Immunomodulatory methods, nucleic acid compositions for immunomodulation, and their uses

Abstract

To provide a polynucleotide that makes it possible to produce extracellular vesicles capable of satisfactorily activating, etc., antigen-specific T cells.　Provided is a polynucleotide including at least one sequence selected from the group consisting of (a) a sequence encoding a fusion protein (A) including an antigen-presenting MHC molecule and capable of presenting the antigen-presenting MHC molecule extramembranously of an extracellular vesicle; (b) a sequence encoding a fusion protein (B) including at least one T cell stimulating cytokine or a subunit thereof and capable of presenting the T cell stimulating cytokine extramembranously of an extracellular vesicle; (c) a sequence encoding a fusion protein (C) including a T cell co-stimulatory molecule and capable of presenting the T cell co-stimulatory molecule extramembranously of an extracellular vesicle; (d) a sequence encoding a fusion protein (D) including an antigen-presenting MHC molecule and at least one T cell stimulating cytokine or a subunit thereof and capable of presenting the antigen and the T cell stimulating cytokine extramembranously of an extracellular vesicle; (e) a sequence encoding a fusion protein (E) including an antigen-presenting MHC molecule, at least one T cell stimulating cytokine or a subunit thereof, and a T cell co-stimulatory molecule and capable of presenting the antigen, the T cell stimulating cytokine, and the T cell co-stimulatory molecule extramembranously of an extracellular vesicle.

Description

[Technical Field]

[0001] This invention relates to an immunomodulatory method, a nucleic acid composition for immunomodulation, and its uses. [Background technology]

[0002] Antigen-specific T cells (e.g., cytotoxic T cells, helper T cells, etc.) are known to play a central role in immune responses, such as the elimination of cancer cells by the body and the regulation of responses to self-antigens and allergens. Antigen-specific T cells recognize the binding complex between MHC molecules on the cell surface of antigen-presenting cells such as dendritic cells and macrophages and antigens derived from cancer, allergens, etc., using T cell receptors, and are activated, proliferate, differentiate, etc. Activated antigen-specific T cells specifically damage cancer cells presenting antigens and regulate responses to self-antigens and allergens. Therefore, activating, proliferating, and differentiating antigen-specific T cells is considered particularly important in immune responses.

[0003] In addition to the already practical method of expressing chimeric antigen receptors on T cells, other methods have also been developed to activate antigen-specific T cells. For example, Patent Document 1 discloses that nanoparticles containing MHC molecules and T cell costimulatory molecules on their surface promote the proliferation of antigen-specific T cells. Non-Patent Document 1 discloses that exosomes expressing IL-12 on their membrane via PTGFRN promote the proliferation of tumor antigen-specific CD8-positive T cells. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2016-520518 [Non-patent literature]

[0005] [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, 2020, 34th Annual Meeting of the Society for Immuno-therapy of Cancer [Non-Patent Document 2] Journal of Extracellular Vesicles(2018);7:1535750 [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors of this invention attempted a novel method for activating antigen-specific T cells using extracellular vesicles containing MHC molecules and T cell costimulatory molecules in their membrane. However, when they attempted to activate antigen-specific T cells using these extracellular vesicles, they discovered for the first time that it was not possible to satisfactorily activate antigen-specific T cells.

[0007] Therefore, the present invention aims to provide a novel immunomodulatory method, a nucleic acid composition for immunomodulation, and its uses. [Means for solving the problem]

[0008] In light of the above-mentioned problems, after diligent research, the inventors unexpectedly discovered that antigen-specific T cells can be satisfactorily activated by using polynucleotides capable of producing extracellular vesicles containing MHC molecules and T cell-stimulating cytokines in their membranes, thus completing the present invention.

[0009] Therefore, the present invention includes the following: [0] Extracellular vesicles that present antigen-presenting MHC molecules and T cell-stimulating cytokines outside the membrane. [1] An extracellular vesicle presenting an antigen, the membrane of which contains the following: (A) A protein containing an antigen-presenting MHC molecule that is capable of presenting the antigen outside the membrane; and (B) A protein comprising a first T cell-stimulating cytokine or a subunit thereof, capable of presenting the first T cell-stimulating cytokine extramembrane; An extracellular vesicle containing an antigen presenting cell. [2] An extracellular vesicle presenting an antigen as described in [1], wherein its membrane contains the following: (A) A fusion protein or protein complex capable of presenting the antigen extracellularly, comprising an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; and (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extracellularly, comprising a first T cell-stimulating cytokine or its subunit and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; An extracellular vesicle containing an antigen presenting cell. [3] An antigen-presenting extracellular vesicle as described in [1] or [2], wherein its membrane contains the following: (A) A fusion protein or protein complex capable of presenting the antigen extramembrane, comprising an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain; and (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, comprising a first T cell-stimulating cytokine or a subunit thereof and a partial sequence of tetraspanin, wherein the partial sequence of tetraspanin has at least two transmembrane domains, and the first T cell-stimulating cytokine is positioned between the two transmembrane domains, or (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extracellularly, comprising the first T cell-stimulating cytokine or its subunit and MFG-E8 or its domain; An antigen-presenting extracellular vesicle containing the same. 〔4〕 The antigen-presenting extracellular vesicle according to any one of 〔1〕 to 〔3〕, wherein on its membrane: (A) A fusion protein capable of presenting an antigen peptide extracellularly, from the N-terminal side, (A-1) An MHC molecule-restricted antigen peptide, (A-2) An optional spacer sequence, (A-3) A single-chain MHC molecule, (A-4) An optional spacer sequence, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, or (A) A protein complex capable of presenting an antigen peptide extracellularly, From the N-terminal side, (A-1) An MHC molecule-restricted antigen peptide, (A-2) An optional spacer sequence, (A-3) MHC class I α chain, β2 microglobulin, MHC class II α chain, or MHC class II β chain (A-4) An optional spacer sequence, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, and (A-6) A protein containing the amino acid sequence of β2 microglobulin, MHC class I α chain, MHC class II β chain, or MHC class II α chain A protein complex containing; and (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 from the N-terminal side, (B-2) An optional spacer sequence, (B-3) The first T cell-stimulating cytokine, (B-4) A spacer arrangement that may exist, and (B-5) Partial sequence of tetraspanin containing transmembrane domain 4 A fusion protein comprising an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane, or (B) From the N-terminal side, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may be present, and (B-5)MFG-E8 A fusion protein comprising an amino acid sequence comprising the above, capable of presenting the first T cell-stimulating cytokine extramembrane; An extracellular vesicle containing an antigen presenting cell. [5] An antigen-presenting extracellular vesicle as described in [4], wherein the membrane has the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of the following: An extracellular vesicle containing an antigen presenting cell. [6] An antigen-presenting extracellular vesicle as described in [4], wherein the membrane has the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of the MHC class IIα chain protein complex An extracellular vesicle containing an antigen presenting cell. [7] An antigen-presenting extracellular vesicle according to any one of [1] to [6], wherein the first T cell-stimulating cytokine is IL-2, IL-4, IL-6, IL-12, a subunit of IL-12, or TGF-β. [8] An antigen-presenting extracellular vesicle described in any of [1] to [7], wherein its membrane contains the following: (C) A protein containing a T cell costimulatory molecule, which is capable of interacting with the T cell costimulatory molecule and T cells; An extracellular vesicle containing an antigen presenting cell. [9] An antigen-presenting extracellular vesicle described in any of [1] to [8], wherein its membrane contains the following: (C) A fusion protein that allows interaction between the T cell costimulatory molecule and T cells, comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; An extracellular vesicle containing an antigen presenting cell.

[10] An antigen-presenting extracellular vesicle described in any of [1] to [9], wherein its membrane contains the following: (C) A fusion protein comprising a T cell costimulatory molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain, wherein the T cell costimulatory molecule and T cells can interact; An extracellular vesicle containing an antigen presenting cell.

[11] An antigen-presenting extracellular vesicle described in any of [1] to

[10] , wherein its membrane contains the following: (C) From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanine A fusion protein comprising an amino acid sequence comprising the above, which is capable of interacting with the T cell costimulatory molecule and T cells; An extracellular vesicle containing an antigen presenting cell.

[12] An antigen-presenting extracellular vesicle described in any of [1] to

[11] , wherein the extracellular vesicle is an exosome.

[13] Polynucleotides, the following: (i) A fusion protein or protein complex of (A) as defined in any of [2] to [6]; (ii) A fusion protein of (B) as defined in any of [2] to [4]; or (iii) A fusion protein of (C) as defined in any of [9] to

[11] ; A polynucleotide that codes for one of the following.

[14] A vector comprising at least one polynucleotide selected from the polynucleotides described in

[13] .

[15] Below: (i) Polynucleotides encoding a fusion protein or protein complex of (A) as defined in any of [2] to [6]; and (ii) A polynucleotide encoding the fusion protein of (B) as defined in any of [2] to [4], and optionally (iii) A polynucleotide encoding a fusion protein of (C) as defined in any of [9] to

[11] . Cells transformed by a single vector or a combination of two or more vectors comprising the above.

[16] The culture supernatant obtained by culturing the cells described in

[15] .

[17] An antigen-presenting extracellular vesicle obtained from the culture supernatant described in

[16] .

[18] A method for producing an antigen-presenting extracellular vesicle according to any of [1] to

[12] , comprising the step of culturing the cells according to

[15] and recovering the culture supernatant obtained.

[19] A pharmaceutical composition comprising an antigen-presenting extracellular vesicle described in any of [1] to

[12] and

[17] , or a culture supernatant described in

[16] .

[20] A pharmaceutical composition for treating or preventing an infectious disease, comprising an antigen-presenting extracellular vesicle according to any of [1] to

[12] and

[17] , or a culture supernatant according to

[16] .

[21] A pharmaceutical composition comprising an antigen-presenting extracellular vesicle according to any of [5] and [7] to

[12] for the treatment or prevention of cancer.

[22] A pharmaceutical composition comprising an antigen-presenting extracellular vesicle according to any one of [6] to

[12] for the treatment or prevention of an autoimmune disease.

[23] A pharmaceutical composition comprising an antigen-presenting extracellular vesicle according to any one of [6] to

[12] for the treatment or prevention of allergic diseases.

[24] A method for activating and / or proliferating T cells in response to a specific antigen, comprising contacting an antigen-presenting extracellular vesicle described in any of [1] to

[12] and

[17] with T cells in vitro or ex vivo.

[0010] 〔twenty five〕 An antigen-presenting extracellular vesicle according to any of [1] to

[11] , wherein the protein or protein complex specified in (A) is fused with the protein or protein complex specified in (B).

[26] An antigen-presenting extracellular vesicle according to any of [8] to

[11] , wherein the protein or protein complex specified in (A) is fused with the protein or protein complex specified in (C).

[27] An antigen-presenting extracellular vesicle according to any of [8] to

[11] , wherein a protein or protein complex as defined in (B) is fused with a protein or protein complex as defined in (C).

[28] An antigen-presenting extracellular vesicle according to any of [8] to

[11] , wherein a protein or protein complex as defined in (A) is fused with a protein or protein complex as defined in (B) and a protein or protein complex as defined in (C).

[29] An antigen-presenting extracellular vesicle described in any of

[25] to

[27] , wherein the extracellular vesicle is an exosome.

[0011]

[30] The pharmaceutical composition according to

[21] , comprising an immune checkpoint inhibitor.

[31] The pharmaceutical composition according to

[30] , wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle.

[32] The pharmaceutical composition according to

[30] or

[31] , wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof.

[0012] [1A] An antigen-presenting extracellular vesicle, whose membrane contains the following: (D) An antigen-presenting extracellular vesicle comprising an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine or its subunit, and a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine outside the membrane. [2A] An antigen-presenting extracellular vesicle as described in [1A], An antigen-presenting extracellular vesicle, wherein the fusion protein comprises the antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or its subunit, and a membrane protein or its transmembrane domain capable of localizing to the membrane of the extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of the extracellular vesicle. [3A] An antigen-presenting extracellular vesicle as described in [2A], An antigen-presenting extracellular vesicle in which 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 is tetraspanin or MFG-E8. [4A] An antigen-presenting extracellular vesicle as described in [3A], The aforementioned fusion protein, from the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one T cell stimulating cytokine or its subunit, An extracellular vesicle that presents an antigen and contains an amino acid sequence that codes for this in this order. [5A] An antigen-presenting extracellular vesicle as described in [3A], The aforementioned fusion protein, from the N-terminus, (D-1) A fusion peptide comprising a tetraspanin or its transmembrane domain, or MFG-E8 or its transmembrane domain, and at least one of the T cell-stimulating cytokines or its subunits. (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, An antigen-presenting extracellular vesicle containing an amino acid sequence that codes in this order. [6A] The aforementioned fusion peptide, from the N-terminus, (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) A spacer array that may exist, (3) at least one T cell stimulating cytokine or its subunit, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 An antigen-presenting extracellular vesicle according to [4A] or [5A], comprising an amino acid sequence encoding in this order. [7A] The aforementioned fusion peptide, from the N-terminus, (1) at least one T cell stimulating cytokine or its subunit, (2) A spacer array which may exist, and (3) MFG-E8 An antigen-presenting extracellular vesicle according to [4A] or [5A], comprising an amino acid sequence encoding in this order. [8A] The antigen-presenting extracellular vesicle according to [4A] or [5A], wherein the MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular domain of an MHC class I α chain. [9A] The antigen-presenting extracellular vesicle according to [4A] or [5A], wherein the MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule comprises the extracellular domain of an MHC class II α chain and / or the extracellular domain of an MHC class II β chain.

[0013] [10A] (C) An antigen-presenting extracellular vesicle according to any one of the items [1A] to [9A], further comprising a protein in its membrane that contains at least one T cell costimulatory molecule and that is capable of interacting with the T cell. [11A] The antigen-presenting extracellular vesicle according to [10A], wherein the protein capable of interacting with the T cell comprises at least one T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle, or a protein or its domain that can bind to the membrane of the extracellular vesicle. [12A] The antigen-presenting extracellular vesicle according to [11A], wherein the protein capable of interacting with the T cell comprises at least one T cell costimulatory molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain. [13A] The protein capable of interacting with the aforementioned T cells is (C) From the N-terminus, (C-1) At least one T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanin An extracellular antigen-presenting vesicle, as described in [12A], containing an amino acid sequence encoding in this order. [14A] An antigen-presenting extracellular vesicle according to any one of [10A] to [13A], wherein the (A) fusion protein and the (C) protein capable of interacting with T cells are fused. [15A] An antigen-presenting extracellular vesicle described in any of [1A] to [14A], wherein the extracellular vesicle is an exosome.

[0014] A pharmaceutical composition comprising an antigen-presenting extracellular vesicle described in any of [1B] [1A] to [15A] and a pharmacologically acceptable carrier.

[0015] [1C] A pharmaceutical composition for treating or preventing cancer, comprising an antigen-presenting extracellular vesicle according to any one of [1A] to [15A]; wherein preferably the antigen peptide comprises a cancer antigen peptide. [2C] A pharmaceutical composition for treating or preventing an autoimmune disease, comprising an antigen-presenting extracellular vesicle according to any one of [1A] to [15A]; wherein preferably the antigen peptide comprises an autoantigen peptide. [3C] A pharmaceutical composition for treating or preventing allergic diseases, comprising an antigen-presenting extracellular vesicle according to any one of [1A] to [15A]; wherein preferably the antigen peptide comprises an allergen. [4C] The pharmaceutical composition according to [1C], comprising an immune checkpoint inhibitor. [5C] The pharmaceutical composition according to [4C], wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle. [6C] The pharmaceutical composition according to [4C] or [5C], wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof. [7C] A pharmaceutical composition for treating or preventing an infectious disease, comprising an antigen-presenting extracellular vesicle described in any of [1A] to [15A] and a pharmacologically acceptable carrier; wherein the antigen peptide is preferably derived from an infectious pathogen causing the infectious disease.

[0016] [1D] An antigen-presenting extracellular vesicle according to any of [1A] to [15A] for use in the treatment or prevention of cancer; wherein preferably the antigen peptide comprises a cancer antigen peptide. [2D] An antigen-presenting extracellular vesicle according to any one of [1A] to [15A] for use in the treatment or prevention of autoimmune diseases; wherein the antigen peptide preferably comprises an autoantigen peptide. [3D] An antigen-presenting extracellular vesicle according to any one of [1A] to [15A] for use in the treatment or prevention of allergic diseases; wherein the antigen peptide preferably comprises an allergen. [4D] An antigen-presenting extracellular vesicle for use as described in [1D], used in conjunction with an immune checkpoint inhibitor. [5D] An antigen-presenting extracellular vesicle for use according to [4D], wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle. [6D] An antigen-presenting extracellular vesicle for use according to [4D] or [5D], wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof. [7D] An antigen-presenting extracellular vesicle according to any of [1A] to [15A] for use in the treatment or prevention of infectious diseases; wherein the antigen peptide is preferably derived from an infectious pathogen causing the infectious disease.

[0017] [1E] Use of an antigen-presenting extracellular vesicle according to any of [1A] to [15A] in the manufacture of a pharmaceutical product for treating or preventing cancer; wherein preferably the antigen peptide comprises a cancer antigen peptide. [2E] Use of antigen-presenting extracellular vesicles according to any of [1A] to [15A] in the manufacture of a pharmaceutical product for the treatment or prevention of an autoimmune disease; wherein preferably the antigen peptide comprises an autoantigen peptide. [3E] Use of antigen-presenting extracellular vesicles according to any of [1A] to [15A] in the manufacture of a pharmaceutical product for the treatment or prevention of allergic diseases; wherein preferably the antigen peptide comprises an allergen. [4E] The use described in [1E], wherein the pharmaceutical product is used in combination with an immune checkpoint inhibitor. [5E] The use according to [4E], wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle. [6E] The use according to [4E] or [5E], wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof. [7E] Use of an antigen-presenting extracellular vesicle according to any of [1A] to [15A] in the manufacture of a pharmaceutical product for treating or preventing an infectious disease; wherein the antigen peptide is preferably derived from an infectious pathogen causing the infectious disease.

