated from human blood or surgically removed organs can also be used as a cell population containing the above-mentioned target cells.

[0125] Furthermore, the target cells may be tumor cells. For example, a cell population separated from an organ removed during surgery can also be used as the cell population containing the target cells.

[0126] In addition, a cell population containing tumor cells can be obtained from ATCC or a cell sales company.

[0127] The target cell may be a recombinant cell different from the antibody-producing cell.

[0128] For example, a cDNA library containing a gene encoding a target substance is integrated into an expression vector such as pcDNA, pEF / FRt / V5-DEST, or Mammalian PowerExpress System. Then, the vector is introduced into cells such as CHO cells, COS cells, HEK293 cells, or NS0 cells to obtain a cell population containing recombinant cells. The cell population can also be used as a cell population containing the above-mentioned target cells.

[0129] Furthermore, among the above-mentioned recombinant cells, a cell population including recombinant cells that survive using the drug-resistant gene retained in the vector and stably express the gene can also be used as a cell population including the above-mentioned target cells.

[0130] Furthermore, a cell population containing a cDNA library assembled in a viral vector derived from adenovirus, lentivirus, or the like and infected with CHO cells, HEK293 cells, NIH3T3 cells, or the like can also be used as the cell population containing the target cells.

[0131] Preferably, a gene encoding a target substance is introduced into the recombinant cell.

[0132] There is no particular limitation on the type of animal from which the target cells are derived, but mammalian cells or bird cells are preferably used. Examples of mammals include mice, rats, guinea pigs, rabbits, monkeys, cattle, horses, dogs, cats, goats, sheep, pigs, camels, alpacas, etc. Examples of birds include chickens, ducks, and turkeys.

[0133] <(1)>

[0134] The screening method of the present invention comprises: (1) fixing the vesicles having the target substance on the membrane surface in the micropores.

[0135] [Vesicles with target substances on membrane surface]

[0136] -Target substance-

[0137] The target substance in the vesicle having the target substance on the membrane surface is preferably a membrane protein. The membrane protein is not particularly limited to cell membrane proteins, lysosomal membrane proteins, ribosomal proteins, mitochondrial membrane proteins, etc., but is preferably a cell membrane protein.

[0138] Furthermore, the target substance may be any protein other than membrane proteins such as enzymes, or a compound other than polypeptides such as glycolipids, polysaccharides, drugs, and organic chemical substances.

[0139] Furthermore, the target substance is preferably an antigen or a receptor for the target substance (antibody or ligand).

[0140] The cell membrane protein is not particularly limited, and all cell membrane proteins including multiply permeabilizing proteins can be used as the target.

[0141] For example, G protein coupled receptors (GPCR), ion channels, transporters, CD antigens, cell adhesion molecules, cancer antigens, viral antigens and the like can be cited.

[0142] Furthermore, the animal species from which the cell membrane protein originates is not particularly limited.

[0143] In addition, the present invention also includes cell membrane proteins for which purification methods have not been established, for which large amounts of purification are difficult, for which separation and purification are difficult while maintaining their naturally occurring structures, and for which a portion of the proteins is exposed to the outside of the cell from the lipid double membrane layer.

[0144] - Vesicles -

[0145] The vesicle is not particularly limited except that it has a target substance on the surface, but is preferably a vesicle composed of a lipid double membrane.

[0146] Furthermore, from the viewpoint of fixing the vesicles in the micropores, the vesicles preferably have at least one selected from the group consisting of four transmembrane proteins such as CD9, CD63 or CD81 and phosphatidylserine on their membrane surface, and more preferably have phosphatidylserine on their membrane surface.

[0147] The diameter of the vesicle is preferably 30 to 1000 nm, more preferably 50 to 800 nm, further preferably 50 to 500 nm, and further preferably 50 to 200 nm.

[0148] In the present invention, the diameter of the vesicle can be measured, for example, by nanoparticle tracking analysis (NTA) using a nanoparticle analysis system (NanoSight). The diameter of the vesicle refers to the number average particle diameter.

[0149] The vesicle is not particularly limited, but is preferably an extracellular vesicle.

[0150] Extracellular vesicles are small membrane vesicles composed of a lipid double membrane that originate from cells.

[0151] As extracellular vesicles, for example, there can be cited various types of extracellular vesicles as described in Nature Reviews Immunology 9, 581-593 (August 2009), "Obesity Research" Vol. 13 No. 2 2007 by Aoki Naoto et al., which are classified according to their origin or the size of small membrane vesicles. Specifically, there can be cited exosomes, microvesicles, extracellular bodies, membrane particles, exosome-like vesicles, apoptotic vesicles, adiposomes, etc., preferably exosomes, microvesicles or apoptotic vesicles, and more preferably exosomes.

[0152] The exosomes are small membrane vesicles composed of a lipid double membrane derived from cells, and examples thereof include small membrane vesicles having a diameter of 30 nm to 200 nm, preferably small membrane vesicles of 30 nm to 150 nm, and more preferably small membrane vesicles of 30 nm to 100 nm. In addition, exosomes are believed to be derived from late endosomes.

[0153] The microvesicles are small membrane vesicles composed of a lipid double membrane derived from cells, and examples thereof include small membrane vesicles having a diameter of 100 nm to 1000 nm, preferably small membrane vesicles of 100 nm to 800 nm, and more preferably small membrane vesicles of 100 nm to 500 nm. In addition, microvesicles are believed to be derived from cell membranes.

[0154] The apoptotic vesicle is a small membrane vesicle composed of a lipid double membrane derived from cells, and examples thereof include apoptotic vesicles having a diameter of 50 nm to 1000 nm, preferably apoptotic vesicles of 50 nm to 800 nm, and more preferably apoptotic vesicles of 50 nm to 500 nm.

[0155] Vesicles can be obtained, for example, by causing vesicle-producing cells to overexpress a target substance by a known method and then recovering vesicles from the culture supernatant, or by recovering vesicles from body fluids of humans or animals.

