COMPOSITION FOR REGULATING CAR-T CELL ACTIVATION, HEMATOPOIETIC STEM CELL PROLIFERATION, AND iPS CELL DIFFERENTIATION, AND USE THEREOF

Abstract

To provide a novel method for activating cells, a composition for activating cells, and use thereof, which induce activation (i.e., proliferation and / or differentiation) of specific cells more efficiently than co-administration of cytokine molecules.SOLUTION: Provided are [0] extracellular vesicles that present at least one cytokine extramembranously; [1] extracellular vesicles that present at least one target factor extramembranously; or [2] extracellular vesicles that present at least one target factor and at least one cytokine extramembranously.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for controlling CAR-T cell activation, hematopoietic stem cell proliferation, and iPS cell differentiation, and uses thereof.

Background Art

[0002] In immune responses such as the elimination of cancer cells and the like by the living body and the regulation of responses to self-antigens, allergic substances, etc., antigen-specific T cells (for example, cytotoxic T cells, helper T cells, etc.) are known to play a central role. Antigen-specific T cells recognize the binding complex of MHC molecules on the cell surface of antigen-presenting cells such as dendritic cells and macrophages and antigens derived from cancer, allergic substances, etc. with the T cell receptor and are activated, proliferated, differentiated, etc. The activated antigen-specific T cells specifically damage cancer cells presenting antigens and regulate responses to self-antigens, allergic substances, etc. Therefore, it is considered particularly important to activate, proliferate, differentiate, etc. antigen-specific T cells in immune responses.

[0003] As methods for activating antigen-specific T cells, not only the method of expressing a chimeric antigen receptor in T cells that has already been put into practical use, but also other methods have been developed. For example, Patent Document 1 discloses that nanoparticles containing MHC molecules and T cell costimulatory molecules on their surface proliferate antigen-specific T cells. Non-Patent Document 1 also discloses that exosomes expressing IL-12 on the membrane via PTGFRN proliferate model antigen-specific CD8-positive T cells.

[0004] The inventors of the present application have also disclosed a method for activating various T cells using exosomes containing MHC molecules presenting antigens and T cell costimulatory molecules on their surface (Patent Documents 2 and 3).

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2016-520518 [Patent Document 2] International Publication No. 2021 / 172595 [Patent Document 3] International Publication No. 2021 / 172596 [Non-Patent Document]

[0006] [Non-Patent Document 1] Katherine Kirwin, et al., “Exosome Surface Display of IL-12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared to Recombinant IL-12”, November 6, 2019, 34th Annual Meeting of the Society for Immuno-therapy of Cancer [Non-Patent Document 2] Journal of Extracellular Vesicles(2018);7:1535750 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a novel cell activation method, a composition for cell activation, and uses thereof that can activate (i.e., proliferate and / or differentiate) specific cells more efficiently than co-administering cytokine molecules. [Means for Solving the Problems]

[0008] As a result of intensive research, the present inventors have newly found that extracellular vesicles presenting cytokines or target factors outside the membrane can activate (i.e., proliferate and / or differentiate) specific cells (e.g., CAR-T cells, hematopoietic stem cells, iPS cells) without using MHC molecules, and have completed the present invention.

[0009] Therefore, the present invention includes the following: [0] Extracellular vesicles that present at least one cytokine outside the membrane. [1] Extracellular vesicles that present at least one target factor outside the membrane. [2] Extracellular vesicles that present at least one target factor and at least one cytokine outside the membrane.

[0010] [0A] The extracellular vesicle according to [1], wherein the target factor is an antigen. [1A] The extracellular vesicle according to [2], wherein the target factor is an antigen and the cytokine is a T cell-stimulating cytokine. [2A] The extracellular vesicle according to [0A], wherein its membrane contains the following: (B) A protein containing the antigen and capable of presenting the antigen outside the membrane; An extracellular vesicle containing the same. [3A] The extracellular vesicle according to [1A], wherein its membrane contains the following: (1) (A) A protein containing the T cell-stimulating cytokine or a subunit thereof and capable of presenting the T cell-stimulating cytokine outside the membrane; and (B) A protein containing the antigen and capable of presenting the antigen outside the membrane; Or (2) (C) A protein containing the antigen and the T cell-stimulating cytokine or a subunit thereof and capable of presenting the antigen and the T cell-stimulating cytokine outside the membrane An extracellular vesicle containing the same. [4A] The protein (B) is A fusion protein of an antigen and A membrane protein capable of being localized on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle The extracellular vesicle according to [2A] or [3A]. [5A] The protein (A) is the T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of being localized on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle The extracellular vesicle according to [3A], which is a fusion protein with [6A] The protein (C) is the antigen, the T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of being localized on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle The extracellular vesicle according to [3A], which is a fusion protein with [7A] The extracellular vesicle according to any one of [4A] to [6A], wherein the membrane protein capable of being localized on the membrane of the extracellular vesicle or the protein capable of binding to the membrane of the extracellular vesicle contains tetraspanin or its transmembrane domain or MFG-E8 or its membrane-binding domain. [8A] The protein (C) is from the N-terminal side, (C-1) an antigen peptide, (C-2) an optional spacer sequence, and (C-3) a fusion peptide containing tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the T cell-stimulating cytokine or a subunit thereof, and the extracellular vesicle according to [3A], which contains an amino acid sequence encoding them in this order. [9A] The protein (C) is from the N-terminal side, (C-1) a fusion peptide containing tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the T cell-stimulating cytokine or a subunit thereof (C-2) an optional spacer sequence, and (C-3) an antigen peptide, An extracellular vesicle according to [3A], comprising an amino acid sequence encoding amino acids to be encoded in this order. [10A] The fusion peptide, from the N-terminal side, (1) A partial sequence of tetraspanin containing transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (2) An optional spacer sequence, (3) The T cell-stimulating cytokine or a subunit thereof, (4) An optional spacer sequence, and (5) A partial sequence of tetraspanin containing transmembrane domain 4 An extracellular vesicle according to [8A] or [9A], comprising an amino acid sequence encoding the above in this order. [11A] The fusion peptide, from the N-terminal side, (1) The T cell-stimulating cytokine or a subunit thereof, (2) An optional spacer sequence, and (3) MFG-E8 An extracellular vesicle according to [8A] or [9A], comprising an amino acid sequence encoding the above in this order. [12A] The extracellular vesicle according to any one of [0A] to [11A], wherein the extracellular vesicle is an exosome.

[0011] [13A] (a) A polynucleotide encoding protein (A) defined in [3A]; (b) A polynucleotide encoding protein (B) defined in [2A] or [3A]; or (c) A polynucleotide encoding protein (C) defined in [3A]. [14A] A vector comprising the polynucleotide according to [13A]. [15A] (a) A polynucleotide encoding protein (A) defined in [3A]; and (b) A polynucleotide encoding protein (B) defined in [2A] or [3A] Cells transformed by a single vector or a combination of two or more vectors, which contain [16A] (c) Cells transformed by a vector containing a polynucleotide encoding protein (C) as defined in [3A]. [17A] A culture supernatant obtained by culturing the cells described in [15A] or [16A]. [18A] Extracellular vesicles contained in the culture supernatant described in [17A]. [19A] A method for producing the extracellular vesicles described in [1A], comprising: 1) A step of culturing the cells described in [15A] or [16A]; 2) A step of recovering the culture supernatant after culturing; and 3) Optionally, a method for purifying extracellular vesicles from the recovered culture supernatant.

[0012] [20A] A pharmaceutical composition comprising the extracellular vesicles described in [0A] or [1A]. [21A] A pharmaceutical composition for proliferating in vivo or in vitro the chimeric antigen receptor gene-transferred T cells (CAR-T cells) specific for the antigen, which comprises the extracellular vesicles described in [0A] or [1A]. [22A] A pharmaceutical composition for treating cancer comprising cancer cells expressing the antigen, which comprises the extracellular vesicles described in [0A] or [1A] and is administered to a patient administered with the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells). [23A] The pharmaceutical composition according to [21A] or [22A], wherein the antigen is the Her2 protein or a fragment thereof.

[0013] [24A] A method for activating and / or proliferating the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells) in a subject administered with the CAR-T cells, the method comprising administering to the subject the extracellular vesicles described in [0A] or [1A], wherein the chimeric antigen receptor of the CAR-T cells reacts with the antigen presented outside the membrane of the extracellular vesicles, and preferably further the T cell-stimulating cytokine receptor on the CAR-T cells reacts with the T cell-stimulating cytokine presented outside the membrane of the extracellular vesicles, whereby the CAR-T cells are activated and / or proliferated in the subject. [25A] A method for treating cancer comprising cancer cells expressing the antigen in a subject, the method comprising administering to the subject the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells), and then administering the extracellular vesicles described in [0A] or [1A], wherein the chimeric antigen receptor of the CAR-T cells reacts with the antigen presented outside the membrane of the extracellular vesicles, and preferably further the T cell-stimulating cytokine receptor on the CAR-T cells reacts with the T cell-stimulating cytokine presented outside the membrane of the extracellular vesicles, whereby the CAR-T cells are activated and / or proliferated in the subject, and the activated and / or proliferated CAR-T cells attack the cancer cells, thereby suppressing the growth of the cancer cells and treating the cancer. [26A] The method according to [24A] or [25A], wherein the antigen is the Her2 protein or a fragment thereof.

[0014] [27A] Use of the extracellular vesicles described in [1A] in the manufacture of a medicament for activating and / or proliferating the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells) in a subject administered with the CAR-T cells. [28A] Use of the extracellular vesicles described in [1A] in the manufacture of a medicament for treating cancer comprising cancer cells expressing the antigen in a subject, wherein the antigen-specific chimeric antigen receptor gene-transduced T cells (CAR-T cells) have been administered to the subject. [29A] The method according to [27A] or [28A], wherein the antigen is the Her2 protein or a fragment thereof.

[0015] [1B] The extracellular vesicles according to [0], wherein the cytokine is thrombopoietin (TPO) and / or stem cell factor (SCF). [2B] The extracellular vesicles according to [1B], wherein on its membrane are the following: (A)-1 A protein containing the TPO and capable of presenting the TPO outside the membrane; and (A)-2 A protein containing the SCF and capable of presenting the SCF outside the membrane; An extracellular vesicle comprising the same. [3B] The extracellular vesicles according to [1B], wherein on its membrane are the following: (A)―3 A protein containing the TPO and the SCF and capable of presenting the TPO and the SCF outside the membrane An extracellular vesicle comprising the same. [4B] The extracellular vesicles according to any one of [1B] to [3B], further comprising a protein containing L-selectin and / or CXCL12 and capable of presenting L-selectin and / or CXCL12 outside the membrane. [5B] The extracellular vesicles according to [2B], wherein the protein (A)―1 is a fusion protein of TPO and a membrane protein capable of localizing on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle. [6B] The extracellular vesicles according to [2B], wherein the protein (A)―2 is a fusion protein of SCF and a membrane protein capable of localizing on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle. [7B] The protein (A)-3 is the TPO, the SCF, a membrane protein capable of localizing to the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles and is a fusion protein therewith, the extracellular vesicle according to [3B]. [8B] The protein (B) is the L-selectin or CXCL12, a membrane protein capable of localizing to the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles and is a fusion protein therewith, the extracellular vesicle according to [4B]. [9B] A membrane protein capable of localizing to the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles contains tetraspanin or its transmembrane domain or MFG-E8 or its membrane-binding domain, the extracellular vesicle according to any one of [5B] to [8B]. [10B] The extracellular vesicle is an exosome, the extracellular vesicle according to any one of [1B] to [8B]. [11B] (a)-1 A polynucleotide encoding the protein (A)-1 defined in [2B]; (a)-2 A polynucleotide encoding the protein (A)-2 defined in [2B]; (a)-3 A polynucleotide encoding the protein (A)-3 defined in [3B]; or (b) A polynucleotide encoding the protein (B) defined in [4B]. [12B] A vector containing the polynucleotide according to [11B]. [13B] (a)-1 A polynucleotide encoding the protein (A)-1 defined in [2B]; and / or (a) A cell transformed by a single vector or a combination of two or more vectors containing a polynucleotide encoding protein (A)-2 defined by -2 [2B]. [14B] (a) A cell transformed by a vector containing a polynucleotide encoding protein (A)-3 defined by -3 [3B]. [15B] (d) The cell according to [13B] or [14B], further comprising a polynucleotide encoding protein (B) defined by [4B]. [16B] A culture supernatant obtained by culturing the cell according to any one of [13B] to [15B]. [17B] An extracellular vesicle contained in the culture supernatant according to [16B]. [18B] A method for producing the extracellular vesicle according to [1B], comprising: 1) A step of culturing the cell according to any one of [13B] to [15B]; 2) A step of recovering the culture supernatant after culturing; and 3) Optionally, a method for purifying extracellular vesicles from the recovered culture supernatant. [19B] A pharmaceutical composition comprising the extracellular vesicle according to [1B] or the culture supernatant according to [16B].

[0016] [20B] A pharmaceutical composition for activating and / or proliferating hematopoietic stem cells in vivo or in vitro, comprising the extracellular vesicle according to [1B]. [21B] A pharmaceutical composition for treating aplastic anemia in a subject, comprising the extracellular vesicle according to [1B] and administered to a subject administered with hematopoietic stem cells. [22B] A pharmaceutical composition for treating blood cancer or immunodeficiency in a subject, comprising the extracellular vesicles described in [1B], wherein the subject has been administered hematopoietic stem cells after chemotherapy and / or radiotherapy treatment.

[0017] [23B] A method for activating and / or proliferating hematopoietic stem cells in a subject administered with hematopoietic stem cells, comprising administering to the subject the extracellular vesicles described in [1B], wherein the hematopoietic stem cells are activated and / or proliferated in the subject by the reaction of a cytokine receptor on the hematopoietic stem cells with a cytokine presented outside the membrane of the extracellular vesicles. [23B] The method according to [22B], wherein the subject suffers from aplastic anemia. [24B] A method for treating blood cancer or immunodeficiency in a subject, comprising administering hematopoietic stem cells to the subject after chemotherapy and / or radiotherapy treatment, and then administering the extracellular vesicles described in [1B], wherein the hematopoietic stem cells are activated and / or proliferated in the subject by the reaction of a cytokine receptor on the administered hematopoietic stem cells with a cytokine presented outside the membrane of the extracellular vesicles, and the hematopoietic function in the subject is restored.

[0018] [25B] Use of the extracellular vesicles described in [1B] in the manufacture of a medicament for activating and / or proliferating hematopoietic stem cells in a subject administered with hematopoietic stem cells. [26B] The use according to [25B], wherein the subject suffers from aplastic anemia. [27B] Use of the extracellular vesicles described in [1B] in the manufacture of a medicament for treating blood cancer or immunodeficiency in a subject, comprising Here, the subject has been administered hematopoietic stem cells after chemotherapy and / or radiotherapy treatment, and the cytokine receptor on the administered hematopoietic stem cells reacts with the cytokine presented outside the membrane of the extracellular vesicles, so that the hematopoietic stem cells are activated and / or proliferated within the subject, and the hematopoietic function in the subject is restored.