[0018] [1F] A method for treating or preventing cancer in a subject, A method for treating or preventing cancer by administering an effective amount of an antigen-presenting extracellular vesicle described in any of [1A] to [15A] to a target, thereby activating and / or proliferating T cells that recognize cancer antigens within the target, and causing the activated and / or proliferated T cells to attack cancer cells; wherein, preferably, the activated and / or proliferated T cells are CD8-positive cytotoxic T cells, and preferably, the antigen peptide contains a cancer antigen peptide. [2F] A method for treating or preventing an autoimmune disease in a subject, comprising administering an effective amount of an antigen-presenting extracellular vesicle described in any of [1A] to [15A] to the subject to activate and / or proliferate T cells that recognize self-antigens within the subject, and desensitizing the immune response to the self-antigen within the subject, thereby treating or preventing an autoimmune disease; wherein, preferably, the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably, the antigen peptide contains a self-antigen peptide. [3F] A method for treating or preventing an allergic disease in a subject, comprising administering an effective amount of an antigen-presenting extracellular vesicle described in any of [1A] to [15A] to the subject to activate and / or proliferate allergen-recognizing T cells in the subject, and desensitizing the immune response to the autoantigen in the subject, thereby treating or preventing an autoimmune disease; wherein, preferably, the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably, the antigen peptide contains an allergen. [4F] The method according to [1F], wherein the antigen-presenting extracellular vesicle is administered together with an immune checkpoint inhibitor. [5F] The method according to [4F], wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle. [6F] The method according to [4F] or [5F], wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof. [7F] A method for treating or preventing an infectious disease in a subject, comprising administering an effective amount of any antigen-presenting extracellular vesicle described in any of [1A] to [15A] to the subject, 1) By inducing the secretion of inflammatory cytokines and activating the target's innate immunity, and / or 2) To provide the subject with acquired immunity to the infectious pathogen that causes the aforementioned infectious disease, A method for treating or preventing an infectious disease, comprising eliminating the infectious pathogen causing the aforementioned infectious disease in the body and / or suppressing its proliferation.

[0019] [1G] A method for activating and / or proliferating T cells in response to a specific antigen, comprising contacting an antigen-presenting extracellular vesicle described in any of [1A] to [15A] with T cells in vitro or ex vivo.

[0020] [1H] (i) A fusion protein or protein complex of (D) as defined in any of [1A] to [9A]; (ii) A protein capable of interacting with T cells as defined in any of [10A] to [13A]; or (iii) A polynucleotide encoding a fusion protein of the fusion protein of (D) described in [14A] and the T cell-interacting protein of (C). A vector containing the nucleic acid described in [2H] and [1G].

[0021] [1I] (a) A sequence comprising an antigen-presenting MHC molecule and encoding a fusion protein (A) capable of presenting the antigen-presenting MHC molecule outside the membrane of an extracellular vesicle; (b) A sequence encoding a fusion protein (B) capable of presenting the T cell-stimulating cytokine (B) outside the membrane of an extracellular vesicle, comprising at least one T cell-stimulating cytokine or a subunit thereof; (c) A sequence encoding a fusion protein (C) that includes a T cell costimulatory molecule and is capable of presenting the T cell costimulatory molecule outside the membrane of an extracellular vesicle; (d) A sequence encoding a fusion protein (D) comprising an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine or its subunit, the antigen and the T cell-stimulating cytokine capable of presenting the antigen and the T cell-stimulating cytokine outside the membrane of an extracellular vesicle; and (e) A sequence encoding a fusion protein (E) that comprises an antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or its subunit, and a T cell-costimulating molecule, and is capable of presenting the antigen, the T cell-stimulating cytokine, and the T cell-costimulating molecule outside the membrane of an extracellular vesicle; A polynucleotide containing at least one sequence selected from the group consisting of the following. [2I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) above comprises an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. [3I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) above comprises at least one T cell-stimulating cytokine or a subunit thereof, and a membrane protein or a transmembrane domain thereof that can be expressed on the membrane of an extracellular vesicle, or a protein or a domain thereof that can bind to the membrane of an extracellular vesicle. [4I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) above comprises an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain. [5I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) above comprises at least one T cell stimulating cytokine or a subunit thereof and a partial sequence of tetraspanin, the partial sequence of tetraspanin having at least two transmembrane domains, and at least one T cell stimulating cytokine is positioned between the two transmembrane domains. [6I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) above comprises at least one T cell-stimulating cytokine or a subunit thereof and MFG-E8 or a domain thereof. [7I] The fusion protein defined in (A) above: From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A polynucleotide as described in [1I], comprising an amino acid sequence consisting of the following. [8I] The fusion protein defined in (A) above From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β2 microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin The amino acid sequence consists of, The polynucleotide described in [1I]. [9I] The polynucleotide according to [8I] further comprising the amino acid sequence of β2 microglobulin, MHC class Iα chain, MHC class IIβ chain, or MHC class IIα chain. [10I] The fusion protein defined in (B) above From the N-terminus, (B-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, from the N-terminus. (B-2) A spacer arrangement that may exist, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may exist, and (B-5) Partial sequence of tetraspanin containing transmembrane domain 4 A polynucleotide as described in [1I], comprising an amino acid sequence consisting of the following. [11I] The fusion protein defined in (B) above From the N-terminus, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may be present, and (B-5)MFG-E8 A polynucleotide as described in [1I], comprising an amino acid sequence consisting of the following. [12I] The fusion protein defined in (A) above, From the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A polynucleotide described in [1I] comprising an amino acid sequence consisting of the above. [13I] The fusion protein defined in (A) above, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A polynucleotide as described in [1I], comprising an amino acid sequence consisting of the following: [14I] The polynucleotide described in [13I] further comprising the amino acid sequence of the (A-6)MHC class IIα chain. [15I] The polynucleotide according to [1I], wherein the T cell stimulating cytokine is IL-2, IL-4, IL-6, IL-12, a subunit of IL-12, or TGF-β. [16I] The fusion protein defined in (C) above The polynucleotide according to [1I], comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. [17I] The fusion protein defined in (C) above The polynucleotide according to [1I], comprising a T cell costimulatory molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain. [18I] The fusion protein defined in (C) above From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanine A polynucleotide as described in [1I], comprising an amino acid sequence consisting of the following.

[0022] [19I] The fusion protein defined in (D) above is The polynucleotide according to [1I], comprising the antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or its subunit, and a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle. [20I] The polynucleotide according to [19I], 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 is tetraspanin or MFG-E8. [21I] The fusion protein defined in (D) above, From the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one T cell stimulating cytokine or its subunit, A polynucleotide as described in [1I], comprising an amino acid sequence that codes for in this order. [22I] The fusion protein defined in (D) above, From the N-terminus, (D-1) A fusion peptide comprising a tetraspanin or its transmembrane domain, or MFG-E8 or its transmembrane domain, and at least one of the T cell-stimulating cytokines or its subunits. (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, A polynucleotide as described in [1I], comprising an amino acid sequence encoding in this order. [23I] A fusion peptide comprising the tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and at least one T cell stimulating cytokine or its subunit, From the N-terminus, (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) A spacer array that may exist, (3) at least one T cell stimulating cytokine or its subunit, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 A polynucleotide according to [21I] or [22I], comprising an amino acid sequence encoding in this order. [24I] A fusion peptide comprising the tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and at least one T cell stimulating cytokine or its subunit, From the N-terminus, (1) at least one T cell stimulating cytokine or its subunit, (2) A spacer array which may exist, and (3) MFG-E8 A polynucleotide according to [21I] or [22I], comprising an amino acid sequence encoding in this order. [25I] The polynucleotide according to [21I] or [22I], wherein the MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular region of an MHC class I α chain. [26I] The polynucleotide according to [21I] or [22I], wherein the MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule comprises the extracellular domain of an MHC class II α chain and / or the extracellular domain of an MHC class II β chain.

[0023] [1J] The polypeptide according to [1I], comprising the sequence defined in (a) and the sequence defined in (b). [2J] Furthermore, the polynucleotide described in [1J] includes the sequence defined in (c) above. [3J] The polypeptide according to [1I], comprising the sequence defined in (d) above. [4J] Furthermore, the polypeptide described in [3J] includes the sequence defined in (c) above. [5J] The polypeptide according to [1I], comprising the sequence specified in (e) above. A vector containing a polynucleotide as described in any one of the following sections: [6J], [1I], to [5J].

[0024] A pharmaceutical composition comprising a polynucleotide described in any one of items [1K] [1I] to [5J] or a vector described in [6J] and a pharmacokinetically acceptable carrier.

[0025] [1L] A pharmaceutical composition for treating or preventing cancer, comprising a polynucleotide as described in any one of [1I] to [5J] or a vector as described in [6J]. [2L] A pharmaceutical composition for treating or preventing an autoimmune disease, comprising a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J]; wherein preferably the antigen peptide comprises an autoantigen peptide. [3L] A pharmaceutical composition for treating or preventing allergic diseases, comprising a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J]; wherein preferably the antigen peptide comprises an allergen. [4L] A pharmaceutical composition comprising a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] for treating or preventing an infectious disease, and a pharmacokinetically acceptable carrier; wherein the antigen peptide is preferably derived from an infectious pathogen that causes an infectious disease.

[0026] [1M] A polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] for use in the treatment or prevention of cancer; wherein preferably the antigen peptide comprises a cancer antigen peptide. [2M] A polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] for use in the treatment or prevention of autoimmune diseases; wherein the antigen peptide preferably comprises an autoantigen peptide. [3M] A polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] for use in the treatment or prevention of allergic diseases; wherein the antigen peptide preferably comprises an allergen. [4M] A polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] for use in the treatment or prevention of infectious diseases; wherein the antigen peptide is preferably derived from an infectious pathogen that causes the infectious disease.

[0027] [1N] Use of a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] in the manufacture of a pharmaceutical product for treating or preventing cancer; wherein preferably the antigen peptide comprises a cancer antigen peptide. [2N] Use of a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] in the manufacture of a pharmaceutical product for treating or preventing an autoimmune disease; wherein preferably the antigen peptide comprises an autoantigen peptide. [3N] Use of a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] in the manufacture of a pharmaceutical product for treating or preventing an allergic disease; wherein the antigen peptide preferably contains an allergen. [4N] Use of a polynucleotide according to any one of [1I] to [5J] or a vector according to [6J] in the manufacture of a pharmaceutical product for treating or preventing an infectious disease; wherein the antigen peptide is preferably derived from an infectious pathogen causing the infectious disease.

[0028]

[10] A method for treating or preventing cancer in a subject, A method for treating or preventing cancer by administering an effective amount of a polynucleotide described in any one of [1I] to [5J] or a vector described in [6J] to a subject to activate and / or proliferate T cells in the subject that recognize cancer antigens, and to cause the activated and / or proliferated T cells to attack cancer cells; wherein, preferably, the activated and / or proliferated T cells are CD8-positive cytotoxic T cells, and preferably, the antigen peptide contains a cancer antigen peptide. [2O] A method for treating or preventing an autoimmune disease in a subject, comprising administering an effective amount of a polynucleotide described in any one of [1I] to [5J] or a vector described in [6J] to the subject to activate and / or proliferate T cells that recognize a self-antigen within the subject, and desensitizing the immune response to the self-antigen within the subject; wherein, preferably, the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably, the antigen peptide contains a self-antigen peptide. [3O] A method for treating or preventing an allergic disease in a subject, comprising administering an effective amount of a polynucleotide described in any one of [1I] to [5J] or a vector described in [6J] to the subject to activate and / or proliferate allergen-recognizing T cells in the subject and desensitize the immune response to the autoantigen in the subject, thereby treating or preventing an autoimmune disease; wherein, preferably, the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably, the antigen peptide contains an allergen. [4O] A method for treating or preventing an infectious disease in a subject, comprising administering an effective amount of a polynucleotide described in any one of the items [1I] to [5J] or a vector described in [6J] to the subject. 1) By inducing the secretion of inflammatory cytokines and activating the target's innate immunity, and / or 2) To provide the subject with acquired immunity to the infectious pathogen that causes the aforementioned infectious disease, A method for treating or preventing an infectious disease, comprising eliminating the infectious pathogen that causes the aforementioned disease infection in the body and / or suppressing its proliferation.

[0029] [1P] A method for activating and / or proliferating T cells against a specific antigen, comprising introducing a polynucleotide described in any one of [1I] to [5J] or a vector described in [6J] into cells in vitro or ex vivo, generating antigen-presenting cells and / or antigen-presenting extracellular vesicles, and contacting the generated antigen-presenting cells and / or antigen-presenting extracellular vesicles with T cells in vitro or ex vivo. [Effects of the Invention]

[0030] According to the present invention, by using polynucleotides for producing cells (antigen-presenting cells) and extracellular vesicles (antigen-presenting extracellular vesicles) containing MHC molecules and T cell-stimulating cytokines in their membranes, antigen-specific T cells can be satisfactorily activated. [Brief explanation of the drawing]

[0031] [Figure 1A] A model diagram of an antigen peptide-single-chain MHC class I molecule (sc-Trimer)-CD81 fusion protein is shown. [Figure 1B] The amino acid sequence of the antigen peptide-single-chain MHC class I molecule (sc-Trimer)-CD81 fusion protein is shown. [Figure 1C] A model diagram of the CD80-CD9 fusion protein is shown. [Figure 1D] The amino acid sequence of the CD80-CD9 fusion protein is shown. [Figure 1E] A model diagram of the CD63-IL-2 fusion protein is shown. [Figure 1F] The amino acid sequence of the CD63-IL-2 fusion protein is shown. [Figure 1G] A model diagram of an antigen peptide-MHC class IIβ chain (sc-Dimer)-CD81 fusion protein is shown. [Figure 1H] The amino acid sequence of the antigen peptide-MHC class IIβ chain (sc-Dimer)-CD81 fusion protein is shown. [Figure 1I] The amino acid sequence of the MHC class IIα chain is shown. [Figure 1J] A model diagram of the TGF-β-MFG-E8 fusion protein is shown. [Figure 1K] The amino acid sequence of the TGF-β-MFG-E8 fusion protein is shown. [Figure 1L] A model diagram of the CD81-IL-4 fusion protein is shown. [Figure 1M] The amino acid sequence of the CD81-IL-4 fusion protein is shown. [Figure 1N] The nucleic acid sequence of the sc-Trimer-CD81-IL-2 fusion protein is shown. [Figure 10] The nucleic acid sequence of the CD81-IL-4 fusion protein is shown. [Figure 2A] A model diagram of the antigen-presenting extracellular vesicle in Example 1 is shown. [Figure 2B] A model diagram of the antigen-presenting extracellular vesicle in Example 2 is shown. [Figure 2C] A model diagram of the antigen-presenting extracellular vesicle in Example 3 is shown. [Figure 2D] A model diagram of the antigen-presenting extracellular vesicle in Example 4 is shown. [Figure 2E] A model diagram of the antigen-presenting extracellular vesicle in Example 5 is shown. [Figure 2F] A model diagram of the antigen-presenting extracellular vesicle in Example 6 is shown. [Figure 2G] A model diagram of the antigen-presenting extracellular vesicle in Example 7 is shown. [Figure 2H] A model diagram of the antigen-presenting extracellular vesicle in Example 8 is shown. [Figure 2I] A model diagram of the antigen-presenting extracellular vesicle in Example 9 is shown. [Figure 2J] Model diagrams of antigen-presenting extracellular vesicles in other embodiments are illustrated. [Figure 3A]In Test Example 1-1, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 2 are shown. [Figure 3B] In Test Examples 1-2, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 3 are shown. [Figure 3C] In Test Examples 1-3, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 4 are shown. [Figure 3D] In Test Examples 1-4, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 5 are shown. [Figure 3E] Examples 1-5 show the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 6. [Figure 3F] In Test Examples 1-6, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 7 are shown. [Figure 3G] Examples 1-7 show the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 8. [Figure 3H] Examples 1-8 show the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicle of Example 9. [Figure 4] In Test Example 2, the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Examples 1 and 2 activate antigen-specific CD8-positive T cells (OT-1 T cells) are shown. [Figure 5] In Test Example 3, we show the results of an in vivo evaluation of whether the antigen-presenting extracellular vesicles of Example 2 activate antigen-specific CD8-positive T cells (OT-1), etc. [Figure 6] In Test Example 4, the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 3 activate antigen-specific CD4-positive T cells are shown. [Figure 7]In Test Example 5, we show the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 4 induce differentiation of antigen-specific CD4-positive T cells (OT-2 T cells) into regulatory T cells. [Figure 8] In Test Example 6, we show the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Examples 3 and 5 induce differentiation of antigen-specific CD4-positive T cells (OT-2 T cells) into Th2 T cells. [Figure 9] In Test Example 7, we show the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 6 induce differentiation of antigen-specific CD4-positive T cells into Th1 cells. [Figure 10] In Test Example 8, we show the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 7 induce differentiation of antigen-specific CD4-positive T cells into Th17 cells. [Figure 11] In Test Example 9, the antigen-presenting extracellular vesicles from Example 1 and Example 8 significantly increased the proliferation of antigen-specific CD8-positive T cells. [Figure 12] In Test Example 10, the antigen-presenting extracellular vesicles of Example 8 significantly suppressed the proliferation of B16 melanoma cells. [Figure 13] In Test Example 11, the results of in vivo evaluation of whether the mRNA from Example 10 activates antigen-specific CD8-positive T cells (OT-1) are shown. [Figure 14] In Test Example 12, the results of an in vivo evaluation of whether the mRNA from Example 10 activates endogenous antigen-specific CD8-positive T cells are shown. [Modes for carrying out the invention]

[0032] definition

[0033] Extracellular vesicles

[0034] As used herein, the term "extracellular vesicle" is not particularly limited as long as it is a vesicle secreted by a cell, but examples include exosomes, microvesicles (MVs), and apoptotic bodies.