[0156] Vesicle-producing cells are not particularly limited, and examples thereof include cells known to have good gene transfer efficiency, such as 293T cells and CHO cells, etc. Also, known cells such as other antibody-producing cells can be used without particular limitation.

[0157] As a method for causing vesicle-producing cells to overexpress a target substance, a known method can be used without particular limitation.

[0158] For example, a gene structure (or vector) encoding a target substance can be introduced into a vesicle-producing cell by a known transfection method, etc. As the introduction method, conventional methods such as naked DNA expression technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated transfection, physical or chemical reagents or treatments, and electroporation can be cited.

[0159] There is no particular limitation on the method for recovering vesicles, as long as a known method can be used. Examples include a method in which a sample containing vesicles, such as a culture supernatant or body fluid, is subjected to ultracentrifugation separation to obtain extracellular membrane vesicles as a precipitate component; a method in which a membrane having a pore size selected according to the diameter of the vesicles to be obtained is used to recover the vesicles by ultrafiltration; a method in which an antibody against a surface antigen protein of the vesicle (e.g., an anti-CD63 antibody) is used to obtain vesicles through the affinity between the surface antigen protein and the antibody; a method in which a Tim family protein bound to a carrier (e.g., magnetic beads) is used as described in Japanese Patent Application Publication No. 2021-12200, etc.

[0160] In addition, in the method of recovering vesicles, a commercially available kit such as MagCapture (registered trademark) Exosome Isolation Kit PS Ver. 2 (manufactured by FUJIFILM Wako Pure Chemical Corporation, 290-84103) can be used.

[0161] -Micropores-

[0162] A micropore is a hole (depression, recess) of a small size that can accommodate about 1 to 3 mammalian cells or avian cells. A micropore is a small hole with a bottom, and the inner diameter of the opening is preferably about 10 μm to 50 μm, more preferably 10 to 30 μm, and further preferably 15 to 25 μm. The depth of the micropore is not particularly limited, and is preferably the same as the inner diameter of the opening.

[0163] Furthermore, the diameter (inner diameter) of the opening of the microwell can be appropriately determined in consideration of the type of cells (ie, candidate cells) stored in the microwell.

[0164] The shape of the micropore is preferably cylindrical. In addition, it can also be a shape in which the cross-sectional area of ​​a polygonal tube such as a square tube or a hexagonal tube, or an inverted cone tube or an inverted pyramid tube decreases from the opening to the bottom surface. In addition, it can also be a shape in which two or more of these shapes are combined and connected.

[0165] When the micropore is in a shape other than a cylinder or an inverted cone, the inner diameter of the opening can be calculated as a circle equivalent diameter from the area of ​​the opening. The circle equivalent diameter is the diameter of a circle having an area equal to the area of ​​the pattern.

[0166] It is preferred that a plurality of micropores are formed on the substrate.

[0167] The number of micropores per unit area (density) on the substrate is not particularly limited, but is preferably 1 cm 2 The number of cells is in the range of 20,000 to 200,000, more preferably per 1 cm 2 The number of the samples is in the range of 25,000 to 75,000.

[0168] By using micropores with a small inner diameter or the like, the density can be increased, thereby improving the screening efficiency.

[0169] The material of the substrate is not particularly limited, but for example, when the detection in (3) is performed by fluorescence detection as described later, a transparent material with low autofluorescence is preferably used.

[0170] As the substrate having micropores formed therein, commercially available products can also be used. For example, a substrate having a plurality of micropores (micropore chambers) having an inner diameter of 10 μm, 20 μm, or 30 μm is commercially available from AS ONE Corporation.

[0171] -Method for fixing vesicles in micropores-

[0172] The method for fixing the vesicles in the micropores is not particularly limited, but it is preferred to fix the vesicles in the micropores using a substance that binds to the vesicles.

[0173] The substance that binds to the vesicle can be appropriately determined by taking into account the type of vesicle, the type of protein expressed on the surface of the vesicle, etc., but examples thereof include Tim family proteins, annexin V that binds to phosphatidylserine, anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, and other antibodies that bind to tetramers present on the surface of the vesicle.

[0174] Among these, in (1), it is preferred to use any one or more of the group consisting of Tim family proteins, annexin V, anti-CD9 antibodies, anti-CD63 antibodies and anti-CD81 antibodies to fix the above-mentioned vesicles in the micropores. From the perspective of screening sensitivity, it is more preferred to use Tim family proteins (for example, Tim 4) to fix the above-mentioned vesicles in the micropores.

[0175] Specifically, for example, after coating the microwell with the substance that binds to the vesicle, the vesicle is placed in the microwell to react, thereby fixing the vesicle to the microwell.

[0176] The coating can be performed, for example, by contacting a solution or suspension containing a substance that binds to the vesicles with the micropores. The contact time and temperature conditions are not particularly limited, and for example, a method of contacting the solution or suspension with the micropores at 4°C for about one night can be cited.

[0177] The concentration of the substance bound to the vesicles in the solution or suspension is not particularly limited and can be determined by considering the type of substance, detection sensitivity, the likelihood of false positives and false negatives, etc. For example, it is preferably set to 1 to 100 μg / mL, and more preferably to 10 to 50 μg / mL.

[0178] When the vesicles are placed in the microwells to react, it is preferred that a suspension containing the vesicles be brought into contact with the coated microwells.

[0179] The content of vesicles in the suspension is not particularly limited, and can be determined by taking into account the type of substance, detection sensitivity, the probability of false positives and false negatives, etc., and is preferably 1×10 9 ~1×10 13particles / mL, more preferably 1×10 10 ~1×10 12 Particles / mL.

[0180] The reaction time and conditions are not particularly limited and can be determined by taking into account the type and amount of the substance to be coated and bound to the vesicles, the type and concentration of the vesicles, etc. For example, the reaction can be carried out at room temperature for 2 hours.