[0019] [1C] The extracellular vesicles according to [0], wherein the cytokine is ActivinA. [2C] The extracellular vesicles according to [1], wherein the target factor is Bc2Lc and the cytokine is ActivinA. [3C] The extracellular vesicles according to [1C], wherein the following are on its membrane: (A) The extracellular vesicles containing a protein capable of presenting the ActivinA outside the membrane, which contains the ActivinA. [4C] The extracellular vesicles according to [2C], wherein the following are on its membrane: (A) A protein containing the ActivinA and capable of presenting the ActivinA outside the membrane; and (B) A protein containing the Bc2Lc and capable of presenting the Bc2Lc outside the membrane; The extracellular vesicles containing the above. [5C] The extracellular vesicles according to [2C], wherein the following are on its membrane: (C) A protein capable of presenting Bc2Lc and ActivinA outside the membrane, which contains the Bc2Lc and the ActivinA. The extracellular vesicles containing the above. [6C] The extracellular vesicles according to [4C], wherein the protein (B) is a fusion protein of Bc2Lc and a membrane protein capable of localizing on the membrane of the extracellular vesicles or a protein capable of binding to the membrane of the extracellular vesicles. [7C] The extracellular vesicle according to [3C] or [4C], wherein the protein (A) is a fusion protein of Activin A and a membrane protein capable of being localized on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle. [8C] The protein (C) is said Bc2Lc and said Activin A and a membrane protein capable of being localized on the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle is a fusion protein with, and is the extracellular vesicle according to [5C]. [9C] The extracellular vesicle according to any one of [6C] to [8C], wherein the membrane protein capable of being localized on the membrane of the extracellular vesicle or the protein capable of binding to the membrane of the extracellular vesicle contains tetraspanin or its transmembrane domain or MFG-E8 or its membrane-binding domain. [10C] The extracellular vesicle according to any one of [1C] to [9C], wherein the extracellular vesicle is an exosome. [11C] (a) A polynucleotide encoding the protein (A) defined in [3C] or [4C]; (b) A polynucleotide encoding the protein (B) defined in [4C]; or (c) A polynucleotide encoding the protein (C) defined in [5C]. [12C] A vector containing the polynucleotide according to [11C]. [13C] (a) A polynucleotide encoding the protein (A) defined in [3C] or [4C]; and / or (b) A cell transformed by a single vector or a combination of two or more vectors containing a polynucleotide encoding the protein (B) defined in [4C]. [14C] (c) A cell transformed by a vector containing a polynucleotide encoding the protein (C) defined in [5C]. [15C] A culture supernatant obtained by culturing the cells according to any one of [11C] or [12C]. [16C] Extracellular vesicles contained in the culture supernatant according to [15C]. [17C] A method for producing the extracellular vesicles according to [1C], comprising: 1) A step of culturing the cells according to [11C] or [12C]; 2) A step of recovering the culture supernatant after culturing; and 3) Optionally, a method for purifying extracellular vesicles from the recovered culture supernatant. [18C] An inducer for differentiating iPS cells or ES cells, comprising the extracellular vesicles according to [1C] or the culture supernatant according to [13C].

Effect of the Invention

[0020] According to the present invention, even without using a major histocompatibility complex (hereinafter also referred to as "MHC") molecule, extracellular vesicles that present cytokines outside the membrane can activate specific cells (i.e., proliferation and / or differentiation). Furthermore, by co-existing a target factor that recognizes specific cells, specific cells can be selectively activated (i.e., proliferation and / or differentiation).

Brief Description of the Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] As used herein, "comprising" includes "substantially comprising", "essentially comprising", "consisting essentially of", and "consisting of".

[0023] As used herein, "extracellular vesicles" are not particularly limited as long as they are vesicles secreted from cells, and examples include Exosomes, Microvesicles (MV), Apoptotic Bodies, etc.

[0024] As used herein, "exosome" refers to vesicles derived from the endocytic-cis pathway, having a size of about 20 to about 500 nm (preferably about 20 to about 200 nm, more preferably about 25 to about 150 nm, still more preferably about 30 to about 100 nm). Components of exosomes include, for example, proteins, nucleic acids (mRNA, miRNA, non-coding RNA), and the like. Exosomes can have a function of mediating intercellular communication. Marker molecules of exosomes include, for example, Alix, Tsg101, tetraspanin, flotillin, phosphatidylserine, and the like.

[0025] As used herein, "microvesicle" refers to vesicles derived from the plasma membrane, having a size of about 50 to about 1000 nm. Components of microvesicles include, for example, proteins, nucleic acids (mRNA, miRNA, non-coding RNA, etc.), and the like. Microvesicles can have functions such as mediating intercellular communication. Marker molecules of microvesicles include, for example, integrin, selectin, CD40, CD154, and the like.

[0026] As used herein, "apoptotic body" refers to vesicles derived from the plasma membrane, having a size of about 500 to about 2000 nm. Components of apoptotic bodies include, for example, fragmented nuclei, organelles, and the like. Apoptotic bodies can have functions such as inducing phagocytosis. Marker molecules of apoptotic bodies include, for example, Annexin V, phosphatidylserine, and the like.

[0027] "Cytokine" refers to a physiologically active substance of a protein secreted from cells. Without particular limitation, examples include interferon (IFN), interleukin (IL), chemokine (such as CCL), stem cell factor (SCF), hematopoietic factors (such as colony-stimulating factor (CSF), erythropoietin (EPO), thrombopoietin (TPO)), tumor necrosis factor (TNF), growth factors (such as EGF, FGF, TGF-β), activin, inhibin, and the like. In this specification, cytokines may include not only immature (inactive form) or mature (active form) cytokines, but also their partial sequences and subunits of active cytokines. Cytokines may be derived from any animal species. For example, rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; mammals such as primates including humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. The cytokines described in this specification are preferably derived from rodents or mammalian animals, more preferably from mice or humans. As long as the cytokines described in this specification can exert their functions, their amino acid sequence identity may be 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more with respect to the wild-type amino acid sequence. Alternatively, as long as the cytokines described in this specification can exert their functions, they may be those in which one or more amino acids are deleted, inserted, added, and / or substituted with respect to the wild-type amino acid sequence.

[0028] A "T cell-stimulating cytokine" is a cytokine that can stimulate (e.g., activate, suppress, etc.) T cells via receptors expressed on the membrane of T cells. Examples of T cell-stimulating cytokines include, but are not limited to, IL-2, IL-4, IL-6, IL-7, IL-12, TGF-β, IFN-α, IFN-γ, etc. Among these, those that can form homo- or hetero-multimers of subunits (e.g., IL-12, TGF-β, etc.) may, as long as they are functional (i.e., as long as they can have the desired pharmacological activity), be in the form of a continuous amino acid sequence linked via a peptide linker or the like in some cases.

[0029] As used herein, the "target factor" refers to a molecule that can bind to a molecule present on the surface of a cell stimulated by extracellular vesicles according to the invention. Examples of molecules present on the cell surface include, but are not limited to, antibodies, receptors, cell adhesion molecules, sugar chains (and glycoproteins). When the molecule present on the cell surface is an antibody, the antigen recognized by the antibody; when the molecule present on the cell surface is a receptor, its ligand (e.g., a chemokine such as CXCL12); when it is a cell adhesion factor, the molecule that binds to the cell adhesion molecule; when the molecule present on the cell surface is a sugar chain (and glycoprotein), the lectin that binds to the sugar chain (e.g., selectin such as L-selectin or lectin such as Bc2Lc) corresponds to the target factor.

[0030] As used herein, the "antigen" is not particularly limited as long as it has antigenicity, and includes not only peptide antigens (i.e., antigen peptides) but also non-peptide antigens such as phospholipids and complex carbohydrates (e.g., bacterial membrane components such as mycolic acid and lipoarabinomannan).

[0031] As used herein, the "antigenic peptide" is not particularly limited as long as it is a peptide composed of two or more amino acids that can serve as an antigen (including those composed of more than 50 amino acids), and it may be of natural origin, synthetic origin, or commercially available. The antigenic peptide may contain the full-length amino acid sequence of a gene product or a partial amino acid sequence thereof. The antigenic peptide is not limited thereto, and examples include, but are not limited to, Axl, BAFF-R, B7-H3, BCMA, CAIX, CD19, CD20, CD22, CD38, CD70, CD138, CEA, CLDN6, EpCAM, FAP, Flt3, folate receptor-α, GD2, Glypican 3, GM-CSF receptor, GRP78, GPC1, HGFR, Integrinαvβ6, IL3R, IL13Ra2, TAG72, Mesothelin, MUC1, MUC16, PSCA, PSMA, ROR1, 5T4, WT-1, α-fetoprotein, MAGE-1, MAGE-3, placental alkaline phosphatase sialyl-Lewis X, CA-125, CA-19, TAG-72, epithelial glycoprotein 2, α-fetoprotein receptor, M2A, tyrosinase, Ras, p53, Her-2 / neu, EGF-R, estrogen receptor, progesterone receptor, myc, BCR-ABL, HPV type 16, melanotransferrin, MUC1, CD10, CD37, CD45R, IL-2 receptor α chain, T cell receptor, prostate acid phosphatase, GP100, MelanA / Mart-1, gp75 / brown, BAGE, S-100, keratin, CYFRA21-1 and other tumor-related antigenic peptides (including their full-length sequences and partial sequences);Self-antigen peptides such as insulin, glutamic acid decarboxylase, ICA512 / IA-2 protein tyrosine phosphatase, ICA12, ICA69, preproinsulin, HSP60, carboxypeptidase H, peripherin, GM1-2, vitronectin, β-crystallin, calreticulin, serotransferrin, keratin, pyruvate carboxylase, C1, bilin 2, nucleosome, ribonucleoprotein, myelin oligodendrocyte glycoprotein, myelin-associated glycoprotein, myelin / oligodendrocyte basic protein, oligodendrocyte-specific protein, myelin basic protein, proteolipid apoprotein, etc. (including their full-length sequences and partial sequences); antigen peptides derived from infectious pathogens such as protozoa (e.g., Plasmodium, Leishmania species, Trypanosoma species), bacteria (e.g., Gram-positive cocci, Gram-positive bacilli, Gram-negative bacteria, anaerobic bacteria), fungi (e.g., Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, Sporothrix), viruses (e.g., adenovirus, herpes simplex virus, papillomavirus, respiratory syncytial virus, poxvirus, HIV, influenza virus, coronaviruses such as SARS-CoV and SARS-CoV2), intracellular parasites (e.g., Chlamydiaceae, Mycoplasmataceae, Acholeplasmataceae, Rickettsiaceae), helminths (e.g., nematodes, trematodes, cestodes), etc. (including their full-length sequences and partial sequences); other antigen peptides such as prions (including their full-length sequences and partial sequences), etc. The antigenic peptide may contain an allergen that causes allergic symptoms. Examples of allergens include, in addition to peptides derived from the above-mentioned protozoa, bacteria, fungi, intracellular parasites, and helminths, exogenous peptides such as peptides derived from house dust, mites, animals (e.g., companion animals such as cats and dogs), and pollen (e.g., cedar and cypress). More specifically, proteins contained in cedar pollen such as Cryj1 (including its full-length sequence and partial sequences) are exemplified. Alternatively, the allergen that causes allergic symptoms may be derived from food. Examples of allergens that cause allergic symptoms to food include peptides (including their full-length sequences and partial sequences) derived from chicken eggs, milk, wheat, buckwheat, crab, shrimp, and peanuts. The antigenic peptide may be subjected to any processing or modification (e.g., phosphorylation or sugar chain modification).

[0032] The extracellular vesicles that present cytokines or target factors outside the membrane are defined as follows. By including the proteins defined in (A), (B), and (C) in the membrane, cytokines or target factors may be presented outside the membrane. Alternatively, cytokines or target factors may be subsequently attached to the surface of isolated extracellular vesicles. The method of attachment is not particularly limited, but cytokines or target factors may be attached to the membrane surface by binding phospholipids to the cytokines or target factors, respectively, and incorporating new lipid moieties into the membrane of the extracellular vesicles. Phosphatidylserine is present on the surface of extracellular vesicles. Therefore, proteins in which the cytokines or target factors to be presented are fused to MFG-E8 that binds to phosphatidylserine are each synthesized and purified, and extracellular vesicles presenting cytokines or target factors on the membrane surface can be produced by mixing the fusion proteins with the extracellular vesicles. In addition, cytokines or target factors with PNEtags attached later may be added to extracellular vesicles in which peptide neoepitopes (PNEs) nanobodies have been expressed in advance to be presented on the membrane surface of the extracellular vesicles. Biotinylated cytokines or target factors may be added to extracellular vesicles expressing streptavidin to be presented on the membrane surface of the extracellular vesicles.

[0033] As used herein, the term "protein containing XXX and capable of presenting XXX outside the membrane" means a protein that contains at least XXX and is capable of presenting XXX outside the membrane of extracellular vesicles. The "protein capable of presenting XXX outside the membrane" may be expressed as a fusion protein containing XXX and a fragment containing a membrane protein or its transmembrane domain, etc., using a plasmid or the like so that XXX is expressed on the membrane of cells or extracellular vesicles. Alternatively, when using soluble XXX (not limited to these, for example, XXX itself; a fusion protein of XXX and the Fc portion of an antibody; a complex of XXX and an antibody recognizing XXX, or its antigen-binding fragment (e.g., scFv, Fab or nanobody), etc.), the soluble XXX and extracellular vesicles may be bound to the membrane of extracellular vesicles via a lipid linker, a peptide linker, etc., as necessary (for example, refer to the method described in JP-A-2018-104341, etc.). Alternatively, a desired tag (e.g., His tag, FLAG tag, PNE tag added thereto (the tag may be expressed as a fusion protein together with other components, or may be bound to separately prepared soluble XXX via a linker, etc. as necessary)) is added to the N-terminal side or C-terminal side of soluble XXX, and a protein containing an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab or nanobody), etc. (for example, an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab or nanobody), etc. itself bound to the membrane of extracellular vesicles via a linker, etc. as necessary; a fusion protein, etc. in which a nanobody against the tag is bound to the N-terminal side or C-terminal side of a membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain) may be mixed with extracellular vesicles containing the tag on the membrane under desired conditions (for example, refer to the method using a PNE tag and an antibody against the tag, etc. described in Raj D, et al., Gut., 2019 Jun;68(6):1052-1064, etc.).In the case of XXX formed by multimers of subunits, if one of the subunits is a protein capable of being presented outside the membrane of extracellular vesicles, the remaining subunits do not necessarily need to be in a form capable of being presented outside the membrane. If one of the subunits is a protein capable of being presented outside the membrane of extracellular vesicles, a functional XXX can be constructed outside the membrane of extracellular vesicles by adding or co-expressing other subunits.

[0034] As the "membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain" used in the present specification, any membrane protein or its transmembrane domain can be selected as long as it can be expressed on the membrane of extracellular vesicles. The "membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain" is preferably a membrane protein known to be capable of being expressed on extracellular vesicles (such as exosomes, etc.) (for example, tetraspanin, CD58, ICAM-1, PTGFRN (see, for example, Non-Patent Document 1, International Publication No. 2019 / 183578, etc.)), or their transmembrane domains, etc.

[0035] As the "protein capable of binding to the membrane of extracellular vesicles or its domain" used in the present specification, any protein or its domain can be selected as long as it can bind to the membrane of extracellular vesicles. The "protein capable of binding to the membrane of extracellular vesicles or its domain" is preferably one known to be capable of binding to the membrane of extracellular vesicles (such as exosomes, etc.) (for example, MFG-E8, or its domain (for example, the C1, C2 domains of MFG-E8 described in Alain Delcayre, et al., Blood Cells, Molecules, and Diseases 35 (2005) 158-168)), etc.