[0035] As used herein, "exosome" refers to a vesicle of about 20 to about 500 nm (preferably about 20 to about 200 nm, more preferably about 25 to about 150 nm, and even more preferably about 30 to about 100 nm) derived from the endocytial-cis pathway. Examples of components of exosomes include proteins and nucleic acids (mRNA, miRNA, non-coated RNA). Exosomes may have a function of facilitating intercellular communication. Examples of exosome marker molecules include Alix, Tsg101, tetraspanin, flotillin, and phosphatidylserine.

[0036] As used herein, "microendoplasmic reticulum" refers to vesicles of approximately 50 to 1000 nm in size, derived from the cytoplasmic membrane. Components of the microendoplasmic reticulum include, for example, proteins and nucleic acids (mRNA, miRNA, non-coated RNA, etc.). The microendoplasmic reticulum may have functions such as facilitating intercellular communication. Examples of marker molecules for the microendoplasmic reticulum include integrins, selectins, CD40, and CD154.

[0037] As used herein, "apoptotic bodies" refer to vesicles of approximately 500 to 2000 nm in size, originating from the cytoplasmic membrane. Components of apoptotic bodies include, for example, fragmented nuclei and organelles. Apoptotic bodies may have functions such as inducing phagocytosis. Examples of marker molecules for apoptotic bodies include Annexin V and phosphatidylserine.

[0038] As used herein, "antigen-presenting extracellular vesicle" refers to an extracellular vesicle that presents an antigen outside its membrane.

[0039] Major histocompatibility complex molecule

[0040] The term "Major Histocompatibility Complex (MHC) molecule" as used herein is not particularly limited, as long as it contains an antigen-binding gap and is capable of binding to antigens presented to T cells, T cell precursors, etc. Examples of MHC molecules include MHC class I molecules and MHC class II molecules. MHC molecules may be derived from any animal species. For example, in humans, this refers to human leukocyte antigens (HLA), and in mice, it refers to the H2 system.

[0041] HLA, which corresponds to MHC class I molecules, can be classified into subtypes such as HLA-A, HLA-B, HLA-Cw, HLA-F, and HLA-G. Polymorphisms (alleles) are known for these subtypes. Examples of HLA-A polymorphisms include HLA-A1, HLA-A0201, and HLA-A24; examples of HLA-B polymorphisms include HLA-B7, HLA-B40, and HLA-B4403; and examples of HLA-Cw polymorphisms include HLA-Cw0301, HLA-Cw0401, and HLA-Cw0602.

[0042] HLAs corresponding to MHC class II molecules are sometimes classified into subtypes such as HLA-DR, HLA-DQ, and HLA-DP.

[0043] The MHC molecules described herein may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, with respect to their wild-type amino acid sequence (for example, in the case of MHC class I molecules: e.g., MHC class I α chain such as SEQ ID NO: 9, β2 microglobulin such as SEQ ID NO: 7, single-chain MHC class I molecules such as SEQ ID NO: 65, etc.; in the case of MHC class II molecules: e.g., MHC class II α chain such as SEQ ID NO: 71, MHC class II β chain such as SEQ ID NO: 37, single-chain MHC class II molecules, etc.) with respect to their wild-type amino acid sequence, as long as they can perform their function. Alternatively, the MHC molecules described herein may have one or more amino acid deletions, insertions, additions and / or substitutions with respect to their wild-type amino acid sequence, as long as they can perform their function.

[0044] The term "antigen-presenting MHC molecule" as used herein is not particularly limited as long as it is an MHC molecule that presents an antigen, but examples include antigen-presenting MHC class I molecules, antigen-presenting MHC class II molecules, etc. Examples of "antigen-presenting MHC class I molecules" include: complexes of an antigen with an MHC class I α chain or its extracellular domain and β2 microglobulin; complexes of an antigen with a single-chain MHC class I molecule; fusion proteins in which an antigen and a single-chain MHC class I molecule are bound; complexes of an antigen with a fusion protein of the extracellular domain of an MHC class I α chain and another protein or its domain or fragment, etc. (for example, a fusion protein of the extracellular domain of an MHC class I α chain and the Fc portion of an antibody; a fusion protein of the extracellular domain of an MHC class I α chain and the transmembrane domain of another membrane protein, etc.); etc. Examples of "antigen-presenting MHC class II molecules" include complexes of an antigen with an MHC class II α chain or its extracellular domain and an MHC class II β chain or its extracellular domain; complexes of an antigen with a single-chain MHC class II molecule; complexes of a fusion protein bound to an antigen and an MHC class II β chain with an MHC class II α chain; complexes of an antigen with a fusion protein of the extracellular domain of an MHC class II α chain and another protein or its domain or fragment (e.g., a fusion protein of the extracellular domain of an MHC class II α chain and the Fc portion of an antibody; a fusion protein of the extracellular domain of an MHC class II α chain and the transmembrane domain of another membrane protein); and fusion proteins of the extracellular domain of an MHC class II β chain and another protein or its domain or fragment (e.g., a fusion protein of the extracellular domain of an MHC class II β chain and the Fc portion of an antibody; a fusion protein of the extracellular domain of an MHC class II β chain and an amino acid sequence containing the transmembrane domain of another membrane protein).

[0045] As used herein, "single-chain MHC molecule," "single-chain MHC class I molecule," or "single-chain MHC class II molecule" refers to a fusion protein in which the α chain or its extracellular domain of an MHC molecule (or an MHC class I molecule or an MHC class II molecule) is linked to the β chain or its extracellular domain or β2 microglobulin via a spacer sequence or the like, as necessary. An example of a "single-chain MHC class I molecule" is a fusion protein in which an MHC class I α chain and β2 microglobulin are linked via a spacer sequence or the like, as necessary. An example of a "single-chain MHC class II molecule" is a fusion protein in which an MHC class II α chain and an MHC class II β chain are linked via a spacer sequence or the like, as necessary.

[0046] As used herein, "a protein (or fusion protein, protein complex, etc.) containing an antigen-presenting MHC molecule and capable of presenting the antigen (or antigen peptide) outside the membrane" means a protein (or fusion protein, protein complex, etc.) that contains at least an antigen-presenting MHC molecule and is capable of presenting the antigen (or antigen peptide) outside the membrane to T cells, etc. "A protein (or fusion protein, protein complex, etc.) containing an antigen-presenting MHC molecule and capable of presenting the antigen (or antigen peptide) outside the membrane" may be expressed in the form of a fusion protein, protein complex, etc. using a plasmid, etc., so that it is expressed on the membrane of an extracellular vesicle. Alternatively, the "protein (or fusion protein, protein complex, etc.) capable of presenting an antigen (or antigen peptide) outside the membrane, including an antigen-presenting MHC molecule," may be a protein in which a soluble antigen-presenting MHC molecule is used (but is not limited to these, for example, a fusion protein containing an MHC class I α chain and an immunoglobulin heavy chain as described in Patent Document 1; or a soluble MHC class I molecule as described in Japanese Patent Application Publication No. 2007-161719), and the soluble antigen-presenting MHC molecule and the extracellular vesicle are bound to the membrane of the extracellular vesicle via a lipid linker, peptide linker, etc., as needed (for example, the method described in Japanese Patent Application Publication No. 2018-104341, etc., may be referenced).Alternatively, the extracellular vesicle may be a mixture of an extracellular vesicle containing a protein containing an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody) attached to the N-terminus or C-terminus of a soluble antigen-presenting MHC molecule (the tag may be expressed as a fusion protein together with other components, or it may be conjugated to a separately prepared soluble antigen-presenting MHC molecule via a linker, etc., as needed) and an extracellular vesicle containing a membrane protein containing an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody) (for example, the antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody) itself, conjugated to the membrane of the extracellular vesicle via a linker, etc., as needed; or a membrane protein that can be expressed on the membrane of the extracellular vesicle, or a fusion protein in which a nanobody for the tag is conjugated to the N-terminus or C-terminus of its transmembrane domain), under desired conditions (e.g., Raj D, et al., Gut., 2019). You may also refer to the method using a PNE tag and an antibody against the tag, as described in Jun;68(6):1052-1064, etc.

[0047] antigen

[0048] As used herein, the term "antigen" is not particularly limited as long as it can possess antigenicity, and includes not only peptide antigens but also non-peptide antigens such as phospholipids and complex carbohydrates (for example, bacterial membrane components such as mycolic acid and lipoarabinoannan).

[0049] The term "antigen peptide" as used herein is not particularly limited as long as it is a peptide that can act as an antigen, and may be of natural origin, synthetic origin, or commercially available. Antigen peptides are not limited to these, but include, for example, WT-1, α-fetal protein, MAGE-1, MAGE-3, placental alkaline phosphatase sialyl-Lewis X, CA-125, CA-19, TAG-72, epithelial glycoprotein 2, 5T4, α-fetal protein receptor, M2A, tyrosinase, Ras, p53, Her-2 / neu, EGF-R, estrogen receptor, progesterone receptor, myc, BCR-ABL, HPV type 16, melanotransferrin, MUC1, CD10, CD19, CD20, CD37, CD45R, IL-2 receptor α chain, T cell receptor, prostatic acid phosphatase, GP100, MelanA / Mart-1, gp75 / Braun, BAGE, S-100, itokeratin, CYFR Tumor-associated antigen peptides such as A21-1 and Ep-CAM; insulin, glutamate 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, virin 2, nucleosomes, ribonucleoproteins, myelin oligodendrocyte glycoproteins, myelin-associated glycoproteins, myelin / oligodendrocyte basic proteins, oligodendrocyte-specific proteins, myelin basic proteins, proteolipide apoproteins, and other autoantigen peptides;Antigenic peptides derived from infectious pathogens such as protozoa (e.g., malaria parasites, 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, Sporospirillus), viruses (e.g., adenovirus, herpes simplex virus, papillomavirus, respiratory cynthiavirus, poxvirus, HIV, influenza virus, coronaviruses such as SARS-CoV and SARS-CoV-2), intracellular parasites (e.g., Chlamydiidae, Mycoplasma, Acholeplasmidae, Rickettsiae), and helminths (e.g., nematodes, trematodes, tapeworms); and other antigenic peptides such as prions. Antigen peptides may contain allergens that cause allergic symptoms. Examples of allergens include peptides derived from the protozoa, bacteria, fungi, intracellular parasites, and helminths mentioned above, as well as exogenous peptides, such as 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, are given as examples. Alternatively, the allergens that cause allergic symptoms may be of food origin. Examples of allergens that cause allergic symptoms to food include peptides derived from chicken eggs, milk, wheat, buckwheat, crab, shrimp, and peanuts.

[0050] As used herein, "MHC molecule-restricted antigen peptide" refers to an antigen peptide that can bind to MHC molecules in vitro, in vivo, and / or ex vivo. The number of amino acid residues in an "MHC molecule-restricted antigen peptide" is typically about 7 to 30. Examples of "MHC molecule-restricted antigen peptides" include MHC class I molecule-restricted antigen peptides and MHC class II molecule-restricted antigen peptides.

[0051] As used herein, "MHC class I molecule-restricted antigen peptide" means an antigen peptide that can bind to MHC class I molecules in vitro, in vivo, and / or ex vivo. When an MHC class I molecule-restricted antigen peptide is presented outside the membrane of an extracellular vesicle, for example, the antigen peptide can be recognized by precursor T cells, etc., and induce cytotoxic T cells, etc. The number of amino acid residues of the "MHC class I molecule-restricted antigen peptide" is usually about 7 to about 30, preferably about 7 to about 25, more preferably about 7 to about 20, even more preferably about 7 to about 15, and even more preferably about 7 to about 12.

[0052] As used herein, "MHC class II molecule-restricted antigen peptide" means an antigen peptide that can bind to MHC class II molecules in vitro, in vivo, and / or ex vivo. When an MHC class II molecule-restricted antigen peptide is presented outside the membrane of an extracellular vesicle, for example, the antigen peptide can be recognized by precursor T cells, etc., and induce helper T cells, etc. The number of amino acid residues of the "MHC class II molecule-restricted antigen peptide" is usually about 7 to about 30, preferably about 10 to about 25, and more preferably about 12 to about 24.

[0053] "MHC molecule-restricted antigen peptide," "MHC class I molecule-restricted antigen peptide," or "MHC class II molecule-restricted antigen peptide" are not particularly limited as long as they are antigen peptides capable of binding to MHC molecules, MHC class I molecules, or MHC class II molecules.

[0054] T-cell stimulating cytokines

[0055] The term "T cell-stimulating cytokine" as used herein is not particularly limited as long as it is a cytokine capable of stimulating (e.g., activating, inhibiting, etc.) T cells via receptors expressed on the membrane of T cells. Examples of T cell-stimulating cytokines, though not limited to these, include IL-2, IL-4, IL-6, IL-12, TGF-β, IFN-α, and IFN-γ. Among these, those that can form multimers of homozygous or heterozygous subunits (e.g., IL-12, TGF-β, etc.) may be in the form of a continuous amino acid sequence linked via a peptide linker, etc., as long as they are functional (i.e., have the desired pharmacological activity).

[0056] The T-cell stimulating cytokines described herein may be derived from any animal species. Examples include rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; and mammals such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees. The T-cell stimulating cytokines described herein are preferably derived from rodents or mammals, and more preferably from mice or humans.

[0057] The T cell-stimulating cytokines described herein may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, with respect to their wild-type amino acid sequence (e.g., for IL-2, e.g., SEQ ID NO: 25; for IL-4, e.g., SEQ ID NO: 53), as long as they are able to exert their function. Alternatively, the T cell-stimulating cytokines described herein may have one or more amino acid deletions, insertions, additions and / or substitutions, with respect to their wild-type amino acid sequence, as long as they are able to exert their function.

[0058] As used herein, "a protein containing a (first, second) T cell-stimulating cytokine and capable of presenting the (first, second) T cell-stimulating cytokine extramembranely" means a protein that contains at least a T cell-stimulating cytokine and is capable of presenting the T cell-stimulating cytokine extramembranely. "A protein containing a (first, second) T cell-stimulating cytokine and capable of presenting the (first, second) T cell-stimulating cytokine extramembranely" may be expressed as a fusion protein containing a T cell-stimulating cytokine and a membrane protein or a fragment containing its transmembrane domain, etc., using a plasmid or the like, so that it is expressed on the membrane of an extracellular vesicle. Alternatively, the "protein containing (first, second) T cell-stimulating cytokines, and capable of presenting the (first, second) T cell-stimulating cytokines extracellularly" may be a protein in which soluble T cell-stimulating cytokines (but not limited to these, for example, the T cell-stimulating cytokine itself; a fusion protein of the T cell-stimulating cytokine and the Fc portion of an antibody; a complex of the T cell-stimulating cytokine with an antibody that recognizes the T cell-stimulating cytokine, or its antigen-binding fragment (e.g., scFv, Fab, or nanobody), etc.) are used, and the soluble T cell-stimulating cytokine and the extracellular vesicle are conjugated to the membrane of the extracellular vesicle via a lipid linker, peptide linker, etc., as needed (for example, the method described in Japanese Patent Application Publication No. 2018-104341, etc., may be referenced).Alternatively, the extracellular vesicle may be a mixture of a soluble T cell-stimulating cytokine with a desired tag (e.g., His tag, FLAG tag, PNE tag attached to the N-terminus or C-terminus; the tag may be expressed as a fusion protein together with other components, or may be conjugated to a separately prepared soluble T cell-stimulating cytokine via a linker, etc., as needed) and an extracellular vesicle containing a protein (e.g., an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody) attached to the extracellular vesicle membrane as needed; or a membrane protein expressible on the extracellular vesicle membrane, or a fusion protein in which a nanobody for the tag is conjugated to the N-terminus or C-terminus of its transmembrane domain) on its membrane, under desired conditions (e.g., Raj D, et al., Gut., 2019). (You may also refer to the method using a PNE tag and an antibody against the tag, as described in Jun;68(6):1052-1064, etc.) In the case of T cell-stimulating cytokines formed by a multimer of subunits, if one of the subunits is a protein that can be presented outside the membrane of an extracellular vesicle, the remaining subunits do not need to be in a form that can be presented outside the membrane. If one of the subunits is a protein that can be presented outside the membrane of an extracellular vesicle, a functional T cell-stimulating cytokine can be constructed outside the membrane of an extracellular vesicle by adding or co-expressing other subunits.

[0059] T cell costimulatory molecule

[0060] As used herein, "T cell costimulatory molecule" refers to a molecule that can contribute to T cell activation, etc., by interacting with molecules present on the membrane of T cells, such as CD28 and CD134. Examples of T cell costimulatory molecules are not limited to these, but include, for example, molecules such as CD80 and CD86, or their extracellular domains or functional fragments; antibodies such as anti-CD28 antibodies and anti-CD134 antibodies, or their antigen-binding fragments (e.g., scFv, Fab, or nanobodies); and fusion proteins (or complexes, aggregates) of these with the transmembrane domains of other proteins or the Fc portion of antibodies.