[0181] Unless otherwise specified, room temperature in this specification refers to 23°C.

[0182] Furthermore, the coated and fixed vesicles are preferably present not only on the bottom surface of the micropores but also on the sides (sidewalls) of the micropores. According to this method, when fluorescence detection is performed in (3), it is possible to detect pores where fluorescence is confirmed at the positions of the sidewalls of the micropores.

[0183] Here, for the purpose of inhibiting the non-specific reaction of a substance binding to a target substance in (2) described later, blocking may be further performed in (1).

[0184] For example, in the above-described method in which vesicles are placed in the microwells and reacted after coating the microwells with a substance that binds to the vesicles, blocking may be performed after coating and before the reaction of the vesicles.

[0185] Blocking is not particularly limited, and can be performed using a known blocking agent. For example, when the substance that binds to the target substance in (2) described later is an antibody, skim milk powder or the like can be used.

[0186] The blocking time, blocking temperature, etc. may be determined according to conventional ELISA methods.

[0187] <(2)>

[0188] The screening method of the present invention comprises (2) introducing candidate cells obtained from a cell population containing the target cells and a substance that binds to the target substance and is labeled into the microwell to which the vesicles are immobilized.

[0189] Candidate cells are cells obtained from a cell population including target cells, and may be cells equivalent to target cells (ie, cells that produce target substances) or cells not equivalent to target cells (ie, cells that do not produce target substances).

[0190] The number of candidate cells introduced into the microwell may be selected depending on the purpose of screening, etc. However, from the viewpoint of easily identifying the target cell as a single cell, the candidate cell introduced into the microwell is preferably a single isolated cell.

[0191] The details of the cell population containing target cells are as described above.

[0192] Examples of the substance that binds to the target substance and is labeled include antibodies and receptor proteins, and antibodies are preferred.

[0193] Furthermore, in (2), in order to indirectly detect the label in the labeled substance that is bound to the target substance, the compound that reacts with the label may be further reacted.

[0194] Specifically, an antibody that binds to a target substance and has a label and a compound that reacts with the label can be used.

[0195] Specific examples of this aspect include an antibody that binds to a target substance and is labeled with a biotin-based marker, and fluorescently labeled streptavidin.

[0196] The label in the antibody that binds to the target substance and has a label is not particularly limited, and a known label can be used. For example, a label based on a fluorescent substance, biotin, etc. can be used, and a label based on a fluorescent substance or biotin is preferred.

[0197] When the label is a label made of a fluorescent substance, the label (fluorescent substance) can be directly detected by fluorescence in (3) described later.

[0198] In the case where the label in the antibody having a label among the antibodies binding to the target substance is a label based on biotin, by further reacting with fluorescently labeled avidin, streptavidin, etc., the label (biotin) can be indirectly fluorescently detected in (3) described later.

[0199] The fluorescent substances in these methods are not particularly limited, and examples thereof include Alexa Fluor (registered trademark), Aqua, Texas Red (registered trademark), fluorescein and its derivatives, rhodamine and its derivatives, Cascade Blue (registered trademark), phycoerythrin, and DyLight (registered trademark).

[0200] <(3)>

[0201] The screening method of the present invention comprises (3) identifying the target cell by detecting the marker bound to the target substance bound to the target substance.

[0202] In (3), the label in the complex of the vesicle with the target substance on the membrane surface, the target substance produced from the target cell, and the substance bound to the target substance and labeled is detected. As described above, the label can be directly detected by the label itself, or it can be indirectly detected by detecting the compound bound to the label.

[0203] The detection method in (3) can be selected according to the type of label, but fluorescence detection is preferred.

[0204] The excitation wavelength and fluorescence wavelength in fluorescence detection are not particularly limited and may be selected according to the type of fluorescent label.

[0205] It is also one of the preferred aspects of the present invention to identify the target cells by the above-mentioned fluorescence detection by excitation at one wavelength and fluorescence measurement at one wavelength.

[0206] Identification of target cells by the above-mentioned fluorescence detection through excitation based on one wavelength and fluorescence measurement at one wavelength means that the data used in identifying the target cells is data obtained by excitation based on excitation light including one wavelength range and fluorescence measurement within one wavelength range.

[0207] For example, in the method described in Patent Document 2, irradiation of excitation light and measurement of fluorescence are performed for fluorescence detection of a first cell, and irradiation of excitation light and measurement of fluorescence are performed for fluorescence detection of a second cell. That is, excitation at two wavelengths and fluorescence measurement at two wavelengths are performed.

[0208] However, in the screening method of the present invention, it is sufficient to detect the complex of the vesicle, the target substance, and the substance bound to the target substance, and thus the target cells can be identified by excitation at one wavelength and fluorescence measurement at one wavelength.

[0209] As described above, in the screening method according to the present invention, the number of times of irradiation of excitation light and fluorescence detection can be reduced compared to the method described in Patent Document 2, and thus the screening method can be made more efficient.

[0210] Furthermore, since the number of times of irradiation of excitation light and fluorescence detection can be reduced, the measurement can be performed in a short time, thereby reducing damage to the target cells during the measurement. That is, when the target cells are recovered in (4) described later, the target cells with less damage can sometimes be recovered.

[0211] In (3), the target cells are preferably identified by detecting wells in which fluorescence is confirmed at the side walls of the microwells.

[0212] Here, usually, the coated and immobilized vesicles are also present on the side walls of the micropores.

[0213] Therefore, it is considered that a complex of a vesicle, a target substance, and a substance that binds to the target substance and is labeled is also formed in the side wall of the microwell.

[0214] Furthermore, since a plurality of the above-mentioned complexes are stacked in the height direction at the sidewall of the microwell, it is considered that when observing from the upper surface or the bottom surface of the microwell, the fluorescence intensity at the sidewall is greater than that at the bottom of the microwell.