[0036] The "membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain" or "protein capable of binding to the membrane of extracellular vesicles or its domain" described in this specification may be derived from any animal species. For example, rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; mammals such as primates including humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees, etc. The "membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain" or "protein capable of binding to the membrane of extracellular vesicles or its domain" described in this specification is preferably derived from rodents or mammalian animals, and more preferably derived from mice or humans.

[0037] According to Non-Patent Document 2, the markers of mammalian extracellular vesicles are classified as follows. As the membrane proteins or GPI-anchored proteins that can be used as marker proteins of extracellular vesicles, 1) Tissue-nonspecific ones Tetraspanins (CD63, CD9, CD81, CD82), other multiple transmembrane membrane proteins (such as CD47 and heterotrimeric G proteins (GNA: Guanine nucleotide-binding proteins)), MHC class I (HLA-A / B / C, H2-K / D / Q), Integrins (ITGA / ITGB), transferrin receptor (TFR2); LAMP1 / 2; Heparan sulfate proteoglycan (including syndecan (SDC)); Extracellular matrix metalloproteinase inducer (EMMPRIN) (also called BSG or CD147); ADAM10; CD73 (NT5E), a GPI-anchored 5'-nucleotidase, CD55 and CD59, GPI-anchored complement-binding proteins; Sonic hedgehog protein (SHH) 2) Cell / tissue-specific ones Some tetraspanins: TSPAN8 (epithelial cell-specific), CD37 and CD53 (leukocyte-specific); PECAM1 (endothelial cell-specific); ERBB2 (breast cancer-specific); EPCAM (epithelial-specific); CD90 (THY1) (mesenchymal stem cell-specific); CD45 (PTPRC) (immune cell-specific), CD41 (ITGA2B) or CD42a (GP9) (platelet-specific); Glycophorin A (GYPA) (erythrocyte-specific); CD14 (monocyte-specific), MHC class II (HLA-DR / DP / DQ, H2-A); CD3 (T cell-specific); Acetylcholinesterase / AChE-S (neuron-specific), AChE-E (erythrocyte-specific); Amyloid beta A4 / APP (neuron-specific); And so on. Therefore, without being limited thereto, a protein that is a marker of extracellular vesicles may be used in the present invention as a "membrane protein capable of being expressed on the membrane of extracellular vesicles" or a "protein capable of binding to the membrane of extracellular vesicles".

[0038] As used herein, "tetraspanin" refers to proteins belonging to the tetraspanin family (including, but not limited to, for example, CD9, CD53, CD63, CD81, CD82, CD151, etc.). Tetraspanins usually have, from the N-terminal side, transmembrane domain 1 (hereinafter also referred to as "TM1"), small extracellular loop (hereinafter also referred to as "SEL"), transmembrane domain 2 (hereinafter also referred to as "TM2"), small intracellular loop (hereinafter also referred to as "SIL"), transmembrane domain 3 (hereinafter also referred to as "TM3"), large extracellular loop (hereinafter also referred to as "LEL"), and transmembrane domain 4 (hereinafter also referred to as "TM4"). Therefore, they are four-transmembrane proteins, and both the N-terminal side and the C-terminal side are present on the cytoplasmic side. For example, when the tetraspanin is mouse CD63, usually, in the amino acid sequence from about 1 to about 110, it contains TM1, SEL, TM2, SIL, and TM3; in the amino acid sequence from about 111 to about 200, it contains LEL; and in the amino acid sequence from about 201 to about 238, it may contain TM4.

[0039] Each domain (e.g., TM1, SEL, SIL, LTL, etc.) in the "tetraspanin" described in this specification may be derived from the same tetraspanin, or all or part of them may be derived from different tetraspanins.

[0040] As long as the tetraspanin described in this specification can be expressed on the membrane of extracellular vesicles, its amino acid sequence identity to the wild-type amino acid sequence may be 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more. Alternatively, as long as the tetraspanin described in this specification can be expressed on the membrane of extracellular vesicles, it may have one or more amino acid deletions, insertions, additions, and / or substitutions with respect to its wild-type amino acid sequence.

[0041] The partial sequences of tetraspanin described in this specification (for example, each domain; partial sequences including TM1, SEL, TM2, SIL, and TM3; partial sequences including TM4) may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more with respect to their wild-type amino acid sequences. Alternatively, the partial sequences of tetraspanin described in this specification may have one or more amino acids deleted, inserted, added, and / or substituted with respect to their wild-type amino acid sequences.

[0042] As long as MFG-E8 described in this specification is capable of binding to the membrane of extracellular vesicles, it may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more with respect to its wild-type amino acid sequence. Alternatively, as long as MFG-E8 described in this specification is capable of binding to the membrane of extracellular vesicles, it may have one or more amino acids deleted, inserted, added, and / or substituted with respect to its wild-type amino acid sequence.

[0043] As long as CD58, PTGFRN, etc. described in this specification are capable of being expressed on the membrane of extracellular vesicles or are capable of binding to the membrane of extracellular vesicles, they may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more with respect to their wild-type amino acid sequences. Alternatively, as long as CD58, PTGFRN, etc. described in this specification are capable of being expressed on the membrane of extracellular vesicles or are capable of binding to the membrane of extracellular vesicles, they may have one or more amino acids deleted, inserted, added, and / or substituted with respect to their wild-type amino acid sequences.

[0044] As used herein, the "spacer sequence (hereinafter also referred to as the 'peptide linker sequence' or 'linker sequence')" means any sequence having at least one amino acid residue that exists between two or more proteins or their partial sequences or domains, etc. The spacer sequence can be used, for example, when linking two or more proteins or their partial sequences or domains, etc. The length of the spacer sequence in terms of amino acid residues is usually from 1 to about 50, preferably from about 2 to about 28, and more preferably from about 4 to about 25. Examples of the spacer sequence include, but are not limited to, (GGGXS) n G m (wherein X is independently A or G each time it appears, n is from 1 to 8, and m is from 0 to 3); T a S b (GGX) n G m (wherein X is independently S or T each time it appears, n is from 1 to 8, m is from 0 to 3, a is 0 or 1, and b is 0 or 1); etc.

[0045] As used herein, "polynucleotide" means a single-stranded or double-stranded DNA molecule, RNA molecule, DNA-RNA chimeric molecule, etc. Polynucleotides include genomic DNA, cDNA, hnRNA, mRNA, etc., and all natural or artificially modified derivatives thereof. The polynucleotide may be linear or circular.

[0046] In one embodiment of the present invention, extracellular vesicles are provided that present at least one (1, 2, 3, 4, or 5 kinds) of cytokine (hereinafter may be referred to as the first cytokine, the second cytokine, more cytokines, etc. to distinguish each cytokine) outside the membrane. In one embodiment of the present invention, extracellular vesicles are provided that present at least one (1, 2, 3, 4, or 5 kinds) of target factor (hereinafter may be referred to as the first target factor, the second target factor, more target factors, etc. to distinguish each target factor) outside the membrane. In one embodiment of the present invention, there is provided an extracellular vesicle that presents at least one (1, 2, 3, 4, or 5 types) of cytokine and at least one (1, 2, 3, 4, or 5 types) of target factor outside the membrane.

[0047] In one embodiment of the present invention, there is provided an extracellular vesicle that presents at least one cytokine outside the membrane, and the membrane thereof has the following: (A) A protein that contains at least one cytokine or a subunit thereof and can present the cytokine outside the membrane; is provided. In one embodiment of the present invention, there is provided an extracellular vesicle that presents at least one target factor outside the membrane, and the membrane thereof has the following: (B) A protein that contains at least one target factor or a subunit thereof and can present the target factor outside the membrane; is provided. In one embodiment of the present invention, there is provided an extracellular vesicle that presents at least one cytokine and at least one target factor outside the membrane, and the membrane thereof has the following: (A) A protein that contains at least one cytokine or a subunit thereof and can present the cytokine outside the membrane; and (B) A protein that contains at least one target factor or a subunit thereof and can present the target factor outside the membrane; is provided.

[0048] In one embodiment of the present invention, there is provided an extracellular vesicle that presents a T cell-stimulating cytokine and an antigen outside the membrane, and the membrane thereof has the following: (C) Provided is an extracellular vesicle that contains a first cytokine or a subunit thereof and a target factor, and a protein that can present the first cytokine and the target factor outside the membrane.

[0049] Constituent element (A) The "protein capable of presenting a cytokine outside the membrane, which contains a cytokine or its subunit" in the above (A) may contain other proteins or their domains, etc., as long as it is a protein capable of presenting a cytokine outside the membrane of extracellular vesicles.

[0050] In one embodiment of the present invention, the above (A) is a fusion protein or protein complex capable of presenting the antigen outside the membrane, which contains a cytokine or its subunit and a membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain or a protein capable of binding to the membrane of extracellular vesicles or its domain.

[0051] In one embodiment of the present invention, the above (A) is (A) A fusion protein capable of presenting a first cytokine outside the membrane, which contains a first cytokine or its subunit and a partial sequence of tetraspanin, wherein the partial sequence of tetraspanin has at least two transmembrane domains, and the first cytokine or its subunit is arranged between the two transmembrane domains, or (A) A fusion protein capable of presenting a first cytokine outside the membrane, which contains a first cytokine or its subunit and MFG-E8 or its domain.

[0052] As used herein, "the partial sequence of tetraspanin has at least two transmembrane domains, and the first cytokine or its subunit is arranged between the two transmembrane domains" means, for example, when the partial sequence of tetraspanin contains at least TM1 and TM2 of tetraspanin, and the first cytokine or its subunit is arranged between TM1 and TM2, or when the partial sequence of tetraspanin contains at least TM3 and TM4 of tetraspanin, and the first cytokine or its subunit is arranged between TM3 and TM4, etc.

[0053] In one embodiment of the present invention, the above (A) is 1. (A) From the N-terminal side, (A-1) A partial sequence of tetraspanin containing, from the N-terminal side, transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (A-2) An optional spacer sequence, (A-3) The amino acid sequence of the first cytokine or its subunit, (A-4) An optional spacer sequence, and (A-5) A partial sequence of tetraspanin containing transmembrane domain 4 A protein capable of presenting the first cytokine outside the membrane, comprising an amino acid sequence consisting of: 2. (A) From the N-terminal side, (A-3) The amino acid sequence of the first cytokine or its subunit, (A-4) An optional spacer sequence, and (A-5) The amino acid sequence of MFG-E8; A protein capable of presenting the first cytokine outside the membrane, comprising an amino acid sequence consisting of: Or 3. (A) From the N-terminal side, (A-3) The amino acid sequence of the first cytokine or its subunit, (A-4) An optional spacer sequence, and (A-5) A sequence containing any transmembrane domain and tetraspanin; A protein capable of presenting the first cytokine outside the membrane, comprising an amino acid sequence consisting of: is.

[0054] As disclosed in International Publication No. 2016 / 139354, it has been reported that tetraspanin can be expressed on the membrane even if its large extracellular loop (LEL) is replaced entirely or partially with a different amino acid sequence. Therefore, the first cytokine or its subunit in (A-3) may be inserted in place of the LEL of tetraspanin via an optional spacer sequence, or may be inserted at any position in the LEL of tetraspanin or a partial sequence thereof.

[0055] The "partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3" in (A-1) usually does not contain transmembrane domain 4 of tetraspanin. The "partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3" in (A-1) may contain a large extracellular loop or a partial sequence thereof. In (A-1), each of the domains of transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 may be a sequence derived from a different tetraspanin, or may all be sequences derived from the same tetraspanin. Preferably, in (A-1), each of the domains of transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 is a sequence derived from the same tetraspanin.

[0056] In one embodiment of the present invention, the partial sequences of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 in (A-1) are all partial sequences derived from CD9, CD63 or CD81. In one embodiment of the present invention, the partial sequences of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 in (B-1) are all partial sequences derived from CD63 or CD81.

[0057] The "subsequence of tetraspanin containing transmembrane domain 4" in (A-5) usually does not include transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 of tetraspanin. The "subsequence of tetraspanin containing transmembrane domain 4" in (A-5) may include a large extracellular loop or a subsequence thereof. The transmembrane domain 4 in (A-5) may be a sequence derived from a tetraspanin different from (A-1), or may be a sequence derived from the same tetraspanin as (A-1). Preferably, the transmembrane domain 4 in (A-5) is a sequence derived from the same tetraspanin as (A-1). In one embodiment of the present invention, the subsequence of tetraspanin containing transmembrane domain 4 in (A-5) is a subsequence derived from CD9, CD63, or CD81. In one embodiment of the present invention, the subsequence of tetraspanin containing transmembrane domain 4 in (A-5) is a subsequence derived from CD63 or CD81.

[0058] In one embodiment of the present invention, the "subsequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (A-1) is a subsequence derived from CD63, and the "subsequence of tetraspanin containing transmembrane domain 4" in (A-5) is a subsequence derived from CD63. In one embodiment of the present invention, the "subsequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (A-1) is a subsequence derived from CD81, and the "subsequence of tetraspanin containing transmembrane domain 4" in (A-5) is a subsequence derived from CD81.

[0059] The above-mentioned "MFG-E8" in (A-5) is preferably SEQ ID NO: 37, 63, 73, 83, etc., or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more thereto.

[0060] The N-terminus of tetraspanin is present inside the extracellular vesicles. Therefore, so that the cytokine of (A-3) or its subunit is presented outside the extracellular vesicles, the "sequence containing any transmembrane domain and tetraspanin" of (A-5) preferably has an amino acid sequence encoding any odd-numbered (1, 3, 5) transmembrane domain fused with the sequence of tetraspanin such that the N-terminus of (A-5) is present outside the extracellular vesicles. In one embodiment of the present invention, it is a sequence encoding a fusion protein of CD8 (single-pass transmembrane protein) (SEQ ID NO: 5, 21, 53, etc.; or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto) and CD81 (SEQ ID NO: 7, 13, 23, 55, 99, etc.; or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto).

[0061] In addition to the first cytokine, the extracellular vesicles described herein may further contain a second (or more) cytokine. Therefore, in one embodiment of the present invention, the extracellular vesicles described herein may further contain a second (or more) cytokine.

[0062] The second (or more) cytokine may be inserted, for example, into (A) above (for example, the second (or more) cytokine may be linked to the N-terminal side and / or C-terminal side of the "first cytokine" of (A-3) via a spacer sequence or the like as necessary). Alternatively, the second (or more) cytokine may have the same configuration as the constituent requirement (A) described herein, and as a protein (or fusion protein) separate from the protein (or fusion protein) of the constituent requirement (A) described herein, it may be contained in the membrane of the antigen-presenting extracellular vesicles described herein in the same manner as the first cytokine.

[0063] In the (A-2) and (A-4) of each of the above embodiments, the "optional spacer array", if present, can be independently selected. (A-2), if present, may be, for example, a spacer array such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc. (A-4), if present, may be, for example, a spacer array such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.

[0064] In one embodiment of the present invention, when a cytokine forms a multimer (homo / heteromer) with a plurality of subunits and has activity, (A-6) Subunits necessary for having activity Extracellular vesicles may further contain.

[0065] Constituent element (B) The "protein capable of presenting the target factor outside the membrane, which contains the target factor or its subunit" in the above (B) may contain other proteins or their domains, etc., as long as it is a protein capable of presenting the target factor outside the membrane of extracellular vesicles.

[0066] In one embodiment of the present invention, the above (B) is a fusion protein or protein complex capable of presenting the antigen outside the membrane, which contains the target factor or its subunit and a membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain or a protein capable of binding to the membrane of extracellular vesicles or its domain.