[0061] The T cell costimulatory molecules described herein may be derived from any animal species. Examples include rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; and mammals such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees. The T cell costimulatory molecules described herein are preferably derived from rodents or mammals, and more preferably from mice or humans.

[0062] The T cell costimulatory molecules described herein may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, relative to their wild-type amino acid sequence (for example, in the case of CD80, for example, SEQ ID NO: 67, etc.), as long as they can exert the functions described above. Alternatively, the T cell costimulatory molecules described herein may have one or more amino acid deletions, insertions, additions and / or substitutions, etc., relative to their wild-type amino acid sequence, as long as they can exert the functions described herein.

[0063] As used herein, "a protein containing a T cell costimulatory molecule, which is capable of interacting with the T cell costimulatory molecule and T cells" means a protein that contains at least a T cell costimulatory molecule and is capable of interacting with molecules present on the membrane of T cells. In other words, it means that at least the portion of the T cell costimulatory molecule that is capable of interacting with T cells is located outside the membrane of the extracellular vesicle. "A protein containing a T cell costimulatory molecule, which is capable of interacting with the T cell costimulatory molecule and T cells" may be expressed using a plasmid or the like so that it is expressed on the membrane of the extracellular vesicle. Alternatively, the "protein containing a T cell costimulatory molecule, on which the T cell costimulatory molecule and T cells can interact" may be a soluble T cell costimulatory molecule (but not limited to these, for example, a fusion protein of the extracellular domain of CD80 and the Fc portion of an antibody; an anti-CD28 antibody, or its antigen-binding fragment (e.g., scFv, Fab, or nanobody)) on which the soluble T cell costimulatory molecule and the extracellular vesicle are bound to the membrane of the extracellular vesicle via a lipid linker, spacer sequence, etc., as needed (for example, the method described in Japanese Patent Publication No. 2018-104341 may be used as a reference).Alternatively, the extracellular vesicle may be a mixture of a soluble T cell costimulatory molecule with a desired tag (e.g., His tag, FLAG tag, PNE tag attached to the N-terminus or C-terminus; the tag may be expressed as a fusion protein together with other components, or may be conjugated to a separately prepared soluble T cell costimulatory molecule via a linker, etc., as needed) and an extracellular vesicle containing a protein (e.g., an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody) attached to the extracellular vesicle membrane as needed; or a membrane protein expressible on the extracellular vesicle membrane, or a fusion protein in which a nanobody for the tag is conjugated to the N-terminus or C-terminus of its transmembrane domain) on its membrane, under desired conditions (e.g., Raj D, et al., Gut., 2019). You may also refer to the method using a PNE tag and an antibody against the tag, as described in Jun;68(6):1052-1064, etc.

[0064] In this specification, "membrane proteins or their transmembrane domains that can be expressed on the membrane of extracellular vesicles" can be any membrane protein or its transmembrane domain, as long as it can be expressed on the membrane of extracellular vesicles. Preferably, "membrane proteins or their transmembrane domains that can be expressed on the membrane of extracellular vesicles" are membrane proteins known to be expressible on extracellular vesicles (e.g., exosomes, etc.) (e.g., tetraspanin, CD58, ICAM-1, PTGFRN (see, for example, Non-Patent Literature 1, International Publication No. 2019 / 183578, etc.)), or their transmembrane domains.

[0065] In this specification, any protein or domain capable of binding to the membrane of an extracellular vesicle can be selected as "a protein or domain thereof capable of binding to the membrane of an extracellular vesicle." Preferably, the "protein or domain thereof capable of binding to the membrane of an extracellular vesicle" is one that is known to be able to bind to the membrane of an extracellular vesicle (e.g., exosomes, etc.) (e.g., MFG-E8, or its domain (e.g., the C1 and C2 domains of MFG-E8 described in Alain Delcayre, et al., Blood Cells, Molecules, and Diseases 35 (2005) 158-168)).

[0066] The "membrane proteins or their transmembrane domains that can be expressed on the membrane of extracellular vesicles" or "proteins or their domains that can bind to the membrane of extracellular vesicles" described herein may be derived from any animal species. Examples include rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; and mammals such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees. The "membrane proteins or their transmembrane domains that can be expressed on the membrane of extracellular vesicles" or "proteins or their domains that can bind to the membrane of extracellular vesicles" described herein are preferably derived from rodents or mammals, and more preferably from mice or humans.

[0067] According to Non-Patent Document 2, mammalian extracellular vesicle markers are classified as follows: Examples of membrane proteins or GPI-anchored proteins that can be used as marker proteins for extracellular vesicles include: 1) Tissue-nonspecific Tetraspanins (CD63, CD9, CD81, CD82), and other multi-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 (containing syndecane (SDC)); Extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as BSG or CD147); ADAM10; CD73(NT5E), a GPI-anchored 5' nucleotidase, CD55 and CD59 are GPI-anchored complement-binding proteins; Sonic hedgehog protein (SHH) 2) Cell / tissue specific Several tetraspanins: TSPAN8 (epithelial cell specific), CD37, and CD53 (leukocyte specific); PECAM1 (endothelial cell specific); ERBB2 (breast cancer specific); EPCAM (epithelial specific); CD90(THY1) (specific to mesenchymal stem cells); CD45 (PTPRC) (immune cell specific), CD41 (ITGA2B) or CD42a (GP9) (platelet specific); Glycophorin A (GYPA) (red blood cell specific); CD14 (monocyte-specific), MHC class II (HLA-DR / DP / DQ, H2-A); CD3 (T cell specific); Acetylcholinesterase / AChE-S (neuronal cell specific), AChE-E (red blood cell specific); Amyloid βA4 / APP (neuronal cell specific); These are some examples. Therefore, although not limited to these, proteins that are markers for extracellular vesicles may be used as "membrane proteins that can be expressed on the membrane of extracellular vesicles" or "proteins that can bind to the membrane of extracellular vesicles" in the present invention.

[0068] As used herein, "tetraspanin" refers to proteins belonging to the tetraspanin family (but not limited to these, e.g., CD9, CD53, CD63, CD81, CD82, CD151, etc.). Tetraspanins typically have four transmembrane domains, and both the N-terminus and C-terminus are located on the cytoplasmic side. These domains consist of, from the N-terminus, transmembrane domain 1 (hereinafter also referred to as "TM1"), a small extracellular loop (hereinafter also referred to as "SEL"), transmembrane domain 2 (hereinafter also referred to as "TM2"), a small intracellular loop (hereinafter also referred to as "SIL"), transmembrane domain 3 (hereinafter also referred to as "TM3"), a large extracellular loop (hereinafter also referred to as "LEL"), and transmembrane domain 4 (hereinafter also referred to as "TM4"). For example, if tetraspanin is mouse CD63 (amino acid sequence 1-238: SEQ ID NO: 27), it typically contains TM1, SEL, TM2, SIL, and TM3 in amino acid sequences approximately 1-110, LEL in amino acid sequences approximately 111-200, and TM4 in amino acid sequences approximately 201-238.

[0069] Each domain in the "tetraspanin" described herein (e.g., TM1, SEL, SIL, LTL, etc.) may be derived from the same tetraspanin, or all or part of them may be derived from different tetraspanins.

[0070] The tetraspanins described herein may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, with respect to their wild-type amino acid sequence (for example, in the case of full-length CD9, e.g., SEQ ID NO: 21; in the case of full-length CD63, e.g., SEQ ID NO: 27; in the case of full-length CD81, e.g., SEQ ID NO: 15), as long as they are expressible on the membrane of extracellular vesicles. Alternatively, the tetraspanins described herein may have one or more amino acid deletions, insertions, additions and / or substitutions, with respect to their wild-type amino acid sequence.

[0071] The tetraspanin subsequences described herein (for example, each domain; subsequences containing TM1, SEL, TM2, SIL, and TM3 (for example, SEQ ID NO: 57 for CD63; SEQ ID NO: 61 for CD81); subsequences containing TM4 (for example, SEQ ID NO: 59 for CD63; SEQ ID NO: 63 for CD81)) may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, with respect to their wild-type amino acid sequence. Alternatively, the tetraspanin subsequences described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions with respect to their wild-type amino acid sequence.

[0072] The MFG-E8 described herein may have an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, relative to its wild-type amino acid sequence (e.g., SEQ ID NO: 49), as long as it is capable of binding to the membrane of an extracellular vesicle. Alternatively, the MFG-E8 described herein may have one or more amino acid deletions, insertions, additions and / or substitutions, relative to its wild-type amino acid sequence, as long as it is capable of binding to the membrane of an extracellular vesicle.

[0073] CD58, PTGFRN, etc., as described herein, may have amino acid sequence identity with respect to their wild-type amino acid sequence of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more, as long as they are expressible on the membrane of extracellular vesicles or can bind to the membrane of extracellular vesicles. Alternatively, CD58, PTGFRN, etc., as described herein, may have one or more amino acid deletions, insertions, additions and / or substitutions with respect to their wild-type amino acid sequence, as long as they are expressible on the membrane of extracellular vesicles or can bind to the membrane of extracellular vesicles.

[0074] Spacer array

[0075] As used herein, "spacer sequence" means any sequence having at least one amino acid residue located between two or more proteins or subsequences or domains thereof. Spacer sequences can be used, for example, when linking two or more proteins or subsequences or domains thereof. Spacer sequences include peptide linkers. Spacer sequences typically have a length of 1 to about 50 amino acid residues, preferably about 2 to about 28, and more preferably about 4 to about 25. Spacer sequences are not limited to these, but for example, (GGGXS) n G m (In the formula, X is independently A or G each time it appears, n is 1 to 8, and m is 0 to 3) (e.g., sequence numbers 5, 11, 29, 39, etc.); (GGGS) n G m (In the formula, n is between 1 and 10, and m is between 0 and 3); T a S b (GGX) n G m (In the formula, X is independently S or T each time it appears, n is 1 to 8, m is 0 to 3, a is 0 or 1, and b is 0 or 1) (e.g., sequence number 77); etc.

[0076] Antigen-presenting extracellular vesicles as described herein

[0077] One embodiment of the present invention provides extracellular vesicles that present antigen-presenting MHC molecules and T cell-stimulating cytokines outside the membrane (a model thereof is illustrated in Figure 2J(1)). Such extracellular vesicles may present antigen-presenting MHC molecules and T cell-stimulating cytokines extracellularly by including the proteins specified in (A) and (B) below in their membrane, or they may present antigen-presenting MHC molecules and T cell-stimulating cytokines extracellularly by including the protein specified in (D) in their membrane. Alternatively, antigen-presenting MHC molecules and T-cell-stimulating cytokines may be attached to the membrane surface of isolated extracellular vesicles afterward. The method of attachment is not particularly limited, but antigen-presenting MHC molecules and T-cell-stimulating cytokines may be attached to the membrane surface by binding phospholipids to the antigen-presenting MHC molecules and T-cell-stimulating cytokines, respectively, and incorporating the new lipid portion into the membrane of the extracellular vesicle. Phosphatidylserine is present on the surface of extracellular vesicles. Therefore, by synthesizing and purifying proteins by fusing the antigen-presenting MHC molecule or T-cell-stimulating cytokine to be presented to MFG-E8, which binds to phosphatidylserine, and mixing these fusion proteins with extracellular vesicles, extracellular vesicles that present antigen-presenting MHC molecules and T-cell-stimulating cytokines on their membrane surface can be produced. In addition, antigen-presenting MHC molecules and T-cell-stimulating cytokines with PNE tags may be added to extracellular vesicles that have previously expressed peptide neoepitope (PNE) nanobodies, and then presented on the membrane surface of the extracellular vesicles. Extracellular vesicles expressing streptavidin may be treated by adding biotinylated antigen-presenting MHC molecules and T cell-stimulating cytokines to the membrane surface of the extracellular vesicles.

[0078] In one embodiment of the present invention, the extracellular vesicle may present multiple types (2, 3, 4, or 5 types) of antigen-presenting MHC molecules and multiple types (2, 3, 4, or 5 types) of T-cell stimulating cytokines (hereinafter, these may be referred to as the first T-cell stimulating cytokine, the second T-cell stimulating cytokine, and so on, in order to distinguish each T-cell stimulating cytokine). Alternatively, the extracellular vesicle may present one type of antigen-presenting MHC molecule and multiple types of T-cell stimulating cytokines (Figure 2J(3) illustrates a model of an extracellular vesicle presenting one type of antigen-presenting MHC molecule and two types of T-cell stimulating cytokines).

[0079] In one embodiment of the present invention, an antigen-presenting extracellular vesicle is provided, the membrane of which has the following: (A) A protein containing an antigen-presenting MHC molecule that is capable of presenting the antigen outside the membrane; and (B) A protein comprising a first T cell-stimulating cytokine, capable of presenting the first T cell-stimulating cytokine extramembrane; It provides antigen-presenting extracellular vesicles.

[0080] Configuration requirements (A) The "protein containing an antigen-presenting MHC molecule and capable of presenting the antigen outside the membrane" in (A) above may include other proteins or their domains in addition to the antigen-presenting MHC molecule, as long as it is a protein capable of presenting the antigen outside the membrane of an extracellular vesicle.

[0081] In one embodiment of the present invention, (A) is a fusion protein or protein complex capable of presenting the antigen outside the membrane, comprising an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle.

[0082] In one embodiment of the present invention, (A) is a fusion protein or protein complex capable of presenting the antigen outside the membrane, comprising an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain.

[0083] In one embodiment of the present invention, (A) is, (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein comprising an amino acid sequence consisting of, (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β2 microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of (A-6) β2 microglobulin, MHC class Iα chain, MHC class IIβ chain, or MHC class IIα chain protein complex That is the case.

[0084] In one embodiment of the present invention, (A) is, (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin This is a fusion protein that contains an amino acid sequence consisting of the above and is capable of presenting the antigen peptide outside the membrane. Furthermore, in one embodiment of the present invention, (A) above is (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class II molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin This is a fusion protein that contains an amino acid sequence consisting of the above and is capable of presenting the antigen peptide outside the membrane.

[0085] In one embodiment of the present invention, (A) is, (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) β2 microglobulin, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of the MHC class Iα chain It is a protein complex that includes [the specified element]. Furthermore, in one embodiment of the present invention, (A) above is (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Protein containing the amino acid sequence of β2 microglobulin It is a protein complex that includes [the specified element]. Furthermore, in one embodiment of the present invention, (A) above is (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of the MHC class IIα chain It is a protein complex that includes [the specified element]. Furthermore, in one embodiment of the present invention, (A) above is (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIα chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of MHC class II β chains It is a protein complex that includes [the specified element].

[0086] In one embodiment of the present invention, if (A-3) is a "single-chain MHC class I molecule", the "single-chain MHC class I molecule" consists of, from the N-terminus, β2 microglobulin (for example, SEQ ID NO: 7, or a molecule with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), an optional spacer sequence (if present, for example, SEQ ID NOs: 5, 11, 29, 39, 77, etc.), and an MHC class I α chain (for example, SEQ ID NO: 9, or a molecule with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more). In one embodiment of the present invention, when (A-3) is a "single-chain MHC class I molecule", the "single-chain MHC class I molecule" is SEQ ID NO: 65, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0087] In one embodiment of the present invention, if (A-3) is a "single-chain MHC class II molecule", the "single-chain MHC class II molecule" consists of an MHC class II β chain, an optional spacer sequence, and an MHC class II α chain, starting from the N-terminus.

[0088] In one embodiment of the present invention, if (A-3) and / or (A-6) above is "β2 microglobulin", then "β2 microglobulin" is sequence number 7, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0089] In one embodiment of the present invention, if (A-3) and / or (A-6) above is an "MHC class Iα chain", the "MHC class Iα chain" is sequence number 9, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0090] In one embodiment of the present invention, if (A-3) and / or (A-6) above is an "MHC class IIβ chain", the "MHC class IIβ chain" is sequence number 37, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0091] In one embodiment of the present invention, if (A-3) and / or (A-6) above is an "MHC class IIα chain", the "MHC class IIα chain" is sequence number 71, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0092] The "optional spacer arrays" of (A-2) and (A-4) in each of the above embodiments can be independently selected if they exist. If (A-2) exists, it may be a spacer array such as sequence numbers 5, 11, 29, 39, 77, etc. If (A-4) exists, it may be a spacer array such as sequence numbers 5, 11, 29, 39, 77, etc.

[0093] In one embodiment of the present invention, the tetraspanin of (A-5) in each of the above embodiments is selected from the group consisting of CD9, CD63, and CD81. In one embodiment of the present invention, the tetraspanin of (A-5) in each of the above embodiments is CD81 (preferably SEQ ID NO: 15, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0094] In one embodiment of the present invention, (A) is, (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) It is a fusion protein consisting of the amino acid sequence.

[0095] In one embodiment of the present invention, (A) is, (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) It is a protein complex that includes [the specified element].

[0096] Configuration requirements (B) The "protein capable of presenting the first T cell-stimulating cytokine outside the membrane, which contains the first T cell-stimulating cytokine" in (B) above may contain other proteins or their domains, etc. in addition to the first T cell-stimulating cytokine, as long as it is a protein capable of presenting the first T cell-stimulating cytokine outside the membrane.

[0097] In one embodiment of the present invention, (B) above is a fusion protein capable of presenting the first T cell-stimulating cytokine outside the membrane, which contains the first T cell-stimulating cytokine 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.