[0215] In addition, there is at least an area where no cells or candidate cells exist at the side wall of the micropore, so when observing fluorescence at the side wall, the influence on detection based on fluorescence derived from candidate cells (for example, negative cell autofluorescence, fluorescence observed through nonspecific binding of a substance bound to a target substance, etc.) can be reduced.

[0216] In this way, by detecting the fluorescence at the side wall of the microwell, the fluorescence intensity is high, and the influence of the candidate cells can be reduced, so the detection sensitivity is increased, and it is easy to judge positive and negative, so false positives and false negatives are not easy to occur. That is, the screening sensitivity is excellent.

[0217] In this aspect, the fluorescence detection in (3) is preferably also the detection of fluorescence having the same shape as the shape of the orthographic projection observed from the observation direction of the side wall of the microhole, for example.

[0218] For example, when the microwell is cylindrical, the fluorescence detection in (3) is preferably detection of ring-shaped fluorescence at the position of the side wall.

[0219] <(4)>

[0220] The screening method of the present invention also preferably further includes the following (4).

[0221] (4) Recovering the target cells identified in (3) above.

[0222] The target cells can be recovered from the microwells using, for example, a micromanipulator. For example, a capillary having a diameter of several μm to 50 μm can be inserted into the microwells (positive microwells) where the target cells are confirmed to be present, and the target cells can be sucked and recovered in a living state.

[0223] The operation by the micromanipulator may be automatic or manual. For example, the cells can be collected using a cell collection system (AS ONE Corporation) or CellCelector (Automated Lab Solution).

[0224] Furthermore, when two or more cells including target cells are introduced into the positive microwells, for example, after the two or more cells are collected in a cell culture medium, the two cells can be separated and one of them can be used as the target cell.

[0225] Furthermore, after the two cells are recovered in a buffer or the like, nucleic acids encoding two target substances are obtained and separated by the method described below, and either nucleic acid can be used as the target nucleic acid (target gene).

[0226] Furthermore, microwells containing two or more cells may be regarded as wells in which target cells have not been identified and excluded from the collection targets.

[0227] (Method for producing nucleic acid or method for producing target substance)

[0228] The method for producing a nucleic acid of the present invention comprises the step of obtaining a nucleic acid encoding a target substance from the target cell obtained by the screening method.

[0229] Furthermore, the method for producing a target substance of the present invention includes the step of producing the target substance using the nucleic acid obtained by the method for producing a nucleic acid of the present invention.

[0230] The production of the target substance using the nucleic acid preferably further includes, for example, the steps of introducing the nucleic acid into a host cell to obtain a recombinant cell expressing the target substance, and culturing the recombinant cell to obtain the target substance from the culture.

[0231] As a method for obtaining a nucleic acid encoding a target substance from a target cell, a known method can be used. For example, a method of combining a reverse transcription reaction and a PCR method to synthesize cDNA and isolating the target nucleic acid from the cDNA can be mentioned.

[0232] As a method for obtaining a recombinant cell expressing a target substance, a known method can be used. For example, a method of obtaining a target recombinant cell by appropriately integrating the obtained nucleic acid into a vector and introducing the vector into a host cell such as Escherichia coli, yeast, or mammalian cells (e.g., CHO cells, HEK293 cells, or NS0 cells) can be cited.

[0233] As a method for culturing the recombinant cells and obtaining the target substance from the culture, a known method can be used. For example, the recombinant cells can be cultured and the target substance can be obtained from the culture (eg, culture supernatant).

[0234] When the target substance is an antibody, the antibody gene can be isolated from the target cell by, for example, a combination of the methods described in International Publication Nos. 2009 / 091048, 2009 / 110606 and 2011 / 027808, or the method described in Nobuyuki Kurosawa, Megumi Yoshioka, Rika Fujimoto, Fuminori Yamagishiand Masaharu Isobe, "Rapid production of antigen-specific monoclonal antibodies from a variety of animals", BMC Biology, 10: 80, 2012 (MAGrahd method).

[0235] The isolated antibody gene can be altered to construct a recombinant cell expressing a complete antibody, a functional antibody fragment, a single-chain antibody, or a multi-specific antibody. Similarly, a recombinant cell expressing a complete human antibody, a humanized antibody, or a chimeric antibody can be constructed. Furthermore, a recombinant cell expressing a feline antibody or a dogized antibody can be constructed.

[0236] When the target cells can be stably cultured, it is also possible to culture the target cells themselves and obtain the target substance such as antibodies from the culture.

[0237] That is, the method for producing the target substance is also preferably a method including the steps of culturing the target cells identified by the screening method of the present invention and obtaining the target substance from the culture.

[0238] As a method for purifying target substances such as antibodies from the above-mentioned culture, known methods can be used. For example, various chromatography methods such as affinity, ion exchange, and gel filtration can be used. As the ligand in the affinity chromatography method, protein A, protein G, anti-FLAG antibody, anti-V5 antibody, etc. can be mentioned.

[0239] <Pharmaceutical Composition>

[0240] A first embodiment of the method for producing a pharmaceutical composition of the present invention comprises the step of combining a nucleic acid produced by the method for producing a nucleic acid of the present invention with a pharmaceutically acceptable carrier or additive to contain the nucleic acid as an active ingredient.

[0241] A second embodiment of the method for producing a pharmaceutical composition of the present invention comprises the step of combining a target substance produced by the method for producing a target substance of the present invention with a pharmaceutically acceptable carrier or additive to contain the target substance as an active ingredient.

[0242] The target substance produced by the method for producing a target substance of the present invention, for example, an antibody, is useful as an active ingredient of a pharmaceutical composition (therapeutic agent).

[0243] The pharmaceutical composition can contain a target substance such as an antibody produced by the method for producing a target substance of the present invention and a pharmaceutically acceptable carrier or additive.