[0067] In one embodiment of the present invention, the above (B) is a fusion protein or protein complex capable of presenting the antigen outside the membrane, which contains the target factor or its subunit and a membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain or a protein capable of binding to the membrane of extracellular vesicles or its domain.

[0068] In one embodiment of the present invention, the above (B) is (B) A fusion protein capable of presenting the first target factor or its subunit outside the membrane, comprising the first target factor or its subunit and a partial sequence of tetraspanin, wherein the partial sequence of tetraspanin has at least two transmembrane domains, and the first cytokine is disposed between the two transmembrane domains, or (B) A fusion protein capable of presenting the first target factor outside the membrane, comprising the first target factor or its subunit and MFG-E8 or its domain.

[0069] As used herein, "the partial sequence of tetraspanin has at least two transmembrane domains, and the first target factor or its subunit is disposed between the two transmembrane domains" means, for example, that the partial sequence of tetraspanin contains at least TM1 and TM2 of tetraspanin, and the first target factor or its subunit is disposed between TM1 and TM2; or the partial sequence of tetraspanin contains at least TM3 and TM4 of tetraspanin, and the first target factor or its subunit is disposed between TM3 and TM4, and the like.

[0070] In one embodiment of the present invention, the above (B) is 1. (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin containing, from the N-terminal side, transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3, (B-2) An optional spacer sequence, (B-3) The amino acid sequence of the first target factor or its subunit, (B-4) An optional spacer sequence, and (B-5) A partial sequence of tetraspanin containing transmembrane domain 4 A fusion protein capable of presenting the first target factor outside the membrane, comprising an amino acid sequence consisting of 2. (B) From the N-terminal side, (B-3) The amino acid sequence of the first target factor or its subunit, (B-4) An optional spacer sequence, and (B-5) The amino acid sequence of MFG-E8 A fusion protein capable of presenting the first target factor outside the membrane, comprising an amino acid sequence consisting of Or 3. (B) From the N-terminal side, (B-3) The amino acid sequence of the first target factor or its subunit, (B-4) An optional spacer sequence, and (B-5) A sequence containing any transmembrane domain and tetraspanin; A fusion protein capable of presenting the first target factor outside the membrane, comprising an amino acid sequence consisting of

[0071] As disclosed in International Publication No. WO 2016 / 139354, it has been reported that tetraspanin can be expressed on the membrane even if its large extracellular loop (LEL) is entirely or partially replaced with a different amino acid sequence. Therefore, the first target factor or its subunit in (B-3) may be inserted via an optional spacer sequence in place of the LEL of tetraspanin, or may be inserted at any position in the LEL of tetraspanin or in a partial sequence thereof.

[0072] The "partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (B-1) usually does not contain transmembrane domain 4 of tetraspanin. The "partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (B-1) may contain a large extracellular loop or a partial sequence thereof. Each of the domains of transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 in (B-1) may be a sequence derived from a different tetraspanin, or may all be sequences derived from the same tetraspanin. Preferably, each of the domains of transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 in (A-1) is a sequence derived from the same tetraspanin.

[0073] In one embodiment of the present invention, the partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 in (B-1) are all partial sequences derived from CD9, CD63, or CD81. In one embodiment of the present invention, the partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 in (B-1) are all partial sequences derived from CD63 or CD81.

[0074] The "subsequence of tetraspanin containing transmembrane domain 4" in (B-5) usually does not include transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 of tetraspanin. The "subsequence of tetraspanin containing transmembrane domain 4" in (B-5) may include a large extracellular loop or a subsequence thereof. The transmembrane domain 4 in (B-5) may be a sequence derived from a tetraspanin different from (B-1), or may be a sequence derived from the same tetraspanin as (B-1). Preferably, the transmembrane domain 4 in (B-5) is a sequence derived from the same tetraspanin as (B-1). In one embodiment of the present invention, the subsequence of tetraspanin containing transmembrane domain 4 in (B-5) is a subsequence derived from CD9, CD63 or CD81. In one embodiment of the present invention, the subsequence of tetraspanin containing transmembrane domain 4 in (B-5) is a subsequence derived from CD63 or CD81.

[0075] In one embodiment of the present invention, the "subsequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3" in (B-1) is a subsequence derived from CD63, and the "subsequence of tetraspanin containing transmembrane domain 4" in (B-5) is a subsequence derived from CD63. In one embodiment of the present invention, the "subsequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3" in (B-1) is a subsequence derived from CD81, and the "subsequence of tetraspanin containing transmembrane domain 4" in (B-5) is a subsequence derived from CD81.

[0076] The above-mentioned "MFG-E8" in (B-5) is preferably SEQ ID NO: 37, 63, 73, 83, etc., or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more thereto.

[0077] The N-terminus of tetraspanin is present inside the extracellular vesicles. Therefore, so that the target factor of (B-3) or its subunit is presented outside the extracellular vesicles, the "sequence containing any transmembrane domain and tetraspanin" of (B-5) preferably has an amino acid sequence encoding any odd-numbered (1, 3, 5) transmembrane domain fused with the sequence of tetraspanin such that the N-terminus of (B-5) is present outside the extracellular vesicles. In one embodiment of the present invention, it is a sequence encoding a fusion protein of CD8 (a single-pass transmembrane protein; for example, SEQ ID NO: 5 or 21, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more thereto) and CD81.

[0078] In addition to the first target factor, the extracellular vesicles described herein may further contain a second (or more) target factor. Therefore, in one embodiment of the present invention, the extracellular vesicles described herein may further contain a second (or more) target factor.

[0079] The second (or more) target factor or its subunit may be inserted, for example, into (B) above (for example, the second (or more) target factor or its subunit may be linked to the N-terminal side and / or C-terminal side of the "first target factor or its subunit" of (B-3) via a spacer sequence or the like as necessary). Alternatively, the second (or more) target factor or its subunit may have the same structure as the constituent requirement (B) described herein, and thus, as a protein (or fusion protein) separate from the protein (or fusion protein) of the constituent requirement (B) described herein, it may be contained in the membrane of the antigen-presenting extracellular vesicles described herein in the same manner as the first target factor.

[0080] In the (B-2) and (B-4) of each of the above embodiments, the "optional spacer array", if present, can be independently selected. (B-2), if present, may be, for example, a spacer array such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc. (B-4), if present, may be, for example, a spacer array such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.

[0081] In one embodiment of the present invention, when the target factor forms a multimer (homo / heteromer) with a plurality of subunits and has activity, (B-6) subunits necessary for having activity the extracellular vesicles may further contain.

[0082] In one embodiment of the present invention, regarding the above (A) and (B), (A) and (B) may be fused into one molecule. Such a fusion molecule may be translated as one protein molecule with or without a spacer sequence between (A) and (B), or the proteins of (A) and (B) may be fused into one molecule by chemical cross-linking (for example, disulfide bonds between cysteine residues). Alternatively, the above (A) and (B) may be functionally fused by sharing a portion of an element for localizing the protein to extracellular vesicles, that is, "a membrane protein capable of being expressed on the membrane of extracellular vesicles or its transmembrane domain" or "a protein capable of binding to the membrane of extracellular vesicles or its domain".

[0083] Constituent element (C) The "protein capable of presenting the cytokine and the target factor outside the membrane, which contains the cytokine or its subunit and the target factor or its subunit" in the above (C) may contain other proteins or their domains, etc., as long as it is a protein capable of presenting the cytokine and the target factor outside the membrane of extracellular vesicles. In one embodiment of the present invention, the above (C) may include a cytokine or a subunit thereof, a target factor or a subunit thereof, and a membrane protein capable of localizing to the membrane of extracellular vesicles or its transmembrane domain, or a protein capable of binding to the membrane of extracellular vesicles or its membrane-binding domain.

[0084] In one embodiment of the present invention, the membrane protein capable of localizing to the membrane of the extracellular vesicles or the protein capable of binding to the membrane of the extracellular vesicles may be tetraspanin or MFG-E8. The fusion protein may include, from the N-terminal side, (C-3) the amino acid sequence of the target factor or a subunit thereof, (C-4) an optional spacer sequence, and (C-5) an amino acid sequence encoding a fusion peptide containing tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the at least one cytokine, in this order. The fusion protein may include, from the N-terminal side, (C-1) a fusion peptide containing tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the at least one cytokine (C-2) an optional spacer sequence, and (C-3) the amino acid sequence of the target factor or a subunit thereof, in this order, and may include an amino acid sequence encoding them. Here, the fusion peptide includes, from the N-terminal side, (1) a partial sequence of tetraspanin containing transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (2) an optional spacer sequence, (3) the amino acid sequence of the at least one cytokine, (4) an optional spacer sequence, and (5) a partial sequence of tetraspanin containing transmembrane domain 4 It may contain an amino acid sequence encoding in this order. The fusion peptide, from the N-terminal side, (1) the amino acid sequence of the at least 1 cytokine, (2) an optional spacer sequence, and (3) the amino acid sequence of MFG-E8 or its membrane-binding domain It may contain an amino acid sequence encoding in this order.

[0085] In one embodiment of the present invention, when a cytokine and / or a target factor forms a multi-subunit and a multimer (homo / heteromer) to have activity, (C-6) the subunits necessary for having activity Extracellular vesicles may further contain.

[0086] In one embodiment of the present invention, the cytokine in each of the above embodiments is a T cell-stimulating cytokine. In one embodiment of the present invention, the T cell-stimulating cytokine is IL-2 (preferably, SEQ ID NO: 89, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto), IL-4, TGF-β, IL-7 (preferably, SEQ ID NO: 93, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto). Among these, those that can form homo- or hetero-multimers of subunits (e.g., IL-12, TGF-β, etc.) may be in the form of a continuous amino acid sequence linked via a peptide linker or the like as long as they are functional (i.e., as long as they can have the desired pharmacological activity). These T cell-stimulating cytokines bind to corresponding receptors present on the cell surface of cells such as T cells, B cells, and NK cells, and signals are transmitted into the cells, thereby differentiating and proliferating T cells, B cells, NK cells, monocytes, macrophages, etc.

[0087] In one embodiment of the present invention, the cytokines of the above embodiments are thrombopoietin (TPO; also referred to as Megakaryocyte Stimulating Factor) and / or stem cell factor (SCF; also referred to as Kit ligand). In one embodiment of the present invention, the first cytokine in the above embodiments is TPO (preferably, SEQ ID NO: 19, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto), and the second cytokine is SCF (preferably, SEQ ID NO: 29 or 33, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto). The TPO gene encodes a protein of 353 amino acid residues, but 21 residues are signal sequences, which are removed and then secreted as a glycoprotein of 60-70 kDa. By binding to the c-mpl, which is the TPO receptor, and transmitting signals into the cell, it stimulates the proliferation of hematopoietic stem cells, the maturation and proliferation of megakaryocytes, and promotes platelet formation. In this specification, fragment peptides of TPO having only the receptor-binding domain are also included in TPO. The SCF gene encodes a protein of 273 residues, but the 25 N-terminal residues are signal sequences, and residues 26-273 are secreted as a membrane protein type of SCF with sugar modification. Its extracellular domain is released as a soluble type of SCF by processing. SCF binds as a homodimer to receptors (two molecules) known as c-Kit (CD117) (heterotetramerization), and the KIT molecule autophosphorylates and transmits signals into the cell, thereby promoting the proliferation and maintenance of hematopoietic stem cells. In this specification, all of the membrane protein type, soluble type, and their homodimers linked by a linker are included in SCF.

[0088] In one embodiment of the present invention, the cytokine of each of the above embodiments is Activin or its subunit. Activin is the C-terminal peptide cleaved from the inhibin βA chain preproprotein (also referred to as inhibin βA subunit precursor) gene product by a processing enzyme, β A Activin A in which the subunit is homodimerized by an SS bond; β which is the C-terminal peptide cleaved from the inhibin βB chain preproprotein (also referred to as inhibin βB subunit precursor) gene product by a processing enzyme B Activin B in which the subunit is homodimerized by an SS bond; β A subunit and β B There is Activin AB in which the subunit is heterodimerized by an SS bond. In one embodiment of the present invention, the cytokine is the inhibin β A chain preproprotein gene product itself or a fragment thereof (for example, SEQ ID NO: 59, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto) that can function as Activin without undergoing processing due to having a mutation; or β A subunit (for example, SEQ ID NO: 69, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto). These can function as active Activin A in cells. Activin A specifically binds to two forms of the Activin type I receptor (RI-A and RI-B) and two forms of the Activin type II receptor (RII-A and RII-B) to transmit a signal into the cell, thereby inducing the differentiation of iPSCs (induced pluripotent stem cells) / ESCs (embryonic stem cells).

[0089] In one embodiment of the present invention, the target factor in each of the above embodiments is an antigen. In one embodiment of the present invention, the target factor is an antigenic peptide. In one embodiment of the present invention, the target factor is HER2 or a fragment thereof (preferably, SEQ ID NO: 77 or 97, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto). Among the antigenic peptides, those capable of forming homo- or hetero-multimers of subunits may be those of a continuous amino acid sequence, which may be linked via a peptide linker or the like as long as they are functional (i.e., capable of having a desired pharmacological activity).

[0090] In one embodiment of the present invention, the target factor in each of the above embodiments is L-selectin and / or CXCL12. In one embodiment of the present invention, the target factor is L-selectin (also referred to as CD62L) (preferably, SEQ ID NO: 11, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto) or CXCL12 (also referred to as SDF1) (preferably, SEQ ID NO: 3, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more thereto). Further, in one embodiment of the present invention, the first target factor may be L-selectin and the second target factor may be CXCL12. L-selectin recognizes CD34 on hematopoietic stem cells (CD34 positive), and CXCL12 recognizes chemokine (C-X-C motif) receptor 4.

[0091] In one embodiment of the present invention, the target factor of each of the above embodiments is lecithin or its subunit. In one embodiment of the present invention, it is the lectin (carbohydrate-binding protein) Bc2Lc of Burkholderia cenocepacia. In one embodiment of the present invention, the target factor may be the Bc2Lc gene product or its multimer (e.g., SEQ ID NO: 43, 47, 51; or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more to these; or multimers thereof) bound via a linker. Bc2Lc is a multimer that recognizes a carbohydrate-modified protein (e.g., podocalyxin with Fucα1-2Galβ1-3GalNAc) on the surface of iPSC (induced pluripotent stem cell) / ESC (embryonic stem cell) cells.

[0092] In one embodiment of the present invention, the extracellular vesicle is an exosome.

[0093] The extracellular vesicles described in the present specification may contain or be bound to a substance (e.g., a low-molecular compound, nucleic acid, etc.) that may be therapeutically beneficial inside or in the membrane thereof. The method of encapsulating the substance inside the membrane of the extracellular vesicle is not limited to these, and examples include a method of mixing the substance and the extracellular vesicles described in the present specification in a suitable solvent. In one embodiment of the present invention, the extracellular vesicles may contain any protein preparation. The protein preparation is not particularly limited, and may be a protein that may also exist naturally, such as erythropoietin, a synthetic protein that does not exist naturally, such as an immunoglobulin-CTLA4 fusion protein, or a monoclonal antibody or an active fragment thereof. These protein preparations may be localized on the surface of extracellular vesicles as a fusion protein with a membrane protein that can be localized on the membrane of extracellular vesicles or its transmembrane domain, or a protein that can bind to the membrane of extracellular vesicles or its membrane-binding domain. Such extracellular vesicles can be obtained by transfecting a cell producing extracellular vesicles with a vector for expressing the fusion protein and secreting the extracellular vesicles from the cell.