[0098] In one embodiment of the present invention, (B) above is (B) A fusion protein capable of presenting the first T cell-stimulating cytokine outside the membrane, which contains the first T cell-stimulating cytokine and a partial sequence of tetraspanin, wherein the partial sequence of tetraspanin has at least two transmembrane domains, and the first T cell-stimulating cytokine is arranged between the two transmembrane domains, or (B) A fusion protein capable of presenting the first T cell-stimulating cytokine outside the membrane, which contains the first T cell-stimulating cytokine and MFG-E8 or its domain.

[0099] As used herein, "the partial sequence of tetraspanin has at least two transmembrane domains, and the first T cell-stimulating cytokine 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 T cell-stimulating cytokine is arranged between TM1 and TM2, or when the partial sequence of tetraspanin contains at least TM3 and TM4 of tetraspanin, and the first T cell-stimulating cytokine is arranged between TM3 and TM4.

[0100] In one embodiment of the present invention, (B) above is (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin containing transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3, from the N-terminal side, (B-2) An optional spacer sequence, (B-3) The first T cell-stimulating cytokine, (B-4) An optional spacer sequence, and (B-5) A partial sequence of tetraspanin containing transmembrane domain 4 A fusion protein containing an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine outside the membrane, or (B) From the N-terminal side, (B-3) The first T cell-stimulating cytokine, (B-4) An optional spacer sequence, and (B-5) MFG-E8 A fusion protein containing an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine outside the membrane.

[0101] 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 entirely or partially replaced with a different amino acid sequence. Therefore, the first T cell-stimulating cytokine in (B-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 in a partial sequence thereof.

[0102] The "sub-sequence of tetraspanin including transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (B-1) usually does not include the transmembrane domain 4 of tetraspanin. The "sub-sequence of tetraspanin including transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (B-1) may also include a large extracellular loop or a sub-sequence thereof. In (B-1), each of the domains, transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3, may be sequences derived from different tetraspanins, or they may all be sequences derived from the same tetraspanin. Preferably, in (B-1), each of the domains, transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3, are all sequences derived from the same tetraspanin.

[0103] In one embodiment of the present invention, the partial sequence of tetraspanin in (B-1), which includes transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, is entirely derived from CD9, CD63, or CD81. In one embodiment of the present invention, the partial sequence of tetraspanin in (B-1), which includes transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, is entirely derived from CD63 or CD81 (preferably SEQ ID NO: 57 or SEQ ID NO: 61, or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0104] The "sub-sequence of tetraspanin containing transmembrane domain 4" in (B-5) typically does not include the transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 of tetraspanin. The "sub-sequence of tetraspanin containing transmembrane domain 4" in (B-5) may include a large extracellular loop or a sub-sequence thereof. The transmembrane domain 4 in (B-5) may be a sequence derived from a different tetraspanin than that in (B-1), or it may be a sequence derived from the same tetraspanin as in (B-1). Preferably, the transmembrane domain 4 in (B-5) is a sequence derived from the same tetraspanin as in (B-1). In one embodiment of the present invention, the sub-sequence of tetraspanin containing transmembrane domain 4 in (B-5) is a sub-sequence derived from CD9, CD63, or CD81. In one embodiment of the present invention, the partial sequence of the tetraspanin containing the transmembrane domain 4 of (B-5) is a partial sequence derived from CD63 or CD81 (preferably SEQ ID NO: 59 or SEQ ID NO: 63, or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0105] In one embodiment of the present invention, the "sub-sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (B-1) is a sub-sequence derived from CD63 (preferably SEQ ID NO: 57, or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) of the same, and the "sub-sequence of tetraspanin comprising transmembrane domain 4" of (B-5) is a sub-sequence derived from CD63 (preferably SEQ ID NO: 59, or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more). In one embodiment of the present invention, the "sub-sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (B-1) is a sub-sequence derived from CD81 (preferably SEQ ID NO: 61, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) of the same, and the "sub-sequence of tetraspanin comprising transmembrane domain 4" of (B-5) is a sub-sequence derived from CD81 (preferably SEQ ID NO: 63, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0106] If the fusion protein in (B) above is a fusion protein containing a partial sequence of tetraspanin, and one or more of the (A) and (C) present in the cases described later contain a fusion protein containing the amino acid sequence of tetraspanin, then the fusion protein in (B) above may be a separate fusion protein from the fusion protein in (A) and / or (C) present in the cases described later, or it may constitute a part of the fusion protein in (A) and / or (C) present in the cases described later. When the fusion protein in (B) above "constitutes a part of the fusion protein in (A) and / or (C) present in the cases described later", examples include cases where the tetraspanin in (A-5) constitutes the fusion protein in (B), and / or cases where the tetraspanin in (C-3) present in the cases described later constitutes the fusion protein in (B).

[0107] The "MFG-E8" in (B-5) above is preferably SEQ ID NO: 49, or one with an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0108] In one embodiment of the present invention, the first T cell stimulating cytokine in (B-3) of each embodiment is IL-2, IL-4, IL-6, IL-12, or TGF-β.

[0109] The "optional spacer arrays" in (B-2) and (B-4) of each of the above embodiments can be independently selected if they exist. If (B-2) exists, it may be a spacer array such as sequence numbers 5, 11, 29, 39, 77, etc. If (B-4) exists, it may be a spacer array such as sequence numbers 5, 11, 29, 39, 77, etc.

[0110] In one embodiment of the present invention, (B) above is (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) This is a fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, comprising the amino acid sequence shown.

[0111] In one embodiment of the present invention, (B) is a fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, which is sequence number 31 (or a protein with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0112] In one embodiment of the present invention, the above (B) is (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 61 (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), (B-2) The spacer sequence of SEQ ID NO: 29, (B-3) The first T cell-stimulating cytokine which is IL-4 of SEQ ID NO: 53 (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), (B-4) The spacer sequence of SEQ ID NO: 29, and (B-5) A partial sequence of tetraspanin of SEQ ID NO: 63 (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) is an amino acid sequence consisting of, and is a fusion protein capable of presenting the first T cell-stimulating cytokine outside the membrane.

[0113] In one embodiment of the present invention, the above (B) is a fusion protein of SEQ ID NO: 55 (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) capable of presenting the first T cell-stimulating cytokine outside the membrane.

[0114] In one embodiment of the present invention, the above (B) is (B) From the N-terminal side, (B-3) The first T cell-stimulating cytokine which is TGF-β of SEQ ID NO: 73 (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), (B-4) The spacer sequence of SEQ ID NO: 29, and (B-5) MFG-E8 of SEQ ID NO: 49 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) This is a fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, comprising the amino acid sequence shown.

[0115] In one embodiment of the present invention, (B) is a fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, which is sequence number 75 (or a protein with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0116] Second (or more) T-cell stimulating cytokines The antigen-presenting extracellular vesicles described herein may further comprise a second (or more) T-cell stimulating cytokine in addition to the first T-cell stimulating cytokine. Therefore, in one embodiment of the present invention, the antigen-presenting extracellular vesicles described herein further comprise a second T-cell stimulating cytokine. In particular, if the antigen-presenting MHC molecule is an antigen-presenting MHC class II molecule, the antigen-presenting extracellular vesicles described herein preferably comprise a second T-cell stimulating cytokine.

[0117] The second (or more) T cell-stimulating cytokine may, for example, be inserted into (B) above (for example, the second (or more) T cell-stimulating cytokine may be ligated to the N-terminal and / or C-terminal side of the "first T cell-stimulating cytokine" in (B-3) via a spacer sequence or the like, as necessary). Alternatively, the second (or more) T cell-stimulating cytokine may be contained in the membrane of the antigen-presenting extracellular vesicle described herein as a separate protein (or fusion protein) from the protein (or fusion protein) of component (B) described herein, having a structure similar to that of component (B) described herein, similar to the first T cell-stimulating cytokine.

[0118] In one embodiment of the present invention, the second T cell-stimulating cytokine is IL-2, IL-4, IL-6, IL-12, or TGF-β. In one embodiment of the present invention, the second T cell-stimulating cytokine is TGF-β (preferably SEQ ID NO: 73, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0119] In one embodiment of the present invention, the first T cell stimulating cytokine is IL-2 or IL-4 (preferably SEQ ID NO: 25 or SEQ ID NO: 53, or a cytokine with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and the second T cell stimulating cytokine is TGF-β (preferably SEQ ID NO: 73, or a cytokine with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0120] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein or protein complex capable of presenting the antigen extracellularly, comprising an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; and (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extracellularly, comprising a first T cell-stimulating cytokine and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; It is an extracellular vesicle that presents antigens.

[0121] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein or protein complex capable of presenting the antigen extramembrane, comprising an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain; and (B) A fusion protein comprising a first T cell stimulating cytokine and a sub-sequence of tetraspanin, wherein the sub-sequence of tetraspanin has at least two transmembrane domains, and the first T cell stimulating cytokine is positioned between the two transmembrane domains, or (B) A fusion protein comprising a first T cell-stimulating cytokine and MFG-E8 or its domain, capable of presenting the first T cell-stimulating cytokine extramembrane; It is an extracellular vesicle that presents antigens.

[0122] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein comprising an amino acid sequence consisting of, (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β2 microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of (A-6) β2 microglobulin, MHC class Iα chain, MHC class IIβ chain, or MHC class IIα chain Protein complexes including; and (B) From the N-terminal side, (B-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, from the N-terminus. (B-2) A spacer arrangement that may exist, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may exist, and (B-5) Partial sequence of tetraspanin containing transmembrane domain 4 A fusion protein comprising an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane, or (B) From the N-terminal side, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may be present, and (B-5)MFG-E8 A fusion protein comprising an amino acid sequence comprising the above, capable of presenting the first T cell-stimulating cytokine extramembrane; It is an extracellular vesicle that presents antigens.

[0123] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of the following: It is an extracellular vesicle that presents antigens.

[0124] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of the MHC class IIα chain A protein complex containing; It is an extracellular vesicle that presents antigens.

[0125] One embodiment of the present invention provides an antigen-presenting extracellular vesicle as described herein, wherein the first T cell-stimulating cytokine is IL-2, IL-4, IL-6, IL-12, or TGF-β.

[0126] In one embodiment of the present invention, the extracellular vesicle described herein further presents T cell costimulatory molecules outside its membrane (a model thereof is illustrated in Figure 2J(2)). Such extracellular vesicles may present T cell costimulatory molecules extracellularly by including the protein specified in (C) below in their membrane. Alternatively, T cell costimulatory molecules may be attached to the membrane surface of isolated extracellular vesicles afterward. The method of attachment is not particularly limited, but antigen-presenting MHC molecules and T cell-stimulating cytokines may be attached to the membrane surface by binding phospholipids to each T cell costimulatory molecule and having the new lipid portion incorporated into the membrane of the extracellular vesicle. In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (C) A protein containing a T cell costimulatory molecule, which is capable of interacting with the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0127] Configuration requirements (C) The "protein containing a T cell costimulatory molecule, which is capable of interacting with the T cell costimulatory molecule and T cells" in (C) above may include other proteins or their domains, in addition to the T cell costimulatory molecule, as long as it is a protein capable of interacting with the T cell costimulatory molecule and T cells.

[0128] In one embodiment of the present invention, (C) is a fusion protein that can interact with T cells, comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle.

[0129] In one embodiment of the present invention, (C) is a fusion protein that can interact with a T cell, comprising a T cell costimulatory molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain.

[0130] In one embodiment of the present invention, (C) above is, (C) From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanine This is a fusion protein containing an amino acid sequence comprising the above, which allows interaction between the T cell costimulatory molecule and T cells.

[0131] In one embodiment of the present invention, the T cell costimulatory molecule in (C-1) is CD80 or CD86. In one embodiment of the present invention, the T cell costimulatory molecule in (C-1) is CD80 (preferably SEQ ID NO: 67, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0132] The "optional spacer sequence" in (C-2) above may be, if present, a spacer sequence such as sequence numbers 5, 11, 29, 39, 77, etc.

[0133] In one embodiment of the present invention, the tetraspanin in (C-3) is selected from the group consisting of CD9, CD63, and CD81. In one embodiment of the present invention, the tetraspanin in (C-3) is CD9 (preferably SEQ ID NO: 21, or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more).

[0134] In one embodiment of the present invention, (C) above is, (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) This is a fusion protein consisting of an amino acid sequence, which allows the T cell costimulatory molecule to interact with T cells.

[0135] In one embodiment of the present invention, (C) is a fusion protein of Sequence ID No. 69 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) that can interact with the T cell costimulatory molecule and T cells.

[0136] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of; and (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) The first T cell stimulating cytokine is IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane; It is an extracellular vesicle that presents antigens.

[0137] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of; and (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, corresponding to Sequence ID No. 31 (or a protein with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more); It is an extracellular vesicle that presents antigens.

[0138] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein consisting of an amino acid sequence; (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) The first T cell stimulating cytokine is IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane; and (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, which allows interaction between the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0139] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein consisting of an amino acid sequence; (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, such as SEQ ID NO: 31 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith); and (C) A fusion protein of Sequence ID No. 69 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith) that can interact with the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0140] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane; and (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, which allows interaction between the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0141] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, such as SEQ ID NO: 31 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith); and (C) A fusion protein of Sequence ID No. 69 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith) that can interact with the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0142] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) The first T cell stimulating cytokine is IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane; (B') Starting from the N-terminus, (B-3) TGF-β of sequence number 73 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is a second T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) MFG-E8 of SEQ ID NO: 49 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, capable of presenting the second T cell-stimulating cytokine extramembrane; and (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, which allows interaction between the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0143] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, corresponding to Sequence ID No. 31 (or a protein with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more); (B') A fusion protein capable of presenting the second T cell-stimulating cytokine extramembrane, corresponding to Sequence ID No. 75 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more); and (C) A fusion protein of Sequence ID No. 69 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith) that can interact with the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0144] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 61 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) IL-4 of SEQ ID NO: 53 (or IL-4 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 63 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane; and (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, which allows interaction between the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0145] In one embodiment of the present invention, an antigen-presenting extracellular vesicle described herein, wherein its membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein and an amino acid sequence consisting of (A-6) MHC class IIα chain of SEQ ID NO: 71 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A protein complex consisting of; (B) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, corresponding to Sequence ID No. 55 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more); and (C) A fusion protein of Sequence ID No. 69 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith) that can interact with the T cell costimulatory molecule and T cells; It is an extracellular vesicle that presents antigens.

[0146] In one embodiment of the present invention, (A), (B), and (C) may be fused together to form one molecule, (B) and (C) may be fused together to form one molecule, or (A), (B), and (C) may be fused together to form one molecule. Such a fused molecule may be translated as a single protein molecule with or without spacer sequences between (A), (B), and (C), or the proteins of (A), (B), and (C) may be fused together to form a single molecule by chemical cross-linking (for example, by disulfide bonds between cysteine ​​residues). Alternatively, (A), (B), and (C) above may be functionally fused by sharing a portion of the element necessary for the protein to localize to an extracellular vesicle, namely, a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. For example, in one embodiment of the present invention, (A) and (B) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) at least one type of T cell stimulating cytokine; and (3) A fusion protein (D) may include "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (A) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) T cell costimulatory molecules; and (3) A fusion protein (F) may include "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (B) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) at least one type of T cell-stimulating cytokine; (2) T cell costimulatory molecules; and (3) A fusion protein (G) may include "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; or (A) to (C) are fused in a manner that they share a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle". (1) Antigen-presenting MHC molecules; (2) at least one type of T cell-stimulating cytokine; (3) T cell costimulatory molecules; and (4) The fusion protein (E) may include "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle."

[0147] In one embodiment of the present invention, instead of constituent element (A) "a membrane protein that can be expressed on the membrane of an extracellular vesicle or its transmembrane domain or a protein that can bind to the membrane of an extracellular vesicle", constituent element (B) "a protein containing a first T cell-stimulating cytokine and capable of presenting the first T cell-stimulating cytokine outside the membrane" may be used, resulting in an antigen-presenting extracellular vesicle cell containing a fusion protein (D) that possesses the functions of constituent element (A) and constituent element (B).

[0148] A fusion protein (D) possessing the functions of constituent element (A) and constituent element (B) is, (D) A fusion protein comprising an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine, which is capable of presenting the antigen and the T cell-stimulating cytokine extramembrane. The fusion protein may include the antigen-presenting MHC molecule, at least one T cell-stimulating cytokine, and a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle.

[0149] In a fusion protein (D) possessing the functions of constituent element (A) and constituent element (B), 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 may be tetraspanin or MFG-E8. The aforementioned fusion protein, from the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may exist, (D-5) The amino acid sequence may include an amino acid sequence encoding a fusion peptide containing (D-5) tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one of the T cell-stimulating cytokines, in this order. The aforementioned fusion protein, from the N-terminus, (D-1) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one of the aforementioned T cell-stimulating cytokines. (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, It may also include amino acid sequences that code in this order. Here, the fusion peptide is, from the N-terminus, (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) A spacer array that may exist, (3) at least one T cell stimulating cytokine, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 It may also include an amino acid sequence that codes for these in this order. The aforementioned fusion peptide, from the N-terminus, (1) At least one T cell stimulating cytokine as described above, (2) A spacer array which may exist, and (3) MFG-E8 It may also include an amino acid sequence that codes for these in this order.

[0150] In one embodiment of the present invention, the MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of an MHC class Iα chain, or the MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of an MHC class IIα chain and / or the extracellular domain of an MHC class IIβ chain.

[0151] In an embodiment comprising a fusion protein (D) having the functions of constituent element (A) and constituent element (B); (C) The membrane may further contain at least one T cell costimulatory molecule, and a protein that can interact with the T cell and the T cell; The protein capable of interacting with the T cell may include at least one T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; The protein capable of interacting with the T cell may include at least one T cell costimulatory molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain.