[0244] Preferably, the pharmaceutical composition blocks or activates a specific intracellular signal transduction mechanism in a cell membrane protein as a target substance.

[0245] The pharmaceutical composition can be administered systemically or topically or orally or parenterally. As the mode of administration, injection dosage form, nasal administration dosage form, pulmonary administration dosage form, transdermal administration dosage form, etc. can be cited, and there is no particular limitation. In the case of injection dosage form, for example, systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. In addition, the administration method can be appropriately selected according to the age or symptoms of the patient.

[0246] When the target substance is an antibody, the dosage of the antibody is not particularly limited, and can be selected within the range of 0.0001 mg to 1000 mg per kg of body weight, for example. Alternatively, although not particularly limited, the dosage can be selected within the range of 0.001 to 100,000 mg / body of the antibody per patient, for example.

[0247] The above-mentioned pharmaceutical composition can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). As examples of the above-mentioned carriers or additives, surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, spices, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, flavoring agents, etc. can be enumerated. Specifically, light silicic anhydride, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc. can be enumerated. Furthermore, it may contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulin, etc.; amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc.

[0248] When the above-mentioned pharmaceutical composition is used as an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, can be mentioned, and it can also be used in combination with appropriate cosolvents such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc.

[0249] Furthermore, if necessary, the antibody as an active ingredient can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or provided as a colloidal drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (see "Remingto's Pharmaceutical Science 16th edition", Oslo Ed. (1980) etc.).

[0250] Furthermore, it is known that other drugs can be directly fused to antibodies to enhance therapeutic effects, and this technology can be applied to the above-mentioned pharmaceutical composition.

[0251] In addition, it is also possible to consider integrating the nucleic acid (gene) obtained in the present invention, such as an antibody gene, into a gene therapy vector and setting it as a gene therapy drug. As a method of administering the above-mentioned gene therapy drug (recombinant vector), in addition to direct administration based on naked plasmids, methods of administering by packaging in liposomes, etc., integrating into various viral vectors such as retroviral vectors, adenoviral vectors, poxvirus vectors, vaccinia virus vectors, adeno-associated virus vectors, HVJ vectors, etc. for administration (reference Adolph "Viral Genome Method", CRC Press, Florid (1996)), and methods of administering by coating on bead carriers such as colloidal gold particles (International Publication No. 93 / 17706, etc.).

[0252] That is, as long as the above-mentioned gene therapy drug expresses the antibody as an active ingredient in vivo and can exert its effect, it can be administered by any method. It is preferred to administer a sufficient amount by an appropriate non-oral route. As a non-oral route, intravenous, intraperitoneal, subcutaneous, intradermal, intrafatty tissue, intramammary tissue, inhalation or injection via intramuscular route, injection or gas-induced particle impact method (based on electron guns, etc.), via mucosal routes such as nasal drops, etc. can be cited. Moreover, the above-mentioned gene therapy drug can be administered to cells by utilizing liposome transfection, particle impact method (U.S. Patent No. 4,945,050) or viral infection in ex vivo, and the cells are reintroduced into animals for administration.

[0253] (Target substance detection reagent)

[0254] The target substance detection reagent of the present invention includes a reagent for detecting a desired target substance comprising a target substance manufactured by the above method. For example, a reagent containing an antibody (target substance) manufactured by the manufacturing method of the target substance of the present invention is used to contact the above antibody with a blood cell derived from a human or non-human mammal. Moreover, a labeling substance composed of a fluorescent substance or a pigment is directly or indirectly contacted. Then, the expression of the desired target substance (for example, a cell membrane protein) can be detected by a flow cytometer or an ELISA instrument. Moreover, the above reagent is used to contact the above antibody with a pathological tissue piece derived from a human or non-human mammal, so that the expression of the desired target substance can be detected.

[0255] Furthermore, a target substance detection kit including the above reagents can be constructed. For example, a labeling substance or the like can be combined with the above reagents to prepare a target substance detection kit.

[0256] The present invention includes a method for detecting a target substance using the target substance produced by the above method. The present invention includes use of the target substance produced by the above method for detecting the target substance.

[0257] Example

[0258] Below, give embodiment and the present invention is described in more detail.Material, usage amount, ratio, processing content and processing sequence etc. shown in the following embodiment can be changed suitably without departing from the gist of the present invention.Therefore, the scope of the present invention is not limited to the specific example shown below.

[0259] (Example 1: Preparation of EVs presenting target protein)

[0260] <Experimental methods>

[0261] 293T cells that were forcibly expressed once by lipofectamine 2000 Transfection Reagent (Invitrogen 11668027) were cultured in serum-free medium for 1 to 3 days, respectively, and EVs were purified from the culture supernatant using MagCapture (registered trademark) Exosome Isolation Kit PS Ver.2 (manufactured by FUJIFILM Wako PureChemical Corporation, 290-84103). The purified EVs were measured by NanoSight (nanoparticle analysis system, manufactured by Nikkantam Design Co., Ltd.). As negative controls for each cell, EVs derived from cells treated with transfection reagent only (TF(-)) were also prepared, and the particle size distribution of each cell was analyzed.

[0262] The analysis results are shown in Figure 1 middle.

[0263] <Results>

[0264] Figure 1 This is a graph showing the analysis results of the particle size distribution, in which the horizontal axis is plotted with the particle size and the vertical axis is plotted with the particle number concentration.

[0265] from Figure 1 It was confirmed that, through the purification of EVs from the culture supernatant and the analysis based on NanoSight, particles (EVs) of about 100 to 300 nm could be recovered from the supernatant of cells expressing each membrane-permeable protein and the negative control.

[0266] (Example 2: Confirmation of EVs expressing target protein)

[0267] <Experimental methods>

[0268] Fluorescence detection EV-ELISA was performed as follows using EVs derived from target protein-expressing cells prepared in Example 1. As a negative control, EVs derived from cells treated with only the transfection reagent (TF(-)) were also prepared and analyzed.