[0094] Each fusion protein, protein complex, or protein preparation contained in the membrane of the extracellular vesicles described herein may contain one or more detectable labels. For example, the fusion protein, protein complex, or protein preparation may be labeled with a specific reporter molecule, fluorophore, radioactive material, or enzyme (e.g., peroxidase, phosphatase) or the like by a conventional method. These may be linked, for example, to the N-terminal side or C-terminal side of the fusion protein, protein complex, or protein preparation as a component of the fusion protein, protein complex, or protein preparation.

[0095] In one embodiment of the present invention, there is provided a polynucleotide encoding each protein (or fusion protein) in (A), (B), and (C) contained in the membrane of the extracellular vesicles described herein. In one embodiment of the present invention, (a) a sequence encoding a fusion protein (A) capable of presenting at least one cytokine or its subunit outside the membrane of extracellular vesicles, containing at least one cytokine or its subunit; (b) a sequence encoding a fusion protein (B) capable of presenting at least one target factor or its subunit outside the membrane of extracellular vesicles, containing at least one target factor or its subunit; (c) at least one cytokine or a subunit thereof, and at least one target factor or a subunit thereof, and encodes a fusion protein (C) capable of presenting the cytokine and the target factor outside the membrane of extracellular vesicles; A polynucleotide comprising at least one sequence selected from the group consisting of is provided. The above sequences (a)-(c) include the sequences specifically described in the present specification and sequences with high homology (preferably, homology of 90% or more, more preferably 95% or more, still more preferably 99% or more), but are not particularly limited. Paralogs (gene sequences generated by gene duplication) or orthologs (gene groups with homologous functions existing in different organisms) may be used as long as they have equivalent functions, and sequences with modified sequence information (such as deletions, insertions, substitutions, etc.) are also included.

[0096] Those skilled in the art can appropriately determine the polynucleotide encoding each protein (or fusion protein) in (A)-(C) above with reference to the amino acid sequence of the above fusion protein or protein complex. Note that the amino acid sequence of each fusion protein or protein complex in (A)-(C) can be appropriately determined with reference to the amino acid sequence of each component in each fusion protein or protein complex (for example, in the case of (A), (A-1)-(A-5) or (A-3)-(A-5), and optionally (A-6)). The types of codons used when determining the polynucleotide can be arbitrarily selected. For example, the polynucleotide may be determined in consideration of the codon frequency of the cells to be transformed using the vector containing the polynucleotide.

[0097] A polynucleotide encoding a signal peptide (signal sequence) may be added to the N-terminal side of the polynucleotide encoding each protein (or fusion protein) in (A)-(C) above, if necessary.

[0098] The amino acid sequence of the signal peptide can be any one, and may be determined, for example, in consideration of the amino acid sequence of the fusion protein to be expressed. Examples of the polynucleotide encoding the signal peptide include polynucleotides encoding the signal peptide of CD8 (for example, SEQ ID NO: 1, 17, 41) (for example, SEQ ID NO: 2, 18, 42), polynucleotides encoding the signal peptide of MEG-E8 (for example, SEQ ID NO: 27) (for example, SEQ ID NO: 28), and the like.

[0099] Information on each component of each protein (or fusion protein) in (A) to (C) described above (for example, in the case of (A), (A-1) to (A-5) or (A-3) to (A-5), optionally (A-6)), the amino acid sequence of the signal peptide, etc., and the polynucleotide encoding them can be appropriately obtained, for example, by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Also, the amino acid sequence in the partial sequence of tetraspanin and the polynucleotide encoding it may be referred to WO 2016 / 139354.

[0100] In one embodiment of the present invention, a vector containing at least one polynucleotide selected from the polynucleotides described herein is provided.

[0101] As used herein, "vector" refers to any vector (including, but not limited to, plasmid vectors, cosmid vectors, phage vectors such as phage, adenovirus vectors, baculovirus vectors and other virus vectors, artificial chromosome vectors, etc.). Vectors include expression vectors, cloning vectors, etc. An expression vector generally may contain a desired coding sequence and appropriate polynucleotides necessary for the expression of the coding sequence operably linked in a host organism (such as plants, insects, animals, etc.) or an in vitro expression system. A cloning vector may be used to manipulate and / or amplify a desired polynucleotide fragment. A cloning vector may lack functional sequences required for the expression of a desired polynucleotide fragment.

[0102] In one embodiment of the present invention, the polynucleotides described herein may be inserted into the same vector as long as they can be operably inserted, or two or more polynucleotides may be inserted into separate vectors. In one embodiment of the present invention, there is provided a kit combining two or more vectors comprising at least one polynucleotide selected from the polynucleotides described herein.

[0103] Transformed cells In one embodiment of the present invention, the following: (i) a polynucleotide encoding at least one protein (or fusion protein) of (A) described herein, (ii) a polynucleotide encoding at least one protein (or fusion protein) of (B) described herein, and / or (iii) a cell transformed by a single vector or a combination of two or more vectors comprising a polynucleotide encoding at least one protein (or fusion protein) of (C) described herein is provided.

[0104] In one embodiment of the present invention, the following: (i) At least one polynucleotide of (a) described in this specification, (ii) At least one polynucleotide of (b) described in this specification, and / or (iii) At least one polynucleotide of (c) described in this specification Provided are cells transformed by a single vector or a combination of two or more vectors containing the above.

[0105] "Transformed by a single vector or a combination of two or more vectors" means, for example, that the cells may be transformed by a vector in which all of the polynucleotides of (i) to (iii) above are inserted, or by a combination of two or more vectors in which two or more of these are inserted into separate vectors.

[0106] The cells to be transformed are not particularly limited as long as extracellular vesicles described in this specification can be obtained after transformation, and may be primary cultured cells, subcultured cells, or cell lines, and these may be normal cells or diseased cells including cancerous or tumorous cells. Also, the origin of the cells to be transformed is not particularly limited, and examples include cells derived from animals such as mammals such as rodents such as mice, rats, hamsters, guinea pigs, rabbits such as rabbits, ungulates such as pigs, cows, goats, horses, sheep, carnivores such as dogs, cats, primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees; cells derived from plants; cells derived from insects, etc. Preferably, the cells to be transformed are cells derived from animals. Examples of cells derived from animals include, but are not limited to, human embryonic kidney cells (including HEK293T cells, etc.), human FL cells, Chinese hamster ovary cells (CHO cells), COS-7, Vero, mouse L cells, rat GH3, etc.

[0107] The method for transforming cells is not particularly limited as long as it can introduce the target polynucleotide into the cells. For example, it may be the electroporation method, the microinjection method, the calcium phosphate method, the cationic lipid method, the method using liposomes, the method using non-liposomal substances such as polyethyleneimine, the virus infection method, etc.

[0108] The transformed cells may be transiently expressing transformed cells or stably expressing transformed cells (stable cell lines) that express the protein (or fusion protein) of (A), (B), and / or (C).

[0109] The culture conditions of the transformed cells are not particularly limited. For example, when the transformed cells are animal-derived cells, for example, media generally used for cell culture (e.g., RPMI1640 medium, Eagle's MEM medium, Dulbecco's modified Eagle medium (DMEM medium), Ham F12 medium, or any combination thereof, etc.), or a medium obtained by adding other components such as fetal bovine serum, antibiotics, amino acids, etc. thereto may be used. For example, in the presence of about 1 to about 10% (preferably about 2 to about 5%) CO 2 at about 30 to about 40 °C (preferably about 37 °C) for a desired time (e.g., about 0.5 hours to about 240 hours (preferably about 5 to about 120 hours, more preferably about 12 to about 72 hours)), the cells may be cultured (e.g., under static or shaking conditions).

[0110] The culture supernatant obtained by culturing the transformed cells may contain the extracellular vesicles described herein. Therefore, when culturing the transformed cells for the purpose of obtaining the antigen-presenting extracellular vesicles described herein, if necessary, a medium from which extracellular vesicles such as exosomes have been removed (e.g., Dulbecco's modified Eagle medium containing about 1 to about 5% fetal bovine serum from which exosomes have been removed, etc.) may be used.

[0111] In one embodiment of the present invention, a culture supernatant obtained by culturing the transformed cells described herein is provided.

[0112] The extracellular vesicles contained in the culture supernatant described in this specification can be recovered, for example, by purifying (e.g., centrifugation, chromatography, etc.), concentrating, and isolating the culture supernatant. In one embodiment of the present invention, extracellular vesicles obtained from the culture supernatant described in this specification are provided.

[0113] The extracellular vesicles described in this specification include, but are not limited to, those obtained by means such as gene recombination techniques known to those skilled in the art (e.g., by the methods described below, or by the methods described in the examples, or by methods similar thereto). By ordinary gene recombination techniques, polynucleotides encoding the proteins (or fusion proteins) of (A), (B), or (C) (or the polynucleotides of (a), (b), or (c) above) are obtained, and these can be operably inserted into the same or separate vectors. When inserting two or more polynucleotides into the same vector, each may be operably linked to the same or separate promoters. The obtained single or two or more vectors are used to transform cells simultaneously or sequentially to obtain transformed cells (these transformed cells may be cells that transiently express these fusion proteins or cells that stably express them (stable strains)). The obtained transformed cells are cultured under desired conditions to obtain a culture supernatant, and the obtained culture supernatant is purified as needed (e.g., by centrifugation, antibodies (e.g., antibodies that recognize proteins contained in the membrane of extracellular vesicles), chromatography, flow cytometry, etc.), concentrated (e.g., by ultrafiltration, etc.), dried, etc., to obtain the extracellular vesicles described in this specification.

[0114] The extracellular vesicles described in this specification may be confirmed, for example, by techniques such as flow cytometry, ELISA, Western blotting, etc., to confirm that (A) and / or (B) (or (C) instead of (A) and (B)) are contained in the membrane.

[0115] In one embodiment of the present invention, there is provided a method for producing antigen-presenting extracellular vesicles described herein, the method comprising recovering a culture supernatant obtained by culturing the transformed cells described herein.

[0116] In one embodiment of the present invention, there is provided a method for producing extracellular vesicles described herein, (i) a polynucleotide encoding the protein (or fusion protein) of (A) described herein (or the polynucleotide of (a)), (ii) a polynucleotide encoding the protein (or fusion protein) of (B) described herein (or the polynucleotide of (b)), and / or (iii) a polynucleotide encoding the protein (or fusion protein) of (C) described herein (or the polynucleotide of (c)), transforming a cell with a single vector or a combination of two or more vectors comprising the same, either simultaneously or sequentially (preferably, simultaneously), recovering a culture supernatant obtained by culturing the resulting transformed cells, and including a method.

[0117] In one embodiment of the present invention, there is provided a composition (e.g., a pharmaceutical composition) comprising the antigen-presenting extracellular vesicles, polynucleotides, and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, described herein. In one embodiment of the present invention, there are provided pharmaceuticals and reagents comprising the antigen-presenting extracellular vesicles described herein or the culture supernatants described herein.

[0118] The compositions described herein (e.g., for pharmaceutical or reagent use), although not limited thereto, can include additives such as excipients, lubricants, binders, disintegrants, pH adjusters, solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners, surfactants, etc. The types of these additives, their usage amounts, etc. can be appropriately selected by those skilled in the art according to the purpose. When used as a pharmaceutical composition, these additives are preferably pharmacologically acceptable carriers. Further, when the composition described herein contains a polynucleotide, although not essential, it preferably contains a carrier suitable for nucleic acid DD (drug delivery), and examples of these carriers include lipid nanoparticles (LNP) and polymers (e.g., PEI).

[0119] The compositions described herein (e.g., for pharmaceutical or reagent use), together with the above-described additives, can be formulated into tablets, coated tablets, orally disintegrating tablets, chewable agents, pills, granules, fine granules, powders, hard capsules, soft capsules, liquids (including, for example, syrups, injections, lotions, etc.), suspensions, emulsions, jellies, patches, ointments, creams, inhalants, suppositories, etc. by methods known per se. These can be oral preparations or parenteral preparations. The formulated products may further contain other beneficial components (e.g., other therapeutically beneficial components) according to the purpose.

[0120] In one embodiment of the present invention, as shown in Example 3, when an antigen is used as a target factor, a genetically modified T cell that recognizes the antigen, for example, a T cell that forcibly expresses a T cell receptor (TCR) that recognizes and binds to the antigen (TCR-T cell); a fusion protein (Chimeric antigen receptor: CAR) containing a target antigen recognition portion of an antibody (such as a single-chain variable region scFv (single chain Fv) containing VH and VL or a nanobody containing a variable region of an immunoglobulin (antibody) consisting only of a heavy chain) that recognizes and binds to the antigen and a lymphocyte activation molecule (such as an intracellular domain of CD28 or an intracellular domain of mCD3z) can specifically proliferate T cells (CAR-T cells), etc. Therefore, by administering the pharmaceutical composition according to one embodiment of the present invention to a subject, in the body of the subject administered with TCR-T cells or CAR-T cells, the TCR of the TCR-T cells or the chimeric antigen receptor of the CAR-T cells reacts with the antigen presented outside the membrane of the extracellular vesicle, and preferably further reacts with the T cell-stimulating cytokine presented outside the membrane of the extracellular vesicle and the T cell-stimulating cytokine receptor on the TCR-T cells or CAR-T cells, whereby the TCR-T cells or CAR-T cells are activated and / or proliferated in the subject. The activated and / or proliferated TCR-T cells or CAR-T cells attack cancer cells that express the antigen or a fragment thereof on the surface, thereby suppressing the growth of cancer cells and treating cancer.

[0121] The cancers include, but are not limited to, any solid cancer and blood cancer, for example, small cell lung cancer, non-small cell lung cancer, breast cancer, esophageal cancer, gastric cancer, small intestine cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, prostate cancer, myeloma, kidney cancer (including renal cell cancer, etc.), parathyroid cancer, adrenal cancer, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, ovarian cancer (for example, its histological type is serous adenocarcinoma, mucinous adenocarcinoma, clear cell adenocarcinoma, etc.), testicular cancer, bladder cancer, vulvar cancer, penile cancer, thyroid cancer, head and neck cancer, skull base pharyngeal cancer, pharyngeal cancer, tongue cancer, skin cancer, Merkel cell cancer, melanoma (malignant melanoma, etc.), epithelial cancer, squamous cell carcinoma, basal cell carcinoma, pediatric cancer, cancer of unknown primary origin, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, Kaposi sarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, mesothelioma, Ewing tumor, seminoma, Wilms tumor, brain tumor, glioma, glioblastoma, astrocytoma, medulloblastoma, meningioma, neuroblastoma, medulloblastoma, retinoblastoma, spinal tumor, malignant lymphoma (for example, non-Hodgkin lymphoma, Hodgkin lymphoma, etc.), chronic or acute lymphocytic leukemia, adult T-cell leukemia, etc.

[0122] In addition, as shown in Example 1, the composition which is one embodiment of the present invention can activate / proliferate endogenous or externally transplanted hematopoietic stem cells in vitro or in vivo. Therefore, it can be used for the recovery treatment of hematopoietic stem cells for aplastic anemia and blood cell reduction after cancer radiotherapy. In aplastic anemia, various symptoms appear due to the decrease in red blood cells, neutrophils, and platelets. Aplastic anemia is treated or prevented by transplanting healthy hematopoietic stem cells to regenerate the patient's hematopoietic ability. Hematopoietic stem cell transplantation is also performed for the purpose of complete cure for blood cancers and immunodeficiency diseases that are difficult to treat with only conventional chemotherapy or immunosuppressive therapy. In hematopoietic stem cell transplantation, after pre-transplant conditioning consisting of high-dose chemotherapy and total body radiotherapy, etc., hematopoietic stem cells previously collected from oneself or a donor are administered by intravenous drip. Cancers such as blood and lymph cancers, which are sensitive to chemotherapy and radiotherapy, are suitable for treatment. The purpose of pre-transplant conditioning is to reduce tumor cells and suppress the patient's own immune cells. As a result, the transplanted hematopoietic stem cells take root (engraft) in the patient's bone marrow, and normal hematopoietic function is restored. Also, in the case of allogeneic hematopoietic stem cell transplantation (allotransplantation) in which hematopoietic stem cells provided by a donor are transplanted, a graft-versus-leukemia effect (GVL effect) in which the donor's lymphocytes attack the patient's tumor cells can also be expected.