[0152] In one embodiment of the present invention, the extracellular vesicle is an exosome.

[0153] The antigen-presenting extracellular vesicles described herein may contain or bind therapeutically beneficial substances (e.g., small molecule compounds, nucleic acids, etc.) within or within their membrane. Methods for encapsulating such substances within the membrane of the extracellular vesicles are not limited to those described herein, but include, for example, mixing the substance with the extracellular vesicles described herein in a suitable solvent. In one embodiment of the present invention, the antigen-presenting extracellular vesicle may contain any protein preparation. The protein preparation is not particularly limited, but may be a naturally occurring protein such as erythropoietin, a synthetic protein that does not exist in nature such as immunoglobulin-CTLA4 fusion protein, or a monoclonal antibody or its active fragment. These protein preparations may be localized to the surface of the antigen-presenting extracellular vesicle as a fusion protein with a membrane protein or its transmembrane domain that can localize to the membrane of the extracellular vesicle, or a protein or its membrane-binding domain that can bind to the membrane of the extracellular vesicle. Such an antigen-presenting extracellular vesicle can be produced by transfecting cells that produce antigen-presenting extracellular vesicles with a vector for expressing the fusion protein.

[0154] Each fusion protein, protein complex, or protein preparation contained within the membrane of an antigen-presenting extracellular vesicle described herein may contain one or more detectable labels. For example, the fusion protein, protein complex, or protein preparation may be labeled by conventional methods with a specific reporter molecule, fluorophore, radiomaterial, or enzyme (e.g., peroxidase, phosphatase). These may be linked to the N-terminal or C-terminal side of the fusion protein, protein complex, or protein preparation, for example, as components of the said fusion protein, protein complex, or protein preparation.

[0155] Polynucleotides

[0156] One embodiment of the present invention provides polynucleotides encoding each fusion protein or protein complex in (A) and (B), and optionally present in (C), contained within the membrane of an antigen-presenting extracellular vesicle as described herein. One embodiment of the present invention provides polynucleotides encoding each fusion protein or protein complex in (A) to (G) as defined herein.

[0157] In one embodiment of the present invention, (a) A sequence encoding a fusion protein (A) that includes an antigen-presenting MHC molecule and is capable of presenting the antigen-presenting MHC molecule outside the membrane of an extracellular vesicle; (b) A sequence encoding a fusion protein (B) capable of presenting the T cell-stimulating cytokine (B) outside the membrane of an extracellular vesicle, comprising at least one T cell-stimulating cytokine or a subunit thereof; (c) A sequence encoding a fusion protein (C) that includes a T cell costimulatory molecule and is capable of presenting the T cell costimulatory molecule outside the membrane of an extracellular vesicle; (d) A sequence encoding a fusion protein (D) that comprises an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine or its subunit, and is capable of presenting the antigen and the T cell-stimulating cytokine extramembrane; and (e) A sequence encoding a fusion protein (E) that comprises an antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or its subunits, and a T cell-costimulating molecule, and is capable of presenting the antigen, the T cell-stimulating cytokine, and the T cell-costimulating molecule extramembrane; A polynucleotide containing at least one sequence selected from the group consisting of the following: To provide. The above sequences (a) to (e) include sequences specifically described in this specification and sequences with high homology (preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology), but are not particularly limited. Paralogs (gene sequences resulting from gene duplication) and orthologs (groups of genes with homologous functions existing in different organisms) may also be used as long as they have equivalent function, and sequences with altered sequence information (such as non-duplication, deletion, or substitution) are also included.

[0158] As used herein, "polynucleotide" means single-stranded or double-stranded DNA molecules or RNA molecules, etc. Polynucleotides include genomic DNA, cDNA, hnRNA, mRNA, etc., and all naturally occurring or artificially modified derivatives thereof. Polynucleotides may be in chain form or circular form.

[0159] The polynucleotides encoding each fusion protein or protein complex in (A) to (G) described above can be appropriately determined by a person skilled in the art by referring to the amino acid sequence of the fusion protein or protein complex. The amino acid sequences of each fusion protein or protein complex in (A) to (G) can be appropriately determined by referring to the amino acid sequences of each component of the fusion protein or protein complex (for example, in the case of (A), (A-1) to (A-5), and optionally (A-6)). Any type of codon can be selected when determining the polynucleotide. For example, the polynucleotide may be determined by considering the frequency of codons in cells to be transformed using a vector containing the polynucleotide.

[0160] In the polynucleotides encoding each fusion protein or protein complex described above in (A) to (G), a polynucleotide encoding a signal peptide (signal sequence) may be added to the N-terminus as needed.

[0161] The amino acid sequence of the signal peptide can be any sequence, and may be determined by considering, for example, the amino acid sequence of the fusion protein to be expressed. Examples of polynucleotides encoding the signal peptide include a polynucleotide encoding the β2 microglobulin signal peptide (e.g., SEQ ID NO: 1) (e.g., SEQ ID NO: 2), a polynucleotide encoding the MHC class I α chain signal peptide, a polynucleotide encoding the MHC class II α chain signal peptide, and a polynucleotide encoding the MHC class II β chain signal peptide (e.g., SEQ ID NO: 33) (e.g., SEQ ID NO: 34).

[0162] Information on each component of each fusion protein or protein complex in (A) to (G) above (for example, in the case of (A), (A-1) to (A-5), and (A-6) if present), as well as the amino acid sequences of signal peptides, etc., and the polynucleotides encoding them, may be obtained as appropriate by searching, for example, known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). In addition, for the amino acid sequences of partial sequences of tetraspanins (for example, partial sequences in (C-1) and (C-5)) and the polynucleotides encoding them, referencing International Publication No. 2016 / 139354 may be used.

[0163] In one embodiment of the present invention, a polynucleotide is, hereafter: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC molecule, (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A) A fusion protein constituting a protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β2 microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) Spacer arrangements that may exist, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of the following: (B) From the N-terminal side, (B-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, from the N-terminus. (B-2) A spacer arrangement that may exist, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may exist, and (B-5) Partial sequence of tetraspanin containing transmembrane domain 4 A fusion protein comprising an amino acid sequence consisting of the above, capable of presenting the first T cell-stimulating cytokine extramembrane, or (B) From the N-terminal side, (B-3) The first T cell stimulating cytokine, (B-4) A spacer arrangement that may be present, and (B-5)MFG-E8 A fusion protein comprising an amino acid sequence comprising the above, capable of presenting the first T cell-stimulating cytokine extramembrane; or (C) From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanine A fusion protein comprising an amino acid sequence comprising the above, which is capable of interacting with the T cell costimulatory molecule and T cells; It is a polynucleotide that codes for one of the following:

[0164] In one embodiment of the present invention, a polynucleotide is, hereafter: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein, or an amino acid sequence consisting of the above. (A) A fusion protein constituting a protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein consisting of an amino acid sequence; (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 57 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) IL-2 of SEQ ID NO: 25 (or IL-2 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 59 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, having an amino acid sequence consisting of the above, (B) From the N-terminal side, (B-1) A partial sequence of tetraspanin of SEQ ID NO: 61 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 29, (B-3) IL-4 of SEQ ID NO: 53 (or IL-4 with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 63 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein capable of presenting the first T cell-stimulating cytokine extramembrane, having an amino acid sequence consisting of the above, or (B') Starting from the N-terminus, (B-3) TGF-β of sequence number 73 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), which is a second T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 29, and (B-5) MFG-E8 of SEQ ID NO: 49 (or a sequence with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein capable of presenting the second (or first) T cell-stimulating cytokine extramembrane, having an amino acid sequence consisting of the following; or (C) From the N-terminus, (C-1) CD80 of SEQ ID NO: 67 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 21 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein having an amino acid sequence consisting of the following, which allows interaction between the T cell costimulatory molecule and T cells; Provides a polynucleotide that codes for any of the following.

[0165] In one embodiment of the present invention, a polynucleotide is, hereafter: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminus, (A-1) MHC class I molecule-restricted antigen peptide, (A-2) Spacer arrangement of sequence number 5, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 65 (or those with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or a tetraspanin with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A fusion protein, or an amino acid sequence consisting of the above. (A) A fusion protein constituting a protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 39, (A-3) MHC class IIβ chain of SEQ ID NO: 37 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 15 (or one with amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A fusion protein consisting of an amino acid sequence; (B) A fusion protein capable of presenting the first (or second) T cell-stimulating cytokine extramembrane, such as SEQ ID NOs. 31, 75, or 55 (or having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith); or (C) A fusion protein of Sequence ID No. 23 (or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence identity therewith) that can interact with the T cell costimulatory molecule and T cells; Provides a polynucleotide that codes for any of the following.

[0166] In one embodiment of the present invention, a polynucleotide is, hereafter: (A) A polynucleotide encoding a fusion protein capable of presenting an antigen peptide outside the membrane, (A-1) Polynucleotide encoding an MHC class I molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 6, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 66 (or those with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 16 (or a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A polynucleotide, or a sequence consisting of (A) A polynucleotide encoding a fusion protein that constitutes a protein complex capable of presenting an antigen peptide outside the membrane, (A-1) Polynucleotide encoding an MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 40, (A-3) MHC class IIβ chain of SEQ ID NO: 38 (or a sequence identity therewith of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 16 (or a tetraspanin with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A polynucleotide, which is a sequence consisting of the following: (B) A polynucleotide encoding a fusion protein capable of presenting a first T cell-stimulating cytokine extramembrane, (B-1) A partial sequence of tetraspanin of sequence number 58 (or a sequence with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 30, (B-3) The first T cell stimulating cytokine is IL-2 of SEQ ID NO: 26 (or a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more). (B-4) Spacer arrangement of sequence number 30, and (B-5) Partial sequence of tetraspanin of SEQ ID NO: 60 (or a sequence with 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A polynucleotide, which is a sequence consisting of (B) A polynucleotide encoding a fusion protein capable of presenting a first T cell-stimulating cytokine extramembrane, (B-1) A partial sequence of tetraspanin of sequence number 62 (or a sequence with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-2) Spacer arrangement of sequence number 30, (B-3) IL-4 of sequence number 54 (or a sequence with 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) is the first T cell stimulating cytokine. (B-4) Spacer arrangement of sequence number 30, and (B-5) A partial sequence of the tetraspanin of SEQ ID NO: 64 (or a sequence with 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity); A polynucleotide, or a sequence consisting of (B') A polynucleotide encoding a fusion protein capable of presenting a second (or first) T cell-stimulating cytokine extramembrane, (B-3) A second (or first) T cell stimulating cytokine, which is TGF-β of sequence number 74 (or one with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), (B-4) Spacer arrangement of sequence number 30, and (B-5) MFG-E8 of sequence number 50 (or a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A polynucleotide, which is a sequence consisting of; or (C) A polynucleotide encoding a fusion protein that can interact with the T cell costimulatory molecule and T cells, (C-1) CD80 of SEQ ID NO: 68 (or one with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), a T cell costimulatory molecule, and (C-3) Tetraspanin of SEQ ID NO: 22 (or a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A polynucleotide, which is a sequence consisting of the following: To provide.

[0167] In one embodiment of the present invention, a polynucleotide is, hereafter: (A) A polynucleotide encoding a fusion protein capable of presenting an antigen peptide outside the membrane, (A-1) Polynucleotide encoding an MHC class I molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 6, (A-3) Single-chain MHC class I molecules of SEQ ID NO: 66 (or those with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 16 (or a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) A polynucleotide, or a sequence consisting of (A) A polynucleotide encoding a fusion protein that constitutes a protein complex capable of presenting an antigen peptide outside the membrane, (A-1) Polynucleotide encoding an MHC class II molecularly restricted antigen peptide, (A-2) Spacer arrangement of sequence number 40, (A-3) MHC class IIβ chain of SEQ ID NO: 38 (or a sequence identity therewith of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), and (A-5) Tetraspanin of SEQ ID NO: 16 (or a tetraspanin with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more), A polynucleotide, which is a sequence consisting of the following: (B) A polynucleotide encoding a fusion protein capable of presenting the first (or second) T cell-stimulating cytokine extramembrane, such as SEQ ID NOs. 32, 76, or 56 (or any of the following with sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more); or (C) A polynucleotide encoding a fusion protein capable of interacting with the T cell costimulatory molecule and T cells, which is sequence identity with respect to Sequence ID No. 24 (or a sequence identity with respect to Sequence ID No. 24 of 80%, preferably 90%, more preferably 95%, even more preferably 98%, and even more preferably 99%); To provide.

[0168] As one embodiment of the present invention, The fusion protein defined in (D) above is The present invention provides a polynucleotide comprising the antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or its subunit, and a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle. 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 may be tetraspanin or MFG-E8.

[0169] As one embodiment of the present invention, The fusion protein defined in (D) above is From the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one T cell stimulating cytokine or its subunit, A polynucleotide comprising an amino acid sequence encoding in this order (where each component (D-1) to (D-5) includes the embodiments described herein); or The fusion protein defined in (D) above is From the N-terminus, (D-1) A fusion peptide comprising a tetraspanin or its transmembrane domain, or MFG-E8 or its transmembrane domain, and at least one of the T cell-stimulating cytokines or its subunits. (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, We provide a polynucleotide comprising an amino acid sequence encoding in this order (where each component (D-1) to (D-5) includes the embodiments described herein). In this embodiment, the fusion peptide comprising the tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and at least one T cell-stimulating cytokine or its subunit is From the N-terminus, (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) A spacer array that may exist, (3) at least one T cell stimulating cytokine or its subunit, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 It may also contain an amino acid sequence that codes in this order; or The fusion peptide comprising the aforementioned tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, From the N-terminus, (1) at least one T cell stimulating cytokine or its subunit, (2) A spacer array which may exist, and (3) MFG-E8 It may also contain an amino acid sequence that codes for these in this order. Here, the MHC molecule-restricted antigen peptide may be an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of the MHC class Iα chain; or the MHC molecule-restricted antigen peptide may be an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of the MHC class IIα chain and / or the extracellular domain of the MHC class IIβ chain.

[0170] In one embodiment of the present invention, a polynucleotide comprising the sequence defined in (a) and the sequence defined in (b) is provided. Furthermore, it may include the sequence defined in (c) above.

[0171] One embodiment of the present invention, The present invention provides a polynucleotide containing the sequence defined in (d) above. An example of such a sequence is the nucleic acid sequence of sequence number 136, which encodes the amino acid sequence of sequence number 135. In such an embodiment, the sequence may include the sequence defined in (c) above.

[0172] In one embodiment of the present invention, a polynucleotide comprising the sequence defined in (e) above is provided.

[0173] In one embodiment of the present invention, (A), (B), and (C) may be polynucleotides encoding a fusion protein formed by the fusion of (A) and (B) into a single molecule, or (B) and (C) into a single molecule, or (A), (B), and (C) into a single molecule. Such polynucleotides may encode a single fusion protein with or without a spacer sequence between (A), (B), and (C). Alternatively, the polynucleotide in one embodiment of the present invention may encode a functionally fused fusion protein in which (A), (B), and (C) share a portion of the elements necessary for the protein to localize to an extracellular vesicle, namely, a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. For example, in one embodiment of the present invention, (A) and (B) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) at least one type of T cell stimulating cytokine; and (3) A polynucleotide encoding a fusion protein (D) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (A) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) T cell costimulatory molecules; and (3) A polynucleotide encoding a fusion protein (F) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (B) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) at least one type of T cell-stimulating cytokine; (2) T cell costimulatory molecules; and (3) A polynucleotide encoding a fusion protein (G) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; or (A) to (C) are fused in a manner that they share a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle". (1) Antigen-presenting MHC molecules; (2) at least one type of T cell-stimulating cytokine; (2) T cell costimulatory molecules; and (4) A polynucleotide encoding a fusion protein (E) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle."

[0174] One embodiment of the present invention is a fusion protein (D) that possesses the functions of constituent element (A) and constituent element (B), by using a protein that includes the first T cell-stimulating cytokine of constituent element (B) and is capable of presenting the first T cell-stimulating cytokine extramembranely, instead of the membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle of constituent element (A) or a protein that can bind to the membrane of the extracellular vesicle, and replacing it with a membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle of constituent element (A), A fusion protein containing an antigen-presenting MHC molecule and at least one T-cell stimulating cytokine, capable of presenting the antigen and T-cell stimulating cytokine extramembrane. It may be a polynucleotide that codes for [something].

[0175] A fusion protein (D) possessing the functions of constituent element (A) and constituent element (B) is, The fusion protein may contain an antigen-presenting MHC molecule and at least one T-cell stimulating cytokine, and be capable of presenting the antigen and the T-cell stimulating cytokine extramembrane. The fusion protein may include the antigen-presenting MHC molecule, at least one T cell-stimulating cytokine, and a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle.

[0176] In a fusion protein (D) possessing the functions of constituent element (A) and constituent element (B), 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 may be tetraspanin or MFG-E8. The aforementioned fusion protein, from the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may exist, (D-5) The amino acid sequence may include an amino acid sequence encoding a fusion peptide containing (D-5) tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one of the T cell-stimulating cytokines, in this order. The aforementioned fusion protein, from the N-terminus, (D-1) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and at least one of the aforementioned T cell-stimulating cytokines. (D-2) A spacer arrangement that may exist, (D-3) Single-chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, It may also include amino acid sequences that code in this order. Here, the fusion peptide is, from the N-terminus, (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) A spacer array that may exist, (3) at least one T cell stimulating cytokine, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 It may also include an amino acid sequence that codes for these in this order. The aforementioned fusion peptide, from the N-terminus, (1) At least one T cell stimulating cytokine as described above, (2) A spacer array which may exist, and (3) MFG-E8 It may also include an amino acid sequence that codes for these in this order.