[0269] EV-ELISA: Mouse TIM4 / Human Fc Chimera, recombinant (FUJIFILM Wako Pure Chemical Corporation 137-18511) 1.5 μg / mL was applied to a 96-well plate overnight at 4°C. The next day, after blocking with Block Ace (manufactured by KAC Co., Ltd.) for 1 hour, the plate was incubated with each EV particle (2.5×10 8 Particles / hole) were reacted at room temperature for 2 hours. Next, various antibodies (EphA2: BioLegend356802, HA: SIGMA H3663, Claudin-5: FUJIFILM Wako Pure Chemical Corporation014-28101) 1μg / mL were added and allowed to react at room temperature for 1 hour, and then Biotin (biotin) labeled secondary antibody (FUJIFILM Wako Pure Chemical Corporation 512-20531) 10μg / mL was added and allowed to react at room temperature for 1 hour. Finally, 100μL of streptavidin-Alexa Fluor (registered trademark) 488 (FUJIFILM Wako PureChemical Corporation560-77841) 2μg / mL was added and allowed to react at room temperature for 1 hour. Then, the fluorescence intensity was measured using an ELISA reader. The measurement results are shown in Figure 2 middle.

[0270] <Results>

[0271] Figure 2 It is a graph showing the values ​​of fluorescence intensity measured in each membrane-permeabilizing protein-expressing EV and negative control.

[0272] from Figure 2 The results shown confirmed that the target protein signal could be detected from each EV in the results of EV-ELISA in fluorescence detection, and that the target protein was presented on the EV. In addition, the results of comparison with the fluorescence signal of EVs not expressing the target protein (EVs derived from cells without gene introduction) confirmed that the S / B ratio was about 2.5 to 8.5 times.

[0273] (Example 3: Verification in ASONE Cell Picking System Microchamber)

[0274] <Experimental methods>

[0275] In a chamber containing about 200,000 pores with a diameter of 20 μm, Tim4 20 μg / mL was applied overnight at 4°C. The next day, after blocking with Block Ace for 1 hour, 100×10 EV particles expressing EphA2 obtained in Example 1 were 9 The particles / mL were reacted at room temperature for 2 hours. Then, 3.5×10 spleen cells from rabbits immunized with EphA2 were added. 5 cells / mL and secondary antibody (Biotin labeled) 10μg / mL (FUJIFILM Wako Pure Chemical Corporation 564-72501), reacted at 37°C, CO2 concentration 5% for 1 hour, and finally added avidin-labeled Alexa Fluor (registered trademark) 488 (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2μg / mL, reacted at room temperature for 1 hour, and then imaged by ASONE Cell Picking System. Spleen cells were obtained by injecting rabbits with DNA immunization of antigen expression plasmids.

[0276] <Results>

[0277] An example of the results of imaging using the ASONE Cell Picking System is shown in Figure 3 In. According to Figure 3 For the wells where cells producing antibodies that specifically bind to EphA2 on EVs (positive cells) exist, fluorescence is detected as a ring near the sidewall of the wells. If 200,000 chambers are scanned, 8 positive cells can be confirmed. For reference, an example of the results of imaging cells that do not produce antibodies that specifically bind to EphA2 on EVs (negative cells) is shown in Figure 4 In the case of B cells of the type that easily retain antibodies on the membrane, the antibodies on the membrane are bound by labeled secondary antibodies, such as Figure 4 As shown, the B cells themselves were detected by fluorescence, but no fluorescence detection was observed in the rings near the side walls.

[0278] (Example 4: Comparison between EV-ELISA and Cell ELISA in ASONE Cell Picking System)

[0279] <Experimental methods>

[0280] The method described in International Publication No. 2020 / 171020 (Cell-ELISA) was implemented and compared with EV-ELISA. As the analysis cells in Cell-ELISA (the first cells in International Publication No. 2020 / 171020), a stable strain of EphA2-expressing CHO-K1 was used, and as the second cells in International Publication No. 2020 / 171020, the EphA2-immunized rabbit spleen cells used in Example 3 were used. The analysis conditions are as follows.

[0281] 5×10 CHO-K1 cells stably expressing EphA2 were seeded in a chamber containing approximately 85,000 pores with a diameter of 30 μm. 5 cells / mL, and stained with Cytored solution 10 μmol / L (FUJIFILM Wako Pure Chemical Corporation 342-08531). Then, 5×10 spleen cells from EphA2-immunized rabbits used in Example 3 were added. 5 cells / mL and secondary antibody (FITC-labeled) 10 μg / mL (FUJIFILM Wako Pure Chemical Corporation 563-77951) were reacted at 37°C and 5% CO2 for 1 hour, and then imaged using the ASONE Cell Picking System.

[0282] <Results>

[0283] An example of the imaging results of positive cells observed by Cell-ELISA is shown in Figure 5 In. From Figure 5 It can be seen that CHO cells have fluorescence in the Cy3 image, and both small B cells and large CHO cells have fluorescence in the FITC image. In other words, it can be seen that among positive cells, CHO cells can be observed to have fluorescence in both the Cy3 image and the FITC image.

[0284] An example of the imaging results of negative cells observed by Cell-ELISA is shown in Figure 6 In. From Figure 6 It can be seen that CHO cells have fluorescence in the Cy3 image, and only small B cells have fluorescence in the FITC image. In other words, it can be seen that among negative cells, CHO cells have fluorescence in the Cy3 image, but no fluorescence is confirmed in the FITC image.

[0285] Table 1 shows the comparison results between EV-ELISA and Cell ELISA.