[0123] Also, as shown in Example 2, the composition which is one embodiment of the present invention can induce differentiation of iPS cells / embryonic stem cells in vitro or in vivo.

[0124] The subjects to be treated or prevented from the various diseases described above are not limited to these, but for example, rodents such as mice, rats, hamsters, guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, sheep; carnivores such as dogs, cats; primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees; etc. mammals, or plants, etc. are mentioned, but preferably animals, more preferably rodents or primates, and even more preferably mice or humans.

[0125] The dosage of the antigen-presenting cell extracellular vesicles, polynucleotide and / or vector containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing these or formulations thereof described in this specification can be appropriately determined in consideration of the sex, age, body weight, health status, degree of disease condition or diet of the subject to be administered; administration time; administration method; combination with other drugs; and other factors.

Examples

[0126] Hereinafter, the present invention will be described in more detail using examples, but these examples do not limit the scope of the present invention in any way.

[0127] Example 1. Effect on hematopoietic stem cells 1.1. Preparation of plasmid A polynucleotide (SEQ ID NO: 10) encoding a synthetic gene sequence (SEQ ID NO: 9) composed of a polynucleotide (SEQ ID NO: 2) encoding the signal peptide of CD8A (SEQ ID NO: 1), a polynucleotide (SEQ ID NO: 4) encoding the full-length sequence of CXCL12 (SEQ ID NO: 3), a polynucleotide (SEQ ID NO: 6) encoding the full-length sequence of CD8A excluding the signal peptide (SEQ ID NO: 5), and a polynucleotide (SEQ ID NO: 8) encoding the full-length sequence of CD81 (SEQ ID NO: 7) was inserted into pcDNA™ 3.1(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to prepare a vector for expressing CXCL-12 on the membrane of extracellular vesicles. In the same manner, a polynucleotide (SEQ ID NO: 16) encoding a synthetic gene sequence (SEQ ID NO: 15) composed of a polynucleotide (SEQ ID NO: 12) encoding the full-length sequence of L-Selectin (SEQ ID NO: 11) and a polynucleotide (SEQ ID NO: 14) encoding the full-length sequence of CD81 (SEQ ID NO: 13) was inserted into pcDNA™ 3.1(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific). A polynucleotide (SEQ ID NO: 18) encoding the signal peptide of CD8A (SEQ ID NO: 17), a polynucleotide (SEQ ID NO: 20) encoding the full-length sequence of Thrombopoietin (TPO) (SEQ ID NO: 19), a polynucleotide (SEQ ID NO: 22) encoding the full-length sequence of CD8A without the signal peptide (SEQ ID NO: 21), and a polynucleotide (SEQ ID NO: 24) encoding the full-length sequence of CD81 (SEQ ID NO: 23) were used to construct a polynucleotide (SEQ ID NO: 26) encoding a synthetic gene sequence (SEQ ID NO: 25) and inserted into pcDNA™ 3.1 / Zeo(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific). A polynucleotide (SEQ ID NO: 28) encoding the signal peptide of MFG-E8 (SEQ ID NO: 27), a polynucleotide (SEQ ID NO: 30) encoding the full-length sequence of Stem cell factor (SCF) (SEQ ID NO: 29), a polynucleotide (SEQ ID NO: 32) encoding peptide linker 1 (SEQ ID NO: 31), a polynucleotide (SEQ ID NO: 34) encoding the full-length sequence of Stem cell factor (SCF) (SEQ ID NO: 33), a polynucleotide (SEQ ID NO: 36) encoding peptide linker 2 (SEQ ID NO: 35), and a polynucleotide (SEQ ID NO: 38) encoding the full-length sequence of MFG-E8 without the signal peptide with the 48th D changed to E (SEQ ID NO: 37) were used to construct a polynucleotide (SEQ ID NO: 40) encoding a synthetic gene sequence (SEQ ID NO: 39) and inserted into pcDNA™ 3.Each was inserted into the Hygro(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to prepare vectors for expressing L-Selectin, TPO, and SCF on the membrane of extracellular vesicles. Each constructed vector was transformed into E. coli DH5α Competent Cells (manufactured by Takara Bio Inc.). The transformed E. coli was amplified using LB medium, and large-scale preparation of the vector was performed using the EndoFree Plasmid Maxi Kit (manufactured by QIAGEN). The sequence information used is shown in Tables 1 to 3 below.

[0128] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0129] [Table 2-1] [Table 2-2] [Table 2-3]

[0130] [Table 3-1]

Table 3-2

Table 3-3

[0131] 1.2. Preparation of extracellular vesicles 1.2.1. Acquisition of HEK293 cell-derived extracellular vesicles and flow cytometric analysis of fusion proteins contained in the membrane of extracellular vesicles Human embryonic kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were pre-cultured using E-MEM medium (containing L-glutamine and phenol red) (manufactured by FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS. HEK293 was seeded at 1×10 6 cells / 10mL / 10 cm dish, and cultured in a CO 2 incubator (37°C, 5% CO 2 ) in a static state for 24 hours. After 24 hours, using Lipofectamine® 3000 Reagent (manufactured by Thermo Fisher Scientific) and Opti-MEM® I Reduced Serum Medium, no phenol red (manufactured by Thermo Fisher Scientific), the non-vector-introduced group (sample 1), the SCF and TPO expression vector-introduced group (sample 2), the SCF, TPO, and L-Selectin expression vector-introduced group (sample 3), and the SCF, TPO, and CXCL12 expression vector-introduced group (sample 4) were each introduced with 5 μg, and cultured in a CO 2 incubator (37°C, 5% CO 2It was cultured for 24 hours. After 24 hours, 10 mL of E-MEM medium (containing L-glutamine and phenol red) containing 10% Fetal Bovine Serum, exosome-depleted, One Shot™ format (manufactured by Thermo Fisher Scientific) was added to each 10-cm dish washed with D-PBS (manufactured by Fujifilm Wako Pure Chemical Corporation), and it was cultured in a CO 2 incubator (37 °C, 5% CO 2 ) for 48 hours. After 48 hours, the culture supernatant was collected. The collected culture supernatant was concentrated using Vivaspin 20 (100k) (manufactured by Sartorius), and immunostaining was performed according to the manufacturer's instructions using the PS Capture™ Exosome Flow Cytometry Kit (manufactured by Fujifilm Wako Pure Chemical Corporation) with the concentrated solution. The antibodies used for staining were as follows. The staining time was set to react for 60 minutes at 4 °C for both the primary antibody and the secondary antibody. After staining, the expression of each fusion protein was detected using a flow cytometer LSR Fortessa X-20 (manufactured by BD Biosciences).

[0132] [Table 4]

[0133] The results are shown in Figure 1. Expression of each molecule was observed in extracellular vesicles expressing three vectors of SCF, TPO, and L-Selectin or CXCL12.

[0134] 1.2.2. Acquisition of HEK293 cell-derived extracellular vesicles and quantitative evaluation of SCF and TPO Human embryonic kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were pre-cultured using E-MEM medium (containing L-glutamine and phenol red) (manufactured by Fujifilm Wako Pure Chemical Corporation) containing 10% FBS. HEK293 was seeded at 1×10 6 cells / 10 mL / 10-cm dish, and it was cultured in a CO 2 incubator (37 °C, 5% CO 2) were cultured in a static state for 24 hours. After 24 hours, using Lipofectamine 3000 Reagent (manufactured by Thermo Fisher Scientific) and Opti-MEM I Reduced Serum Medium, no phenol red (manufactured by Thermo Fisher Scientific), 5 μg each of the non-vector-introduced group (sample 1), the SCF and TPO expression vector-introduced group (sample 2), the SCF, TPO and L-Selectin expression vector-introduced group (sample 3), and the SCF, TPO and CXCL12 expression vector-introduced group (sample 4) were introduced, and CO 2 incubator (37 °C, 5% CO 2 ) and cultured for 24 hours. After 24 hours, 10 mL of E-MEM medium (containing L-glutamine and phenol red) containing 10% Fetal Bovine Serum, exosome-depleted, One Shot format (manufactured by Thermo Fisher Scientific) was added to each 10 cm dish washed with D-PBS, and CO 2 incubator (37 °C, 5% CO 2 ) and cultured for 48 hours. After 48 hours, the culture supernatant was collected. The collected culture supernatant was centrifuged at 2000 xg for 10 minutes, the supernatant was collected, and then passed through a 0.22 μm filter (manufactured by Millipore). The processed culture supernatant was added to a UC tube (manufactured by Beckman Coulter), set on SW41Ti (manufactured by Beckman Coulter), and centrifuged at 35000 rpm at 4 °C for 70 minutes using Optima L-90K (manufactured by Beckman Coulter). After centrifugation, the supernatant was removed, 10 mL of D-PBS was added to the UC tube, and centrifuged at 35000 rpm at 4 °C for 70 minutes. After centrifugation, the supernatant was removed and suspended in 50 μL of D-PBS. To quantify SCF and TPO expressed on extracellular vesicles, a Human SCF ELISA Kit (manufactured by abcam) and a Human Thrombopoietin ELISA Kit (manufactured by abcam) were used. 50 μL of a 10-fold diluted extracellular vesicle solution or a serially diluted calibration curve solution was added to each plate used. Then, 50 μL of an Antibody Cocktail prepared with Antibody Diluent CPI or Antibody Diluent 5BI to a final concentration of 1× Capture Antibody and 1× Detector Antibody was added, and the mixture was shaken at room temperature for 1 hour. After 1 hour, the solution was discarded and washed 3 times with 350 μL of 1× Wash Buffer PT. Subsequently, 100 μL of TMB Development Solution was shaken at room temperature for 10 minutes. Finally, 100 μL of Stop Solution was added to stop the reaction, and then the absorbance at 450 nm was measured. The concentrations of SCF and TPO contained in Samples 1 to 4 were calculated from the absorbances of each calibration curve.

[0135] As a result, expression levels as shown in Table 5 were observed in Samples 2 to 4. On the other hand, in Sample 1, all were below the lower limit of quantification.

Table 5

[0136] 1.3. Effect in vitro 1.3.1. Cell proliferation evaluation using human bone marrow CD34-positive progenitor cells Human bone marrow CD34-positive progenitor cells (manufactured by Lonza) were lysed in a 37°C water bath and suspended in 10 mL of StemSpan™ Serum-Free Expansion Medium (manufactured by STEMCELL technologies). After centrifugation at 300 x g for 10 minutes, the supernatant was removed, and the cells were resuspended in the medium and seeded into a 96-well clear flat-bottom untreated cell culture plate (manufactured by Falcon) at a density of 5000 cells / 80 μL / well. Each extracellular vesicle solution prepared in 1.2.2 above was added so that the final concentrations of SCF and TPO were SCF: 3 pg / mL, TPO: 1.63 pg / mL (sample 2); SCF: 3 pg / mL, TPO: 3.69 pg / mL (sample 3); and SCF: 3 pg / mL, TPO: 2.92 pg / mL (sample 4). Also, sample 1 was added in a manner consistent with sample 2, which had the highest addition amount. Furthermore, recombinant SCF (manufactured by R&D Systems) and recombinant TPO (manufactured by Peprotech) were also added to samples 5 - 7 so that they had the same final concentrations as samples 2 - 4. As a control, a D-PBS treatment group (control) was set up. The final volume of each group was adjusted with the medium and D-PBS solution to 100 μL. The treated plates were cultured in a CO 2 incubator (37°C, 5% CO 2 ) for 96 hours. After 96 hours, 100 μL of CellTiter-Glo® Luminescent Cell Viability Assay (manufactured by Promega) was added and allowed to react for 10 minutes, and then the luminescence was measured using Enspire (manufactured by PerkinElmer) to measure the number of cells at each time point. Based on the calculated luminescence, the value relative to the control was determined as % of control for each group.

[0137] As a result, an increase in luminescence was observed in the groups treated with samples 2, 3, and 4 compared to the control, sample 1, and samples 5, 6, and 7 (Figure 2).

[0138] Example 2. Induction of iPSC differentiation 2.1. Preparation of plasmid A polynucleotide (SEQ ID NO: 42) encoding the signal peptide of CD8 (SEQ ID NO: 41), a polynucleotide (SEQ ID NO: 44) encoding fragment 1 of Bcl-C (SEQ ID NO: 43), a polynucleotide (SEQ ID NO: 46) encoding a peptide linker (SEQ ID NO: 45), a polynucleotide (SEQ ID NO: 48) encoding fragment 2 of Bcl-C (SEQ ID NO: 47), a polynucleotide (SEQ ID NO: 50) encoding a peptide linker (SEQ ID NO: 49), a polynucleotide (SEQ ID NO: 52) encoding fragment 3 of Bcl-C (SEQ ID NO: 51), a polynucleotide (SEQ ID NO: 54) encoding the extracellular domain, transmembrane domain, and cytoplasmic domain of CD8 (SEQ ID NO: 53), and a polynucleotide (SEQ ID NO: 58) encoding a synthetic gene sequence (SEQ ID NO: 57) composed of a polynucleotide (SEQ ID NO: 56) encoding the full-length sequence of CD81 (SEQ ID NO: 55) were inserted into pcDNA™ 3.1 / Hygro(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to prepare a vector for expressing Bcl-C on the membrane of extracellular vesicles. In the same way, a polynucleotide (SEQ ID NO: 66) encoding a synthetic gene sequence (SEQ ID NO: 65) composed of a polynucleotide (SEQ ID NO: 60) encoding the full-length sequence of Mutation Activin A (SEQ ID NO: 59) with mutations at C35S, C244S, and C247S, a polynucleotide (SEQ ID NO: 62) encoding a peptide linker (SEQ ID NO: 61), and a polynucleotide (SEQ ID NO: 64) encoding the full-length sequence of MFGE8 (SEQ ID NO: 63) excluding the signal peptide was inserted into pcDNA™ 3.1 / Zeo(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific). A polynucleotide (SEQ ID NO: 68) encoding the signal peptide of MFGE8 (SEQ ID NO: 67), a polynucleotide (SEQ ID NO: 70) encoding amino acids 306 - 426 of Mature Activin A (SEQ ID NO: 69), a polynucleotide (SEQ ID NO: 72) encoding a peptide linker (SEQ ID NO: 71), and a polynucleotide (SEQ ID NO: 74) encoding the full-length sequence of MFGE8 (SEQ ID NO: 73) excluding the signal peptide were inserted into pcDNA™ 3.1 / Zeo(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to construct vectors for expressing Mutation Activin A and Mature Activin A on the membrane of extracellular vesicles. Each constructed vector was transformed into E. coli DH5α Competent Cells (manufactured by Takara Bio Inc.). The transformed E. coli was amplified using LB medium, and the vectors were prepared in large quantities using the EndoFree Plasmid Maxi Kit (manufactured by QIAGEN). The sequence information used is shown in Tables 6 - 8 below.