[0177] In one embodiment of the present invention, the MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of an MHC class Iα chain, or the MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule may include the extracellular domain of an MHC class IIα chain and / or the extracellular domain of an MHC class IIβ chain.

[0178] In an embodiment comprising a fusion protein (D) having the functions of constituent element (A) and constituent element (B); (C) The membrane may further contain at least one T cell costimulatory molecule, and a protein that can interact with the T cell and the T cell; The protein capable of interacting with the T cell may include at least one T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; The protein capable of interacting with the T cell may include at least one T cell costimulatory molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain.

[0179] Vector, Kit

[0180] One embodiment of the present invention provides a vector comprising at least one polynucleotide selected from the polynucleotides described herein.

[0181] As used herein, "vector" means any vector (but is not limited to, plasmid vectors, cosmid vectors, phage vectors such as phages, viral vectors such as adenovirus vectors and baculovirus vectors, artificial chromosome vectors, etc.). Vectors include expression vectors, cloning vectors, etc. Expression vectors generally contain a desired coding sequence and appropriate polynucleotides necessary for the expression of the operably linked coding sequence in a host organism (e.g., plants, insects, animals, etc.) or in an expression system in vitro. Cloning vectors may be used to manipulate and / or amplify a desired polynucleotide fragment. Cloning vectors may lack functional sequences required for the expression of a desired polynucleotide fragment.

[0182] In one embodiment of the present invention, the polynucleotides described herein may all be inserted into the same vector, or two or more polynucleotides may be inserted into separate vectors, as long as they can be operably inserted. In one embodiment of the present invention, a kit is provided which combines two or more vectors, each containing at least one polynucleotide selected from the polynucleotides described herein.

[0183] transformed cells

[0184] In one embodiment of the present invention, the following applies: (i) Polynucleotides encoding the fusion protein or protein complex of (A) described herein, (ii) a polynucleotide encoding the fusion protein of (B) described herein, or (iii) Polynucleotide encoding the fusion protein of (C) described herein The present invention provides cells transformed with a vector comprising the following components.

[0185] In one embodiment of the present invention, the following applies: (i) polynucleotides encoding the fusion protein or protein complex of (A) as described herein, and (ii) Polynucleotides encoding the fusion protein of (B) as described herein, and optionally (iii) Polynucleotide encoding the fusion protein of (C) described herein The present invention provides cells transformed by a single vector or a combination of two or more vectors comprising the above.

[0186] In one embodiment of the present invention, cells (A), (B), and (C) above may be transformed with a vector containing a polynucleotide encoding a fusion protein formed by the fusion of (A) and (B) into a single molecule, a vector containing a polynucleotide encoding a fusion protein formed by the fusion of (B) and (C) into a single molecule, or a vector containing a polynucleotide encoding a fusion protein formed by the fusion of (A), (B), and (C) into a single molecule. Such polynucleotides may encode a single fusion protein with or without a spacer sequence between (A), (B), and (C). Alternatively, (A), (B), and (C) above may encode a functionally fused fusion protein by sharing a portion of the protein that localizes to an extracellular vesicle, namely, a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. For example, in one embodiment of the present invention, (A) and (B) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) at least one type of T cell stimulating cytokine; and (3) A vector containing a polynucleotide encoding a fusion protein (D) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (A) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) Antigen-presenting MHC molecules; (2) T cell costimulatory molecules; and (3) A vector containing a polynucleotide encoding a fusion protein (F) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; (B) and (C) fused in a manner that they shared a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle." (1) at least one type of T cell-stimulating cytokine; (2) T cell costimulatory molecules; and (3) A vector containing a polynucleotide encoding a fusion protein (G) comprising "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; or (A) to (C) are fused in a manner that they share a portion of either "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle". (1) Antigen-presenting MHC molecules; (2) at least one type of T cell-stimulating cytokine; (3) T cell costimulatory molecules; and (4) A vector comprising a polynucleotide encoding a fusion protein (E) containing "a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle" or "a protein or its domain that can bind to the membrane of an extracellular vesicle"; It's okay if it's been transformed.

[0187] Alternatively, in one embodiment of the present invention, (iv) A fusion protein possessing the functions of both constituent elements (A) and (B), wherein instead of a membrane protein or its transmembrane domain capable of being expressed on the membrane of an extracellular vesicle of constituent element (A), or a protein capable of binding to the membrane of an extracellular vesicle, a protein containing the first T cell-stimulating cytokine of constituent element (B) and capable of presenting the first T cell-stimulating cytokine extramembrane is used. A polynucleotide encoding a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine extramembrane, comprising the antigen-presenting MHC molecule described in (D) herein and at least one T cell-stimulating cytokine. This provides cells transformed with a vector containing the following:

[0188] "Transformed by a single vector or a combination of two or more vectors" means, for example, that cells may be transformed by a vector in which all of the polynucleotides (i) to (iv) above are inserted into the same vector, or by a combination of two or more vectors in which two or more of these are inserted into separate vectors.

[0189] Examples of "a single vector or a combination of two or more vectors" when (A) is a fusion protein include the following: A vector comprising a polynucleotide encoding the fusion protein of (A) and a polynucleotide encoding the fusion protein of (B); A combination of a vector containing a polynucleotide encoding the fusion protein of (A) and a vector containing a polynucleotide encoding the fusion protein of (B); A vector comprising a polynucleotide encoding the fusion protein of (A), a polynucleotide encoding the fusion protein of (B), and a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding the fusion protein of (A) and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding the fusion protein of (A) and a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding the fusion protein of (B) and a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding the fusion protein of (A); or A combination of a vector containing a polynucleotide encoding the fusion protein of (A), a vector containing a polynucleotide encoding the fusion protein of (B), and a vector containing a polynucleotide encoding the fusion protein of (C).

[0190] Alternatively, "a single vector or a combination of two or more vectors" could include, for example, the following when (A) is a protein complex: A vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein containing (A-6), and a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), and a polynucleotide encoding a protein containing (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding a protein containing (A-6); A combination of a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein containing (A-6), and a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein containing (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (B); A vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein containing (A-6), a polynucleotide encoding the fusion protein of (B), and a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), a polynucleotide encoding a protein containing (A-6), and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), a polynucleotide encoding the fusion protein of (B), and a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding a protein containing (A-6); A combination of a vector comprising a polynucleotide encoding a protein containing (A-6), a polynucleotide encoding a fusion protein of (B), and a polynucleotide encoding a fusion protein of (C), and a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), a polynucleotide encoding a protein containing (A-6), and a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5) and a polynucleotide encoding a protein containing (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (B) and a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5) and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding a protein containing (A-6) and a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5) and a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding a protein containing (A-6) and a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein containing (A-6), a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), a vector comprising a polynucleotide encoding the fusion protein of (B), a polynucleotide encoding a protein containing (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence (A-1) to (A-5), a vector comprising a polynucleotide encoding the fusion protein of (C), a polynucleotide encoding a protein containing (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (B); A combination of a vector comprising a polynucleotide encoding a protein containing (A-6), a vector comprising a polynucleotide encoding a fusion protein of (B), a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), and a vector comprising a polynucleotide encoding a fusion protein of (C); A combination of a vector comprising a polynucleotide encoding a protein containing (A-6), a vector comprising a polynucleotide encoding a fusion protein of (C), a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), and a vector comprising a polynucleotide encoding a fusion protein of (B); A combination of a vector comprising a polynucleotide encoding the fusion protein of (B), a vector comprising a polynucleotide encoding the fusion protein of (C), and a vector comprising a polynucleotide encoding a fusion protein containing the amino acid sequence of (A-1) to (A-5), and a polynucleotide encoding a protein containing (A-6); or A combination of a vector comprising a polynucleotide encoding a fusion protein containing an amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein containing (A-6), a vector comprising a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding the fusion protein of (C).

[0191] The cells to be transformed are not particularly limited as long as they can produce antigen-presenting extracellular vesicles as described herein after transformation, and may be primary cultured cells, passaged cells, or established cell lines, and may be normal cells or diseased cells including cancerous or tumorous cells. Furthermore, the origin of the cells to be transformed is not particularly limited, but examples include animal-derived cells such as rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; mammals such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees; plant-derived cells; and insect-derived cells. Preferably, the cells to be transformed are animal-derived cells. Examples of animal-derived cells, though not limited to these, include human embryonic kidney cells (including HEK293T cells, etc.), human FL cells, Chinese hamster ovary cells (CHO cells), COS-7, Vero, mouse L cells, and rat GH3 cells.

[0192] The method for transforming cells is not particularly limited as long as it can introduce the desired polynucleotide into the cells. For example, it may be electroporation, microinjection, calcium phosphate, cationic lipid, liposome-based methods, non-liposomal methods such as polyethyleneimine, or viral infection methods.

[0193] The transformed cells may be transformed cells that transiently express the fusion protein or protein complex of (A), (B), (C), (D), (E), (F), and / or (G), or transformed cells (stable cell lines) that stably express it.

[0194] The culture conditions for transformed cells are not particularly limited. For example, if the transformed cells are of animal origin, a culture medium commonly 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) or a medium to which other components such as fetal bovine serum, antibiotics, and amino acids are added may be used, and the cells may be cultured (e.g., statically or with shaking) for a desired time (e.g., approximately 0.5 hours to approximately 240 hours (preferably approximately 5 to approximately 120 hours, more preferably approximately 12 to approximately 72 hours) in the presence of approximately 1 to approximately 10% (preferably approximately 2 to approximately 5%) CO2 at approximately 30 to approximately 40°C (preferably approximately 37°C).

[0195] The culture supernatant obtained by culturing transformed cells may contain the antigen-presenting extracellular vesicles described herein. Therefore, when culturing transformed cells for the purpose of obtaining the antigen-presenting extracellular vesicles described herein, a culture medium from which extracellular vesicles such as exosomes have been removed may be used as needed (for example, Dulbecco's modified Eagle medium containing approximately 1-5% fetal bovine serum from which exosomes have been removed).

[0196] Culture supernatant

[0197] In one embodiment of the present invention, a culture supernatant obtained by culturing transformed cells as described herein is provided.

[0198] The antigen-presenting extracellular vesicles contained in the culture supernatant described herein can be further recovered by, for example, purifying (e.g., centrifugation, chromatography, etc.), concentrating, and isolating the culture supernatant.

[0199] One embodiment of the present invention provides an antigen-presenting extracellular vesicle obtained from the culture supernatant described herein.

[0200] Method for producing antigen-presenting extracellular vesicles as described herein.

[0201] The antigen-presenting extracellular vesicles described herein may be obtained, for example, by means known to those skilled in the art, such as genetic engineering techniques (for example, by the methods described below, or by the methods described in the examples, or by similar methods). Using conventional genetic engineering techniques, polynucleotides encoding the proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), can be obtained and operably inserted into the same or separate vectors. When two or more of the polynucleotides encoding the proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are inserted into the same vector, each may be operably linked to the same or separate promoter. The obtained single or multiple vectors can be used to simultaneously or sequentially transform cells to obtain transformed cells (which may be transformed cells that transiently express these fusion proteins, or transformed cells that stably express them (stable strains)). The transformed cells obtained can be cultured under desired conditions to obtain the culture supernatant, and the obtained culture supernatant can be purified as needed (e.g., by centrifugation, purification using 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 antigen-presenting extracellular vesicles described herein.

[0202] Alternatively, when soluble proteins are used as (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), the antigen-presenting extracellular vesicles described herein may be obtained, for example, by the following method. The soluble proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are obtained by conventional genetic engineering techniques or are commercially available. Next, extracellular vesicles are obtained from the desired cells, for example, by known methods, by the methods described herein, or by similar methods. Next, the obtained extracellular vesicles are reacted with one or more of the soluble proteins described above in a desired solvent under desired conditions (for example, the method described in Japanese Patent Application Publication No. 2018-104341 may be used as a reference). This operation is carried out while appropriately changing the conditions until the soluble proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are contained in the membrane of the extracellular vesicles, thereby obtaining the antigen-presenting extracellular vesicles described herein.

[0203] Alternatively, when soluble proteins are used as (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), the antigen-presenting extracellular vesicles described herein may be obtained, for example, by the following method. Soluble proteins of (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are obtained by conventional genetic engineering techniques, with a desired tag (e.g., His tag, FLAG tag, PNE tag of SEQ ID NO: 79, etc., which may all be the same tag or all be different tags) attached to their N-terminal or C-terminal side. Next, extracellular vesicles are obtained from the desired cells, for example, by known methods or by the methods described herein or by similar methods, and an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody, e.g., anti-PNE tag nanobody of SEQ ID NO: 83) is attached to them via a peptide linker, etc., as needed; or an antibody against the tag or its antigen-binding fragment (e.g., scFv, A polynucleotide encoding a fusion protein (e.g., SEQ ID NOs. 88, 90, etc.) to which a Fab or nanobody is bound (e.g., SEQ ID NO: 89, a fusion protein of an anti-PNE tag nanobody (SEQ ID NO: 83), CD8a (SEQ ID NO: 85), and CD81 (SEQ ID NO: 15) is obtained, and cells are transformed using a vector into which this polynucleotide is operably inserted to obtain transformed cells (which may be transformed cells that transiently express the fusion protein, or transformed cells that stably express it (stable strain)). The obtained transformed cells are cultured, and extracellular vesicles are recovered by the method described above. An antigen-presenting extracellular vesicle described herein may be obtained by mixing soluble proteins (A) and (B), and optionally (C), to which the tag is attached, with an extracellular vesicle containing a protein in its membrane that includes an antibody against the tag or its antigen-binding fragment (e.g., scFv, Fab, or nanobody), etc., under desired conditions.

[0204] Alternatively, transformation may be performed using a combination of polynucleotides encoding the fusion proteins described in (A) to (G) above, and antigen-presenting extracellular vesicles as described in the specification may be obtained from the transformed cells.

[0205] Alternatively, the antigen-presenting extracellular vesicles described herein may be obtained by combining two or more of the methods described above.

[0206] The antigen-presenting extracellular vesicles described herein may be confirmed to contain the proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), in their membranes by methods such as flow cytometry, ELISA, and Western blotting.

[0207] One embodiment of the present invention provides a method for producing antigen-presenting extracellular vesicles as described herein, comprising recovering a culture supernatant obtained by culturing transformed cells as described herein.

[0208] In one embodiment of the present invention, a method for producing an antigen-presenting extracellular vesicle as described herein, (i) polynucleotides encoding the fusion protein or protein complex of (A) as described herein, and (ii) Polynucleotides encoding the fusion protein of (B) as described herein, and optionally (iii) Polynucleotide encoding the fusion protein of (C) described herein The process involves simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising the above, The transformed cells obtained are cultured and the resulting culture supernatant is collected. This provides a method that includes [something]. Alternatively, in one embodiment of the present invention, a method for producing an antigen-presenting extracellular vesicle as described herein, (iv) a polynucleotide encoding a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine extramembrane, comprising the antigen-presenting MHC molecule of (D) described herein and at least one T cell-stimulating cytokine, and Depending on the circumstances (iii) Polynucleotide encoding the fusion protein of (C) described herein The process involves simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising the above, The transformed cells obtained are cultured and the resulting culture supernatant is collected. This provides a method that includes [something]. Alternatively, in one embodiment of the present invention, a method for producing an antigen-presenting extracellular vesicle as described herein, (v) A polynucleotide encoding a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine extramembrane, comprising the antigen-presenting MHC molecule described in (E) herein and at least one T cell-stimulating cytokine and a T cell-costimulating molecule. The process involves transforming cells with a vector containing the following: The transformed cells obtained are cultured and the resulting culture supernatant is collected. This provides a method that includes [something].

[0209] One embodiment of the present invention provides an antigen-presenting extracellular vesicle obtained from the culture supernatant described herein.

[0210] In one embodiment of the present invention, an antigen-presenting extracellular vesicle is, hereafter: (i) polynucleotides encoding the fusion protein or protein complex of (A) as described herein, and (ii) Polynucleotides encoding the fusion protein of (B) as described herein, and optionally (iii) Polynucleotide encoding the fusion protein of (C) described herein The process involves simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising the above, The transformed cells obtained are cultured and the resulting culture supernatant is collected. The present invention provides antigen-presenting extracellular vesicles obtained by a method comprising [a specific method]. Alternatively, in one embodiment of the present invention, an antigen-presenting extracellular vesicle is, hereafter: (iv) a polynucleotide encoding a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine extramembrane, comprising the antigen-presenting MHC molecule of (D) described herein and at least one T cell-stimulating cytokine, and Depending on the circumstances (iii) Polynucleotide encoding the fusion protein of (C) described herein The process involves simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising the above, The transformed cells obtained are cultured and the resulting culture supernatant is collected. The present invention provides antigen-presenting extracellular vesicles obtained by a method comprising [a specific method]. Alternatively, in one embodiment of the present invention, an antigen-presenting extracellular vesicle is, hereafter: (v) Transforming cells with a polynucleotide encoding a fusion protein capable of presenting the antigen and the T cell-stimulating cytokine extramembrane, comprising the antigen-presenting MHC molecule described in (E) herein and at least one T cell-stimulating cytokine and a T cell-costimulating molecule. The transformed cells obtained are cultured and the resulting culture supernatant is collected. The present invention provides antigen-presenting extracellular vesicles obtained by a method comprising [a specific method].

[0211] Composition, use

[0212] One embodiment of the present invention provides a composition (e.g., a pharmaceutical composition) comprising an antigen-presenting extracellular vesicle, a polynucleotide and / or a vector containing the same, and / or transformed cells and / or their culture supernatant, as described herein. One embodiment of the present invention provides a pharmaceutical composition comprising an antigen-presenting extracellular vesicle or a culture supernatant as described herein.