[0286] As can be seen from Table 1, the number of analyzable wells when using Cell-ELISA is 43,079 wells, while in EV-ELISA, as shown in Example 3 above, the number of wells is 140,416, which is about 3 times the number of wells. Here, the count of the number of analyzable wells is calculated as follows. In Cell-ELISA, CHO cells are filled in the wells (96%: 82,682 wells), and spleen cells are also filled in the wells (50%: 43,079 wells). In EV-ELISA, the wells filled with spleen cells are directly counted as the number of analyzable wells (71%: 140,416 wells). In addition, among the number of positive cells, there are 46 in EV-ELISA compared to 3 in Cell-ELISA. Moreover, the result of the positive rate relative to the number of analyzable wells was 0.007% in Cell-ELISA, while it was 0.03% in EV-ELISA, which can be detected with a high sensitivity of about 4 times that of existing methods. In addition, in Cell-ELISA, the criteria for setting the obtained merged image as positive or negative are unclear, and it is difficult to visually determine what degree of fluorescence is detected in CHO cells to be positive. However, in EV-ELISA, it can be determined by confirming the fluorescence of the side wall of the well (specifically, confirming the ring fluorescence), so it is easy to visually determine, and the screening sensitivity is significantly improved.

[0287] In addition, in Cell-ELISA, the number of wells filled with CHO cells and spleen cells (i.e., filling rate) was studied. The seeding condition of spleen cells was set to 2.5×10 5 cells, 5×10 5 cells, 10×10 5 cells, but the filling rate did not increase to more than 50%. That is, the ratio of the number of analyzable wells to the total number of wells was about 50%. In contrast, in EV-ELISA, the wells filled with spleen cells directly become analyzable wells, so the ratio of the number of analyzable wells to the total number of wells is about 71% as described above. As can be seen from the above, compared with Cell-ELISA, the ratio of analyzable wells in EV-ELISA is high, and more wells can be used for analysis.

[0288] Furthermore, from the perspective of screening efficiency, the positive rate relative to the number of wells was calculated, and the positive rate was 0.0035% in Cell-ELISA, while it was 0.023% in EV-ELISA, which is about 6 times higher than the conventional method.

[0289] [Table 1]

[0290]

[0291] (Example 5: Advantages of EV solidification in PS (phosphatidylserine)-TIM4 affinity reaction)

[0292] <Experimental methods>

[0293] On a 96-well plate, Tim4 1.5 μg / mL, Anti-CD9 antibody (FUJIFILM Wako Pure Chemical Corporation 013-28171) 1 μg / mL, or Anti-CD81 antibody (FUJIFILM Wako Pure Chemical Corporation 010-28223) 1 μg / mL was coated overnight at 4°C. The next day, after blocking with Block Ace (manufactured by KAC Co., Ltd.) for 1 hour, the plates were incubated with each EV particle (2.5×10 8 Particles / well) were reacted at room temperature for 2 hours. Next, various antibodies (EphA2: BioLegend 356802, HA: SIGMA H3663, Claudin-5: FUJIFILM Wako Pure Chemical Corporation 014-28101) 1μg / mL were added and reacted at room temperature for 1 hour, and then reacted with HRP-labeled secondary antibody (FUJIFILM Wako Pure Chemical Corporation512-20531, 567-80161) at room temperature for 1 hour. Finally, 100μL of TMB solution (FUJIFILM Wako PureChemical Corporation 208-17371) was added and reacted at room temperature for 15 minutes, and then the reaction was stopped with 1M HCl. The absorbance A at 450nm and the absorbance B at 650nm of each well were measured using an enzyme reader, and the absorbance A / B (Abs.450 / 650nm) was calculated. The measurement results are shown in Figure 7 In addition, the TF(-)EV obtained in the above Example 1 was used as a negative control.

[0294] <Results>

[0295] from Figure 7 The results shown show that the absorbance measurement value is the highest when Tim4 is used compared to when Anti-CD9 antibody or Anti-CD81 antibody is used for EV immobilization. In other words, it is believed that the detection sensitivity is the best when Tim4 is used for EV immobilization.

[0296] (Example 6: Changes in the amount of CD marker protein and PS on EV)

[0297] <Experimental methods>

[0298] A 96-well plate was coated with 1.5 μg / mL of Tim4 at 4°C overnight. The next day, after blocking with Block Ace (manufactured by KACC Co., Ltd.) for 1 hour, the plate was incubated with the EphA2-expressing EV particles (0.5×10 8 The cells were reacted at room temperature for 2 hours. After adding various CD marker protein antibodies (FUJIFILM Wako Pure Chemical Corporation CD9: 013-28171, CD63: 012-27063, CD81: 010-28223) or Annexin V Biotin (EXBIO EXB0031) 1 μg / mL, the cells were reacted at room temperature for 1 hour, and then reacted with HRP-labeled secondary antibodies (FUJIFILM Wako Pure Chemical Corporation 512-20531, 567-80161) or Poly-HRP Streptavidin (Thermo N200) 1 μg / mL for 1 hour. Finally, after adding 100 μL of TMB solution (FUJIFILM Wako Pure Chemical Corporation 208-17371) and reacting at room temperature for 15 minutes, the reaction was stopped with 100 μL of 1M (mol / L) HCl. The absorbance A at 450 nm and the absorbance B at 650 nm were measured for each well using an ELISA reader, and the absorbance A / B was calculated. Figure 8 Furthermore, the target protein non-expressing EVs (EVs derived from cells without gene introduction) obtained in Example 1 above were used as controls.

[0299] <Results>

[0300] Figure 8 This is a graph showing absorbance values ​​measured in EphA2 protein-expressing EVs and controls.

[0301] Fig. 9 This is a graph showing the S / B ratio (signal / background ratio) value obtained by dividing the absorbance measurement value measured in EphA2 protein-expressing EVs by the absorbance measurement value measured in controls.

[0302] from Figure 8 The results shown show that the expression level of CD marker protein on EVs decreased by gene introduction, but the amount of PS did not decrease.

[0303] And, from Fig. 9 The results shown show that the S / B ratio relative to the CD-labeled protein is reduced by about 20% to 40% when compared with the absorbance measurement value of EVs not expressing the target protein (EVs derived from cells without gene introduction).