[0139] [Table 6-1] [Table 6-2]

Table 6-3

Table 6-4

Table 6-5

[0140]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

[0141]

Table 8-1

Table 8-2

Table 8-3

[0142] 2.2. Preparation of extracellular vesicles 2.2.1. Acquisition of extracellular vesicles derived from HEK293 cells and flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles Human embryonic kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were pre-cultured using E-MEM medium containing 10% FBS (containing L-glutamine and phenol red) (manufactured by FUJIFILM Wako Pure Chemical Corporation). HEK293 was seeded at 8×10 5 cells / 10 mL / 10 cm dish and cultured in a CO 2 incubator (37 °C, 5% CO 2 ) in a static state for 24 hours. After 24 hours, using Lipofectamine® 3000 Reagent (manufactured by Thermo Fisher Scientific) and Opti-MEM® I Reduced Serum Medium, no phenol red (manufactured by Thermo Fisher Scientific), the Mutation Activin A expression vector-introduced group and the Mature Activin A expression vector-introduced group were introduced at 7.5 μg each and cultured in a CO 2 incubator (37 °C, 5% CO 2 ) for 24 hours. After 24 hours, 10 mL of E-MEM medium containing 10% Fetal Bovine Serum, exosome-depleted, One Shot® format (manufactured by Thermo Fisher Scientific) (containing L-glutamine and phenol red) was added to each 10 cm dish washed with D-PBS (manufactured by FUJIFILM Wako Pure Chemical Corporation), and the cells were cultured in a CO 2 incubator (37 °C, 5% CO 2It was cultured for 48 hours. After 48 hours, the culture supernatant was collected. The collected culture supernatant was used to purify extracellular vesicles with the Capturem (trademark) Extracellular Vesicle Isolation Kit (Maxi) (manufactured by Takara Bio Inc.), and the obtained solution was concentrated using Amicon Ultra-0.5, PLGC Ultracel, 10 kDa (manufactured by Merck Millipore) and replaced with D-PBS(-). Immunostaining was performed on the concentrated solution using the PS Capture (trademark) exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions. For staining, AlexaFluor (registered trademark) 647-conjugated anti-human Activin A antibody (manufactured by Bioss antibodies) was used and reacted at room temperature for 60 minutes. After staining, the expression of each fusion protein was detected with a flow cytometer LSR Fortessa X-20 (manufactured by BD Biosciences).

[0143] The results are shown in Figure 3. Expression of Activin A was observed in extracellular vesicles expressing Activin A with both types of vectors.

[0144] 2.2.2. Acquisition and quantitative evaluation of Activin A in extracellular vesicles derived from HEK293 cells Human embryonic kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were pre-cultured using E-MEM medium containing 10% FBS (containing L-glutamine and phenol red) (manufactured by Fujifilm Wako Pure Chemical Corporation). HEK293 was seeded at 8×10 5 cells / 10 mL / 10 cm dish, and placed in a CO 2 incubator (37 °C, 5% CO 2It was cultured in a static state for 24 hours. After 24 hours, using Lipofectamine 3000 Reagent (manufactured by Thermo Fisher Scientific) and Opti-MEM I Reduced Serum Medium, no phenol red (manufactured by Thermo Fisher Scientific), 7.5 μg was introduced in the Mutation Activin A expression vector-introduced group (Example condition 1), 3.75 μg of each vector in the Bc2l-C and Mutation Activin A expression vector-introduced group (Example condition 2), and 7.5 μg of the vector in the Mature Activin A expression vector-introduced group (Example condition 3), and CO 2 It was cultured for 24 hours in an incubator (37 °C, 5% CO 2 ). A non-vector-introduced group that was operated in the same manner was used as a comparative example. After 24 hours, 10 mL of E-MEM medium (containing L-glutamine and phenol red) containing 10% Fetal Bovine Serum, exosome-depleted, One Shot format (manufactured by Thermo Fisher Scientific) was added to each 10 cm dish washed with D-PBS, and CO 2 It was cultured for 48 hours in an incubator (37 °C, 5% CO 2 ). After 48 hours, the culture supernatant was collected. The collected culture supernatant was used to purify extracellular vesicles using the Capturem (trademark) Extracellular Vesicle Isolation Kit (Maxi) (manufactured by Takara Bio Inc.), and the obtained solution was concentrated using Amicon Ultra-0.5, PLGC Ultracel, 10 kDa (manufactured by Merck Millipore) and replaced with D-PBS (-). To quantify Activin A expressed on the extracellular vesicles, the concentrated solution was measured using Human Activin A ELISA (manufactured by RayBiotech Inc.). Also, the number of particles and the average particle diameter of the extracellular vesicles contained in the same solution were measured using a nanoparticle tracking analyzer ZetaView (manufactured by DKSH Japan Inc.).

[0145] As a result, extracellular vesicles with an average particle size of 50 to 150 nm were obtained in all samples. Regarding the quantification results of Activin A, expression levels as shown in the table were observed in Samples 2 to 5, while in Sample 1, all were below the lower limit of quantification. Thus, it became clear that Activin A could be expressed in extracellular vesicles by introducing the vector.

[0146]

Table 9

[0147] 2.3. Effect in vitro 2.3.1. Induction experiment for iPSC differentiation using Activin A-expressing extracellular vesicles derived from HEK293 cells It was verified whether the extracellular vesicles expressing Activin A obtained above could induce the differentiation of human iPSCs. Human iPSCs (RPChiPS771 strain, manufactured by Reprocell) were cultured in StemFit (registered trademark) AK02N (manufactured by Ajinomoto Healthy Supply Co., Ltd.) in a 6-well plate coated with iMatrix-511 silk (manufactured by Matriksome) at 0.5 μg / cm 2 The obtained cells were incubated with 0.5 mM EDTA solution at 37°C for 10 minutes and detached as single cells. The obtained cells were suspended in StemFit (registered trademark) medium supplemented with 10 μM Y-27632 (manufactured by Fujifilm Wako Pure Chemical Corporation) and seeded at 2×10 2 cells / well in a 24-well plate coated with iMatrix-511 at 0.5 μg / cm 5 and incubated in a CO 2 incubator (37°C, 5% CO 2) were cultured in a static state for 24 hours (Day-1). On the day after seeding, it was confirmed that the cells had adhered, and the medium was changed to a differentiation medium to initiate differentiation (Day0). The differentiation medium used was DMEM (high glucose) (manufactured by Fujifilm Wako Pure Chemical Corporation) containing 2 mM L-glutamine (manufactured by DS Pharma Biomedical), 1% MEM non-essential amino acid solution (×100) (manufactured by Fujifilm Wako Pure Chemical Corporation), 0.1 mM 2-mercaptoethanol (manufactured by Thermo Fisher Scientific), 2% B-27 supplement XenoFreeCTS (manufactured by Thermo Fisher Scientific), and penicillin streptomycin. The HEK293-derived Activin A-expressing extracellular vesicles obtained in Example 2.2.1. were added to this differentiation medium, and CO 2 incubator (37 °C, 5% CO 2 ) and cultured in a static state for 3 days. As a comparison, recombinant Activin A protein (manufactured by Shenandoah) was added to reach the same concentration as the ELISA quantification result of the HEK293-derived Activin A-expressing extracellular vesicles used, and cultured for 3 days. RNA was recovered from the cells obtained 3 days after differentiation using the RNeasy Mini Kit (manufactured by Qiagen). After reverse transcription using PrimeScript™ RT Master Mix (Perfect Real Time) (manufactured by Takara Bio), gene expression was analyzed using Premix Ex Taq (Perfect Real Time) and Applied Biosystems 7500 Real-Time PCR System (manufactured by Thermo Fisher Scientific). TaqMan probes in Table 10 (all manufactured by Thermo Fisher Scientific) were used for the analysis of gene expression, and the expression level of each gene was corrected by the expression level of the housekeeping gene GAPDH. The results are shown in Table 11.

[0148]

Table 10

[0149]

Table 11

[0150] As a result, when iPSC differentiation was induced using recombinant protein and HEK293-derived Activin A-expressing extracellular vesicles so that the addition concentrations were the same, it was revealed that the expressions of the mesoderm marker T, the endoderm markers SOX17 and FOXA2 were significantly increased in the group using extracellular vesicles. Therefore, it was suggested that Activin A-expressing extracellular vesicles can induce iPSC differentiation at a lower concentration compared to recombinant protein.

[0151] 2.3.2. iPSC Differentiation Induction Experiment Using HEK293 Cell-Derived Activin A-Expressing Extracellular Vesicles In the same manner as in the above condition 3, extracellular vesicles obtained by introducing the Mature Activin A vector into HEK293 cells were obtained. In the induction of iPSC differentiation, the addition amount of these extracellular vesicles and the expression of differentiation markers were evaluated. Human iPSCs (RPChiPS771 strain, manufactured by ReproCell) were detached into single cells in the same manner as above. The obtained cells were suspended in StemFit (registered trademark) medium supplemented with 10 μM Y-27632 (manufactured by Fujifilm Wako Pure Chemical Corporation), and iMatrix-511 was coated at 0.5 μg / cm 2 and seeded in a 48-well plate at 2×10 4 cells / well, and placed in a CO 2 incubator (37°C, 5% CO 2) It was cultured in a static state for 24 hours (Day - 1). On the day after seeding, it was confirmed that the cells had adhered, and the medium was changed to a differentiation medium to initiate differentiation (Day 0). In the differentiation medium, HEK293 - derived Mature Activin A - expressing extracellular vesicles, whose Activin A concentration was quantified by ELISA, were added at concentrations of 0.05, 0.1, 0.2, and 0.5 ng / mL and cultured for 3 days. As a comparison control, Activin A recombinant protein (manufactured by Shenandoah) was added at concentrations of 0, 1, 10, and 100 ng / mL, and the medium was changed daily in the comparison control group. The results of evaluating the gene expression 3 days after differentiation in the same manner as above are shown in Figure 4.

[0152] As a result, it became clear that the expression of mesoderm marker T, endoderm markers SOX17 and FOXA2 increased at a lower concentration compared to the recombinant protein. Particularly for the endoderm marker T, at the extracellular vesicle addition concentrations in this study, a high expression was confirmed, greatly exceeding the expression level with the recombinant protein, suggesting that it may be particularly useful for differentiation into mesoderm. Also, in the case of the recombinant protein, during the 3 - day differentiation period, medium exchange was required and the addition of the recombinant protein was needed each time, but in the case of extracellular vesicles, induction of iPSC differentiation was possible without medium exchange with only the addition on the first day. From these points, it was suggested that extracellular vesicles expressing Activin A can stably induce iPSC differentiation at a low concentration.

[0153] Example 3. Effect on CAR-T cells 3.1. Preparation of plasmid 3.1.0.CAR - P2A - Venus A polynucleotide (SEQ ID NO: 106) encoding a chimeric antigen receptor (CAR) (SEQ ID NO: 105) that recognizes Her2, a polynucleotide (SEQ ID NO: 108) encoding P2A (SEQ ID NO: 107), one of the 2A peptides, and a polynucleotide (SEQ ID NO: 110) encoding Venus (SEQ ID NO: 109) were used to construct a polynucleotide (SEQ ID NO: 112) encoding a synthetic gene sequence CAR-P2A-Venus (SEQ ID NO: 111), which was inserted into a pMX vector to construct a vector for producing CAR-T cells. Since the 2A peptide sequence causes ribosome skipping, when the sequence encoding CAR-P2A-Venus is actually translated, a protein containing an independent CAR and a protein containing an independent Venus are translated. 3.1.1.Her2-MEGE8 A polynucleotide (SEQ ID NO: 80) encoding the signal peptide of Her2 (SEQ ID NO: 79), a polynucleotide (SEQ ID NO: 78) encoding Her2 (extracellular domain) (SEQ ID NO: 77), a polynucleotide (SEQ ID NO: 82) encoding a peptide linker (SEQ ID NO: 81), and a polynucleotide (SEQ ID NO: 84) encoding MFG-E8 (SEQ ID NO: 83) were used to construct a polynucleotide (SEQ ID NO: 86) encoding a synthetic gene sequence Her2-MFG-E8 (SEQ ID NO: 85), which was inserted into a pCAG-puro vector to construct a vector for expressing Her2 on the membrane of extracellular vesicles. 3.1.2.Her2-MEGE8-IL-2 and Her2-MEGE8-IL-7 Polynucleotides (SEQ ID NO: 92 or 96) encoding gene sequences Her2-MEGE8-IL-2 (SEQ ID NO: 91) or Her2-MEGE8-IL-7 (SEQ ID NO: 95), in which IL-2 (SEQ ID NO: 89) or IL-7 (SEQ ID NO: 93) was fused to the C-terminus via a linker (SEQ ID NO: 87) to the Her2-MFG-E8 constructed above, were inserted into a pCAG-puro vector to construct vectors for expressing Her2 and IL-2, or Her2 and IL-7 on the membrane of extracellular vesicles. 2.1.3.Her2-CD81 A polynucleotide (SEQ ID NO: 102) encoding an artificially synthesized gene sequence Her2-CD81 (SEQ ID NO: 101), which is composed of a polynucleotide (SEQ ID NO: 98) encoding Her2 (including a signal peptide, an extracellular domain, a transmembrane domain, and a part of the intracellular domain) (SEQ ID NO: 97) and a polynucleotide (SEQ ID NO: 100) encoding CD81 (SEQ ID NO: 99), was inserted into the pCAG-puro vector to prepare a vector for expressing Her2 on the membrane of extracellular vesicles. 2.1.3. Her2-CD81-IL-2 A sequence consisting of linker 8 (SEQ ID NO: 87) ~ the sequence of IL-2 excluding the signal peptide ~ linker 8 (SEQ ID NO: 87) was introduced into the 2nd loop of Her2-CD81 constructed above, and a polynucleotide (SEQ ID NO: 92 or 96) encoding Her2-CD81-IL-2 (SEQ ID NO: 103) was inserted into the pCAG-puro vector to prepare a vector for expressing Her2 and IL-2 on the membrane of extracellular vesicles. A schematic diagram of each gene construct is shown in Figure 5, and its sequence information is shown in Tables 12 to 14.