[0304] This is considered to be because the expression levels of CD marker proteins such as CD9 and CD81 are reduced by gene introduction, but the amount of PS does not decrease or is unlikely to decrease.

[0305] (Example 7: Confirmation of activity of acquired antibodies)

[0306] <Experimental methods>

[0307] The antigen binding of rabbit antibodies (A-1, A-2, A-3) derived from B cells obtained by the screening method described in Example 3 was confirmed by flow cytometric analysis. EphA2-expressing cells prepared by the method shown in Example 1 were used in the analysis, and non-expressing cells were also prepared and analyzed at the same time in order to confirm the specific binding to the antigen. In addition, as a comparison object, mouse anti-EphA2 antibodies (B-1) and rat antibodies (C-1, C-2) prepared by the hybridoma cell method were also analyzed at the same time. The spleen cells used in the preparation of hybridoma cells were obtained by DNA immunization produced by administering antigen expression plasmids to mice or rats.

[0308] Specifically, it is performed as follows.

[0309] For EphA2-expressing cells and non-expressing cells, the culture medium was discarded and the cells were washed with PBS(-). After washing, Cell Dissociation Buffer (6-well plate: 500μL, 150mm culture dish: 3mL) was added and incubated at 37°C for about 5 minutes. After incubation, 3% FBS / PBS (6-well plate: 1mL, 150mm culture dish: 12mL) was added, the cells were suspended with a pipette, and the suspension was recovered in a centrifuge tube. After centrifugation at 1,200rpm for 3 minutes to precipitate the cells, 3% FBS / PBS (6-well plate: 1200μL, 150mm culture dish: 10mL) was added and the cells were resuspended. The cell suspension was added at 200μL per well (approximately 2x10 5 After adding the cells to a 96-well plate (round bottom, Corning 3788), the wells were centrifuged at 400×g for 5 minutes.

[0310] The antibody was diluted with FCM Buffer (3% FBS / PBS) to an antibody concentration of 1 μg / mL, 200 ng / mL, and 40 ng / mL to prepare a primary antibody solution. The cell culture supernatant was removed, 50 μL of the culture supernatant stock solution was added to each well, and 25 μL of the primary antibody solution (diluted with 3% FBS / PBS) was added to each well for suspension, and then incubated at 4°C for 30 minutes. After incubation, 150 μL of 3% FBS / PBS was added to each well of the 96-well plate, and centrifuged at 4°C and 400xg for 5 minutes. The supernatant was removed, and 25 μL of the PE-labeled secondary antibody solution (Southern Biotech, 1030-09 or 4030-09) (diluted with 3% FBS / PBS to an antibody concentration of 1 μg / mL) was added to each well to suspend the cells, and incubated at 4°C for 30 minutes. After incubation, 150 μL of 3% FBS / PBS was added to each well of the 96-well plate, and the plate was centrifuged at 4°C and 400 x g for 5 minutes. The supernatant was removed, and 200 μL of 3% FBS / PBS was added to each well. The fluorescence intensity of the fluorescently labeled cells was measured using a flow cytometer (CytoFLEX (manufactured by Beckman Coulter, Inc.)). The measurement results are shown in Fig.10 middle.

[0311] <Results>

[0312] Fig.10 The median value of the FCM displacement intensity is calculated, and the median value of the non-expressing cells is subtracted from the expressing cells and the graph is plotted. The rabbit antibodies obtained all react specifically to EphA2 expressing cells, confirming that antigen-specific antibodies can be obtained by the screening method of the present invention. In addition, the binding force of the rabbit antibodies obtained by the screening method of the present invention is stronger than that of the mouse antibodies and rat antibodies produced by the hybridoma cell method. According to the screening method of the present invention, it is suggested that antibodies with high binding force to antigens can be obtained.

Claims

1. A method for screening target cells that produce a target substance, comprising the following (1) to (3): (1) Immobilizing vesicles with target substances on the membrane surface into micropores; (2) introducing candidate cells obtained from a cell population containing the target cells and a substance that binds to the target substance and is labeled into the micropores to which the vesicles are fixed; and (3) The target cell is identified by detecting the marker bound to the target substance bound to the target substance.

2. The screening method according to claim 1, wherein The target substance is a membrane protein.

3. The screening method according to claim 1, wherein The target substance is an antibody.

4. The screening method according to claim 1, wherein The target cells are spleen cells or B cells derived from lymphoid tissue.

5. The screening method according to claim 1, wherein The target cell is a CHO cell or a hybridoma cell.

6. The screening method according to claim 1, wherein In (1) above, the vesicles are fixed in the micropores using at least one selected from the group consisting of Tim family proteins, annexin V, anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies.

7. The screening method according to claim 6, wherein: In (1), the vesicles are fixed in the micropores using Tim family proteins.

8. The screening method according to claim 1, wherein In the above (2), the candidate cell introduced into the microwell is a single cell to be separated.

9. The screening method according to claim 1, wherein The vesicles are extracellular vesicles.

10. The screening method according to claim 1, wherein The diameter of the vesicle is 30 to 1000 nm.

11. The screening method according to claim 1, wherein The detection in (3) is fluorescence detection.

12. The screening method according to claim 11, wherein By excitation at one wavelength and fluorescence measurement at one wavelength, identification of target cells based on the fluorescence detection is performed.

13. The screening method according to claim 11, wherein In (3) above, the target cells are identified by detecting the wells where fluorescence is confirmed at the side walls of the microwells.

14. The screening method according to claim 1, further comprising the following (4): (4) Recovering the target cells identified in (3). 15 . A method for producing a nucleic acid, comprising the step of obtaining a nucleic acid encoding a target substance from the target cell obtained by the screening method according to claim 1 . 16 . A method for producing a target substance, comprising producing the target substance using the nucleic acid obtained by the method for producing a nucleic acid according to claim 15 .

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