[0154]

Table 12-1

Table 12-2

Table 12-3

Table 12-4

Table 12-5

Table 12-6

Table 12-7

Table 12-8

Table 12-9

Table 12-10

Table 12-11

Table 12-12

Table 12-13

Table 12-14

Table 12-15

Table 12-16

Table 12-17

[0155]

Table 13-1

Table 13-2

Table 13-3

Table 13-4

Table 13-5

Table 13-6

Table 13-7

Table 13-8

Table 13-9

Table 13-10

[0156]

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Table 14-5

[0157] 3.2. Preparation of CAR-T cells and extracellular vesicles 3.2.1. Preparation of CAR-T cells PLAT-E cells (retroviral packaging cell line) were seeded in a cell culture dish and cultured in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with the pMX vector encoding CAR-P2A-Venus using Polyethylenimine “Max” (manufactured by Polysciences) according to the manufacturer's instructions. The medium was changed 12 hours after transfection, and 60 hours after transfection, the supernatant was collected and centrifuged at 300 g for 5 minutes. The collected supernatant was used as virus particles. The collected virus particles were seeded onto plates coated with RetroNection according to the manufacturer's instructions. Lymph nodes excised from C57BL / 6 mice were disrupted on a 100-μm filter to obtain a lymph node cell suspension. 5 2×10 5 cells were suspended in 200 μL of RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, penicillin / streptomycin, and 10 ng / ml mIL-2, and Dynabeads Mouse T-Activator CD3 / CD28 were added according to the manufacturer's instructions and cultured for 2 days. After culture, the Dynabeads were removed, and 2×10

[0158] 3.2.2. Preparation of extracellular vesicles containing Her2 molecules and T cell-stimulating cytokines on the membrane HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with plasmids (pCAG vectors encoding Her2-CD81, Her2-CD81-IL-2, Her2-MFG-E8, or Her2-MFG-E8-IL-2) using Polyethylenimine “Max” (manufactured by Polysciences) according to the manufacturer's instructions. The medium was replaced 6 hours after transfection, and the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin / streptomycin from which exosomes had been removed 24 hours after transfection. 72 hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and then centrifuged at 300 g for 5 minutes. The supernatant was collected and centrifuged at 2,000 g for 20 minutes. The supernatant was collected and centrifuged at 10,000 g for 30 minutes. The supernatant was collected and centrifuged at 100,000 g for 2 hours, after which the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet, centrifuged at 100,000 g for 2 hours, after which the supernatant was removed, and the pellet suspended in 100 μL of PBS was used as the extracellular vesicles of Example 3. The concentration of the extracellular vesicles was measured using a BCA protein assay kit (manufactured by Thermo Fisher Scientific) according to the manufacturer's instructions. As a control, extracellular vesicles were collected from HEK293 cells not transfected with plasmids under the same conditions (hereinafter referred to as control exosomes or 293 exosomes).

[0159] 3.2.3. Flow cytometry analysis of fusion proteins contained in the membranes of extracellular vesicles The antigen-presenting cell extracellular vesicles prepared in 3.2.2 were immunostained using the PS Capture (trademark) Exosome Flow Cytometry Kit (manufactured by Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions. The antibodies used for staining were as follows (staining time: 15 minutes, temperature: 4°C). After staining, the expression of each fusion protein was detected using the following antibodies with a flow cytometer FACSCantoII (manufactured by BD Biosciences). · APC-conjugated anti-mouse IL-2 antibody (JES6-5H4, manufactured by Biolegend) · PE-conjugated anti-human CD340 (HER2) antibody (24D2, manufactured by Biolegend) The results are shown in Figure 6.

[0160] 3.3. Effect in vitro To examine whether the CAR-T cell-activating extracellular vesicles activate CAR-T cells and so on, the following tests were conducted in vitro. 2 × 10 5 cells of the CAR-T cells prepared in Example 3.2.1 were suspended in 200 μL of RPMI1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin, and the extracellular vesicles (Her2-CD81-IL-2) of Example 3.2.2 or control extracellular vesicles (293 exosome) were added at a final concentration of 20 μg / ml. After culturing in a 96-well round-bottom plate for 4 days, the expression of Venus was detected using a flow cytometer FACSCantoII (manufactured by BD Biosciences). The results are shown in Figure 7. The extracellular vesicles expressing Her2-CD81-IL-2 significantly proliferated the CAR-T cells expressing Venus.

[0161] 3.4. Effect in vivo 3.4.1. Activation of CAR-T cells in vivo To examine whether the extracellular vesicles for activating CAR-T cells activate CAR-T cells and so on, the following tests were conducted in vivo. 4×10 CAR-T cells prepared in Example 3.2.1 were transplanted into CD45.1 / CD45.2 congenic mice. 6 At the same time, 200 μg of extracellular vesicles for CAR-T cell activation (Her2MFGE8 or Her2CD81) or control extracellular vesicles (Control exosome) were transplanted into the recipient mice via the tail vein. Four days after cell transplantation, the spleen was removed from the recipient mice, a lymphocyte suspension was prepared, and immunostaining was performed. The following antibodies were used for staining (staining time: 15 minutes, temperature: 4 °C). After staining, Venus was detected with a flow cytometer FACSCantoII (manufactured by BD Biosciences) to observe the proliferation of the transplanted CAR-T cells. · APCcy7-conjugated anti-mouse CD45.1 antibody (manufactured by A20 Biolegend) · Percpcy5-conjugated anti-mouse CD45.2 antibody (104, manufactured by Biolegend) The results are shown in Figure 8. Extracellular vesicles expressing Her2MFGE8 or Her2CD81 significantly activated and proliferated CAR-T cells expressing Venus.

[0162] 3.4.2. Anti-tumor effect in vivo 1×10 MC38 cells expressing Her2 derived from mouse colon cancer were transplanted into CD45.1 / CD45.2 congenic mice. 5 Subsequently, 4×10 CAR-T cells prepared in Example 3.2.1 were transplanted, and at the same time, 200 μg of extracellular vesicles for CAR-T cell activation (Her2MFGE8, Her2CD81 or Her2-CD81-IL-2) or control extracellular vesicles (Control exosome) were transplanted into the recipient mice via the tail vein. Four days after cell transplantation, the size of the main tissue containing MC38 cells formed in the recipient mice was measured. 6 When extracellular vesicles for CAR-T cell activation are co-administered with CAR-T cells, the CAR-T cells are activated in the recipient mice, the anti-tumor effect against cancer cells expressing Her2 is enhanced, and the size of the tumor tissue is reduced compared to the administration of CAR-T cells alone. ​

[0163] The details of the array information used in the examples are shown in Table 15 below.

Table 15-1

Table 15-2

Claims

1. Extracellular vesicles that present an antigen outside the membrane.

2. The extracellular vesicles according to claim 1, which further present a T cell-stimulating cytokine outside the membrane.

3. The extracellular vesicles according to claim 2, wherein the membrane thereof has the following: (B) a protein capable of presenting the antigen outside the membrane, which contains the antigen; and (A) a protein capable of presenting the T cell-stimulating cytokine outside the membrane, which contains the T cell-stimulating cytokine or a subunit thereof; Extracellular vesicles containing the same.

4. The extracellular vesicles according to claim 2, wherein the membrane thereof has the following: (C) a protein capable of presenting the antigen and the T cell-stimulating cytokine outside the membrane, which contains the antigen and the T cell-stimulating cytokine or a subunit thereof Extracellular vesicles containing the same.

5. The extracellular vesicles according to claim 3, wherein the protein (B) is a fusion protein of the antigen and a membrane protein capable of localizing to the membrane of the extracellular vesicles or a protein capable of binding to the membrane of the extracellular vesicles.

6. The extracellular vesicles according to claim 3, wherein the protein (A) is a fusion protein of the T cell-stimulating cytokine or a subunit thereof and a membrane protein capable of localizing to the membrane of the extracellular vesicles or a protein capable of binding to the membrane of the extracellular vesicles.

7. The protein (C) is the antigen, the T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of localizing to the membrane of the extracellular vesicles or a protein capable of binding to the membrane of the extracellular vesicles The extracellular vesicles according to claim 4, which is a fusion protein therewith.

8. The extracellular vesicles according to any one of claims 5 to 7, wherein the membrane protein capable of localizing to the membrane of the extracellular vesicles or the protein capable of binding to the membrane of the extracellular vesicles contains tetraspanin or a transmembrane domain thereof or MFGE8 or a membrane-binding domain thereof.

9. The protein (C) is from the N-terminal side, (C-1) an antigen peptide, (C-2) an optional spacer sequence, and (C-3) an amino acid sequence encoding a fusion peptide containing tetraspanin or a transmembrane domain thereof or MFGE8 or a transmembrane domain thereof and the T cell-stimulating cytokine or a subunit thereof, in this order. The extracellular vesicles according to claim 4.

10. The extracellular vesicles according to any one of claims 1 to 9, wherein the extracellular vesicles are exosomes.

11. A polynucleotide encoding the protein (A) defined in claim 3; A polynucleotide encoding the protein (B) defined in claim 3; or A polynucleotide encoding the protein (C) defined in claim 4.

12. A vector containing the polynucleotide according to claim 11.

13. (a) A polynucleotide encoding the protein (A) defined in claim 3; and / or (b) A cell transformed by a single vector or a combination of two or more vectors containing a polynucleotide encoding the protein (B) defined in claim 3.

14. A cell transformed by a vector containing a polynucleotide encoding the protein (C) defined in claim 4.

15. A culture supernatant obtained by culturing the cell according to claim 13 or 14.

16. Extracellular vesicles contained in the culture supernatant according to claim 15.

17. A method for producing the extracellular vesicles according to claim 1 or 2, comprising: 1) A step of culturing the cell according to claim 13 or 14; 2) A step of recovering the culture supernatant after culturing; and 3) A method for purifying extracellular vesicles from the optionally recovered culture supernatant.

18. A pharmaceutical composition containing the extracellular vesicles according to claim 1 or 2, or the culture supernatant according to claim 15.

19. A pharmaceutical composition for proliferating the antigen-specific chimeric antigen receptor gene-introduced T cells (CAR-T cells), containing the extracellular vesicles according to claim 1 or 2.

20. A pharmaceutical composition for treating cancer containing cancer cells expressing the antigen, containing the extracellular vesicles according to claim 1 or 2, and administered to a patient administered with the antigen-specific chimeric antigen receptor gene-introduced T cells (CAR-T cells).

21. The pharmaceutical composition according to claim 19 or 20, wherein the antigen is the Her2 protein or a fragment thereof.

22. Extracellular vesicles presenting thrombopoietin (TPO) and stem cell factor (SCF) outside the membrane.

23. The extracellular vesicles according to claim 22, wherein on its membrane are the following: (A)-1 A protein containing the TPO and capable of presenting the TPO outside the membrane; and (A)-2 A protein containing the SCF and capable of presenting the SCF outside the membrane. The extracellular vesicles containing the same.

24. The extracellular vesicle according to claim 22, wherein on its membrane are the following: (A)-3 A protein capable of presenting TPO and SCF outside the membrane, including the said TPO and the said SCF An extracellular vesicle containing the same.

25. (B) An extracellular vesicle according to any one of claims 22 to 24, further comprising a protein capable of presenting L-selectin or CXCL12 outside the membrane and containing L-selectin or CXCL12.

26. The extracellular vesicle according to claim 23, wherein the protein (A)-1 is a fusion protein of TPO and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle.

27. The extracellular vesicle according to claim 23, wherein the protein (A)-2 is a fusion protein of SCF and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle.

28. The protein (A)-3 is the said TPO, the said SCF, and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle The extracellular vesicle according to claim 24, which is a fusion protein therewith.

29. The protein (B) is the said L-selectin or CXCL12, and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle The extracellular vesicle according to claim 25, which is a fusion protein therewith.

30. The extracellular vesicle according to any one of claims 26 to 29, wherein the membrane protein capable of localizing to the membrane of the extracellular vesicle or the protein capable of binding to the membrane of the extracellular vesicle comprises tetraspanin or its transmembrane domain or MFGE8 or its membrane-binding domain.

31. The extracellular vesicle according to any one of claims 22 to 30, wherein the extracellular vesicle is an exosome.

32. (a)-1 A polynucleotide encoding the protein (A)-1 defined in claim 23; (a)-2 A polynucleotide encoding the protein (A)-2 defined in claim 23; (a)-3 A polynucleotide encoding the protein (A)-3 defined in claim 24; or (b) A polynucleotide encoding the protein (B) defined in claim 25.

33. A vector containing the polynucleotide according to claim 22.

34. (a)-1 A polynucleotide encoding the protein (A)-1 defined in claim 23; and (a)-2 A cell transformed by a single vector or a combination of two or more vectors containing a polynucleotide encoding the protein (A)-2 defined in claim 23.

35. (a)-3 A cell transformed by a vector containing a polynucleotide encoding the protein (A)-3 defined in claim 24.

36. (d) The cell according to claim 34 or 35, further comprising a polynucleotide encoding the protein (B) defined in claim 25.

37. A culture supernatant obtained by culturing the cell according to any one of claims 34 to 36.

38. Extracellular vesicles contained in the culture supernatant according to claim 37.

39. A method for producing the extracellular vesicles according to claim 22, comprising: 1) A step of culturing the cell according to any one of claims 34 to 36; 2) A step of recovering the culture supernatant after culturing; and 3) A method comprising a method of purifying extracellular vesicles from the optionally recovered culture supernatant.

40. A pharmaceutical composition comprising the extracellular vesicles according to claim 22.

41. A pharmaceutical composition for activating and / or proliferating hematopoietic stem cells in vivo or in vitro, comprising the extracellular vesicles according to claim 22.

42. A pharmaceutical composition for treating aplastic anemia in a subject, comprising the extracellular vesicles according to claim 22 and administered to a subject administered with hematopoietic stem cells.

43. A pharmaceutical composition for treating blood cancer or immunodeficiency in a subject, comprising the extracellular vesicles according to claim 22, wherein the subject has been administered hematopoietic stem cells after chemotherapy and / or radiotherapy treatment.

44. Extracellular vesicles presenting Activin A outside the membrane.

45. The extracellular vesicles according to claim 44, wherein on its membrane: (A) A protein containing the Activin A and capable of presenting the Activin A outside the membrane is included.

46. The extracellular vesicles according to claim 45, further presenting Bc2Lc outside the membrane.

47. The extracellular vesicles according to claim 46, wherein on its membrane: (A) A protein containing the Activin A and capable of presenting the Activin A outside the membrane; and (B) A protein containing the Bcl2Lc and capable of presenting the Bcl2Lc outside the membrane; An extracellular vesicle containing the same. **Claim 48** The extracellular vesicle according to claim 46, wherein the following are present on its membrane: (C) A protein containing the Bcl2Lc and the Activin A and capable of presenting the Bcl2Lc and the Activin A outside the membrane An extracellular vesicle containing the same. **Claim 49** The extracellular vesicle according to claim 45 or 47, wherein the protein (A) is a fusion protein of Activin A and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle. **Claim 50** The extracellular vesicle according to claim 47, wherein the protein (B) is a fusion protein of Bcl2Lc and a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle. **Claim 51** The protein (C) is the Bcl2Lc, the Activin A, a membrane protein capable of localizing to the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle and is a fusion protein thereof. The extracellular vesicle according to claim 48. **Claim 52** The extracellular vesicle according to any one of claims 49 to 51, wherein the membrane protein capable of localizing to the membrane of the extracellular vesicle or the protein capable of binding to the membrane of the extracellular vesicle contains tetraspanin or its transmembrane domain or MFGE8 or its membrane-binding domain. **Claim 53** The extracellular vesicle according to any one of claims 44 to 52, wherein the extracellular vesicle is an exosome. **Claim 54** (a) A polynucleotide encoding the protein (A) defined in claim 47; (b) A polynucleotide encoding the protein (B) defined in claim 47; or (c) A polynucleotide encoding the protein (C) defined in claim 48. **Claim 55** A vector containing the polynucleotide according to claim 54. **Claim 56** (a) A polynucleotide encoding the protein (A) defined in claim 47; and / or (b) A cell transformed by a single vector or a combination of two or more vectors containing a polynucleotide encoding the protein (B) defined in claim 47. **Claim 57** (c) A cell transformed by a vector containing a polynucleotide encoding the protein (C) defined in claim 48. **Claim 58** A culture supernatant obtained by culturing the cell according to any one of claims 56 or 57.

59. Extracellular vesicles contained in the culture supernatant according to claim 58.

60. A method for producing extracellular vesicles according to claim 44 or 46, comprising: 1) a step of culturing the cell according to claim 56 or 57; 2) a step of recovering the culture supernatant after culturing; and 3) a method of purifying extracellular vesicles from the optionally recovered culture supernatant.

61. A differentiation inducer for iPS cells or ES cells, comprising the extracellular vesicles according to claim 44 or 46.

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