[0213] The compositions described herein (e.g., pharmaceutical compositions) may include, but are not limited to, excipients, lubricants, binders, disintegrants, pH adjusters, solvents, solubilizers, suspending agents, isotonic agents, buffers, analgesics, preservatives, antioxidants, colorants, sweeteners, surfactants, and other additives. The types and amounts of these additives may be appropriately selected by those skilled in the art depending on the purpose. When used as pharmaceutical compositions, these additives are preferably pharmacologically acceptable carriers. Furthermore, if the compositions described herein contain polynucleotides, it is preferable, though not essential, to include carriers suitable for drug delivery (DD) of nucleic acids, and examples of such carriers include lipid nanoparticles (LNPs) and polymers (e.g., PEIs).

[0214] The compositions described herein (e.g., pharmaceutical compositions) can be formulated together with the above-mentioned additives by methods known to the present invention into, for example, tablets, coated tablets, orally disintegrating tablets, chewable tablets, 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. These may be oral or parenteral preparations. Depending on their purpose, the formulations may further contain other beneficial components (e.g., other therapeutically beneficial components).

[0215] A composition according to one embodiment of the present invention can enhance acquired immunity (cellular immunity and / or humoral immunity) against a specific antigen, as shown in the test examples, and if a peptide derived from an infectious pathogen (such as a pathogen or virus) is used as the antigen, it can be used as a pharmaceutical composition for treating or preventing infectious diseases caused by infectious pathogens. Furthermore, as shown in the test examples, a composition that is one embodiment of the present invention can induce inflammatory cytokines and activate innate immunity (including mobilizing and activating neutrophils, monocytes, macrophages, etc., to phagocytose pathogens), thereby eliminating infectious pathogens, and can be used as a pharmaceutical composition for treating or preventing infectious diseases caused by infectious pathogens.

[0216] The antigen-presenting extracellular vesicles described herein (preferably antigen-presenting extracellular vesicles containing MHC class I-restricted antigen peptides and MHC class I molecules in their membrane), polynucleotides thereof and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing the same (e.g., pharmaceutical compositions) may be useful for treating or preventing cancer.

[0217] Accordingly, one embodiment of the present invention provides antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing the same (e.g., pharmaceutical compositions), for treating or preventing cancer. As the test examples show, antigen-presenting extracellular vesicles, etc., which are one embodiment of the present invention, can promote the proliferation and activation of antigen-specific cytotoxic T cells, and if tumor-associated antigen peptides are used as the antigen, the proliferated and activated cytotoxic T cells can recognize and attack cancer cells, thereby killing them.

[0218] Another embodiment of the present invention provides the use of antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising them as described herein, and / or transformed cells and / or their culture supernatants, or compositions comprising them (e.g., pharmaceutical compositions), for the production of pharmaceuticals for treating or preventing cancer.

[0219] Another embodiment of the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of an antigen-presenting extracellular vesicle, polynucleotide and / or a vector comprising the same as described herein, and / or transformed cells and / or their culture supernatant, or a composition comprising the same.

[0220] Cancer includes, but is not limited to, any solid tumor or hematological cancer, such as small cell lung cancer, non-small cell lung cancer, breast cancer, esophageal cancer, stomach cancer, small intestine cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, prostate cancer, bone marrow cancer, kidney cancer (including renal cell carcinoma, etc.), parathyroid cancer, adrenal cancer, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, ovarian cancer (e.g., serous adenocarcinoma, mucinous adenocarcinoma, clear cell adenocarcinoma, etc.), testicular cancer, bladder cancer, vulvar cancer, penile cancer, thyroid cancer, head and neck cancer, craniopharynx cancer, pharyngeal cancer, tongue cancer, skin cancer, and Merkel cell cancer. Examples include melanoma (malignant melanoma, etc.), epithelial carcinoma, squamous cell carcinoma, basal cell carcinoma, childhood cancer, cancer of unknown primary origin, fibrosarcoma, mucosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, intralymphatic sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, synoviomas, mesothelioma, Ewing's tumor, seminomas, Wilms' tumor, brain tumors, gliomas, glioblastomas, astrocytomas, myeloblastomas, meningiomas, neuroblastomas, medulloblastomas, retinoblastomas, spinal tumors, malignant lymphomas (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, etc.), chronic or acute lymphoblastic leukemia, adult T-cell leukemia, etc.

[0221] In one embodiment of the present invention, immune checkpoint inhibitors may be used in combination to treat or prevent cancer. The immune checkpoint inhibitors may be administered to the patient simultaneously or sequentially, or they may be included in the pharmaceutical product according to the present invention. Examples of immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (e.g., anti-PD-1 antibodies such as nibrumab and vembrolidimab), CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies such as ipilimumab), and PD-L1 inhibitors (e.g., anti-PD-L1 antibodies such as durvalumab, atezolizumab, and avelumab). If the immune checkpoint inhibitor is an antibody or an active fragment thereof, the antibody or its active fragment may be conjugated to a membrane protein or its transmembrane domain capable of localizing to the membrane of the extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of the extracellular vesicle, so that it is present on the membrane of the extracellular vesicle according to the present invention. The combined use of such immune checkpoint inhibitors enhances cytotoxicity against cancer cells.

[0222] The antigen-presenting extracellular vesicles described herein (preferably antigen-presenting extracellular vesicles containing MHC class II-restricted antigen peptides and MHC class II molecules on the membrane), their polynucleotides and / or vectors containing them, and / or transformed cells and / or their culture supernatants, or compositions containing them, may be useful for treating or preventing autoimmune diseases. As illustrated by the test examples, an antigen-presenting extracellular vesicle in one embodiment of the present invention can promote the proliferation and activation of antigen-specific regulatory T cells (Tregs), and if an autoantigen peptide is used as the antigen, the proliferated and activated Tregs can induce immune tolerance to the autoantigen, thereby treating or preventing autoimmune diseases.

[0223] Accordingly, one embodiment of the present invention provides antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising the same, and / or transformed cells and / or their culture supernatant, or compositions comprising the same (e.g., pharmaceutical compositions), for treating or preventing autoimmune diseases.

[0224] Another embodiment of the present invention provides the use of antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising them as described herein, and / or transformed cells and / or their culture supernatants, or compositions comprising them (e.g., pharmaceutical compositions), for the manufacture of pharmaceuticals for treating or preventing autoimmune diseases.

[0225] Another embodiment of the present invention provides a method for treating or preventing an autoimmune disease, comprising administering to a subject in need an effective amount of an antigen-presenting extracellular vesicle, polynucleotide and / or a vector comprising the same as described herein, and / or transformed cells and / or their culture supernatant, or a composition comprising the same.

[0226] Autoimmune diseases are not limited to these, but include, for example, asthma, psoriasis, systemic lupus erythematosus, Guillain-Barré syndrome, Sjögren's syndrome, multiple sclerosis, myasthenia gravis, pernicious anemia, Graves' disease, Hashimoto's thyroiditis, type 1 diabetes, Crohn's disease, inflammatory bowel disease, and rheumatoid arthritis.

[0227] The antigen-presenting extracellular vesicles described herein (preferably antigen-presenting extracellular vesicles containing MHC class II-restricted antigen peptides and MHC class II molecules on the membrane), their polynucleotides and / or vectors containing them, and / or transformed cells and / or their culture supernatants, or compositions containing them (e.g., pharmaceutical compositions), may be useful for treating or preventing allergic diseases. As the test examples show, antigen-presenting extracellular vesicles, etc., which are one embodiment of the present invention, can promote the proliferation and activation of antigen-specific regulatory T cells (Tregs), and if an allergen is used as the antigen, the proliferated and activated Tregs can induce immune tolerance to the allergen, thereby treating or preventing allergic diseases.

[0228] Accordingly, one embodiment of the present invention provides antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising the same, and / or transformed cells and / or their culture supernatant, or compositions comprising the same (e.g., pharmaceutical compositions), for treating or preventing allergic diseases.

[0229] Another embodiment of the present invention provides the use of antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising them as described herein, and / or transformed cells and / or their culture supernatants, or compositions comprising them (e.g., pharmaceutical compositions), for the manufacture of pharmaceuticals for treating or preventing allergic diseases.

[0230] Another embodiment of the present invention provides a method for treating or preventing an allergic disease, comprising administering to a subject in need an effective amount of an antigen-presenting extracellular vesicle, polynucleotide and / or a vector comprising the same as described herein, and / or transformed cells and / or their culture supernatant, or a composition comprising the same.

[0231] Allergic diseases are not limited to these, but examples include allergic rhinitis, atopic dermatitis, allergic asthma, allergic conjunctivitis, allergic gastroenteritis, food allergies, drug allergies, and urticaria.

[0232] The subjects targeted for treatment or prevention of the various diseases described above are not limited to those mentioned above, but include animals such as mammals, such as rodents like mice, rats, hamsters, and guinea pigs; animals of the order Lagomorpha, such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; animals of the order Carnivora, such as dogs and cats; and primates such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees; or plants. Preferably, the subjects are animals, more preferably rodents or primates, and even more preferably mice or humans.

[0233] The dosage of antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing them, and / or transformed cells and / or their culture supernatants, or compositions containing them or formulations thereof, as described herein, may be appropriately determined taking into consideration the sex, age, weight, health condition, severity of illness or diet of the subject to be administered; the time of administration; the method of administration; combinations with other drugs; and other factors.

[0234] Methods for activating, proliferating, and / or differentiating T cells against specific antigens.

[0235] The antigen-presenting extracellular vesicles described herein can activate, proliferate, differentiate, etc., T cells in response to specific antigens by contacting T cells (but not limited to, T cells or T cell populations obtained from, for example, peripheral blood or the spleen) in vitro, ex vivo, and / or in vivo.

[0236] One embodiment of the present invention provides a method for activating, proliferating, and / or differentiating T cells in response to a specific antigen, comprising contacting the antigen-presenting extracellular vesicles described herein with T cells in vitro or ex vivo.

[0237] In one embodiment of the present invention, T cells obtained by the method described above are provided.

[0238] T cells obtained by the method described above may be administered to a subject to treat and / or prevent diseases (e.g., cancer, autoimmune diseases, allergic diseases, etc.). [Examples]

[0239] The present invention will be described in more detail below using examples, but these examples do not limit the scope of the present invention in any way.

[0240] Plasmid preparation 1 Using a pCAG-puro vector, we created a vector for expressing MHC class I molecules capable of presenting antigens extracellularly on the membrane of extracellular vesicles. Using established cloning techniques, a single-chain trimer (sc-Trimer) was created consisting of a polynucleotide (SEQ ID NO: 2) encoding the β2-microglobulin signal peptide (amino acids 1-20; SEQ ID NO: 1), a polynucleotide (SEQ ID NO: 4) encoding the OVA peptide, a model antigen peptide (SEQ ID NO: 3), a peptide linker (amino acid sequence: SEQ ID NO: 5, polynucleotide: SEQ ID NO: 6), a polynucleotide (SEQ ID NO: 8) encoding the full-length sequence of β2-microglobulin excluding the signal peptide (amino acids 21-119; SEQ ID NO: 7), a polynucleotide (SEQ ID NO: 12) encoding the peptide linker (SEQ ID NO: 11), and a polynucleotide (SEQ ID NO: 10) encoding the full-length sequence of the MHC class Iα chain excluding the signal peptide (amino acids 22-369; SEQ ID NO: 9). (Amino acid sequence: SEQ ID NO: 13; polynucleotide: SEQ ID NO: 14). Next, a polynucleotide (sequence number 18; corresponding amino acid sequence: sequence number 17) was created by linking sc-Trimer with a polynucleotide (sequence number 16) encoding the full sequence of the tetraspanin CD81 (amino acids 1-236; sequence number 15), and this polynucleotide was inserted into the pCAG-puro vector (Figure 1A, 1B: hereafter referred to as sc-Trimer-CD81). Similarly, to express CD80, one of the T cell costimulatory molecules, on the membrane of an extracellular vesicle, a polynucleotide (SEQ ID NO: 24; corresponding amino acid sequence: SEQ ID NO: 23) was inserted into a pCAG-puro or pMX vector. This polynucleotide consisted of a polynucleotide (SEQ ID NO: 20) encoding the full-length sequence of CD80 (amino acids 1-306; SEQ ID NO: 19) and a polynucleotide (SEQ ID NO: 22) encoding the full-length sequence of the tetraspanin CD9 (amino acids 1-306; SEQ ID NO: 21). (Figure 1C, 1D: Hereafter referred to as CD80-CD9). Similarly, to express IL-2, one of the T cell-stimulating cytokines, on the membrane of an extracellular vesicle, a polynucleotide (SEQ ID NO: 26) encoding the full-length sequence of IL-2 excluding the signal peptide (amino acids 21-169; SEQ ID NO: 25) was inserted between amino acids 170C and 171I in the large extracellular loop of the tetraspanin mouse CD63 (amino acids 1-238: SEQ ID NO: 27; polynucleotide: SEQ ID NO: 28). (That is, the IL-2 sequence was inserted between the polynucleotide (SEQ ID NO: 58) encoding the partial sequence of CD63 in SEQ ID NO: 57 and the polynucleotide (SEQ ID NO: 60) encoding the partial sequence of CD63 in SEQ ID NO: 59.) Furthermore, polynucleotides (SEQ ID NO: 30) encoding peptide linkers (amino acid sequence GGGGS: SEQ ID NO: 29) were added to the N-terminus and C-terminus of IL-2, respectively. This polynucleotide (SEQ ID NO: 32; corresponding amino acid sequence: SEQ ID NO: 31) was inserted into the pCAG-puro vector (Figure 1E, 1F: hereafter referred to as CD63-IL-2).

[0241] Plasmid preparation 2 : Using a pCAG-puro vector, we created a vector for expressing MHC class II molecules capable of presenting antigens extracellularly on the membrane of extracellular vesicles. Using established cloning techniques, a single-chain dimer (sc-Dimer) was created by linking a polynucleotide (sequence number 34) encoding the signal peptide of the MHC class IIβ chain (amino acids 1-27; sequence number 33), a polynucleotide (sequence number 36) encoding the model antigen peptide OVA peptide (sequence number 35), and a polynucleotide (sequence number 38) encoding the full-length sequence of the MHC class IIβ chain excluding the signal peptide (amino acids 28-265; sequence number 37), with a polynucleotide (sequence number 40) encoding the peptide linker (sequence number 39) (amino acid sequence: sequence number 41; polynucleotide: sequence number 42). Next, a polynucleotide (sequence number 44; corresponding amino acid sequence: sequence number 43) was created by linking the sc-Dimer with a polynucleotide (sequence number 16) encoding the full-length sequence of the tetraspanin CD81 (amino acids 1-236; sequence number 15), and this was inserted into a pCAG-puro vector (Figure 1G, 1H: hereafter, sc-Dimer-CD81). A polynucleotide (SEQ ID NO: 46) encoding the full-length sequence (amino acids 1-256; SEQ ID NO: 45) of the MHC class IIα chain, a component of MHC class II molecules, was inserted into another pCAG-puro vector (Figure 1I: hereafter referred to as the MHC class IIα chain). Similarly, to express TGF-β1, a T cell-stimulating cytokine, on the membrane of an extracellular vesicle, a polynucleotide (SEQ ID NO: 48) encoding the full-length sequence of TGF-β1 (amino acids 1-390; SEQ ID NO: 47) in which the 33rd, 223rd, and 225th C atoms in the LAP domain were changed to S atoms, and a polynucleotide (SEQ ID NO: 50) encoding the full-length sequence of MFG-E8 excluding the signal peptide (amino acids 23-463; SEQ ID NO: 49) in which the 89th D atom was changed to E, was linked via a polynucleotide (SEQ ID NO: 30) encoding a peptide linker (SEQ ID NO: 29). This polynucleotide (SEQ ID NO: 52; corresponding amino acid sequence: SEQ ID NO: 51) was inserted into a pCAG-puro vector (Figure 1J, 1K: hereafter, TGF-β-MFG-E8). Similarly, to express IL-4, one of the T cell-stimulating cytokines, on the membrane of an extracellular vesicle, a polynucleotide (SEQ ID NO: 54) encoding the full-length sequence of IL-4 (amino acids 21-140; SEQ ID NO: 53), excluding the signal peptide, was inserted between amino acids 177S and 178G in the large extracellular loop of the tetraspanin mouse CD81 (amino acids 1-236: SEQ ID NO: 15; polynucleotide: SEQ ID NO: 16). (That is, the IL-4 sequence was inserted between the polynucleotide (SEQ ID NO: 62) encoding the partial sequence of CD81 in SEQ ID NO: 61 and the polynucleotide (SEQ ID NO: 64) encoding the partial sequence of CD81 in SEQ ID NO: 63.) Furthermore, polynucleotides (SEQ ID NO: 30) encoding peptide linkers (amino acid sequence GGGGS; SEQ ID NO: 29) were added to the N-terminus and C-terminus of IL-4, respectively. This polynucleotide (SEQ ID NO: 56; corresponding amino acid sequence: SEQ ID NO: 55) was inserted into the pCAG-puro vector (Figure 1L, 1M: hereafter referred to as CD81-IL-4).

[0242] Plasmid preparation 3: sc-Dimer-CD81―IL―12p40 A polynucleotide (SEQ ID NO: 92) encoding a protein (SEQ ID NO: 91) that fuses CD81 and IL-12p40, a subunit of the T cell-stimulating cytokine IL-12, to the above sc-Dimer was inserted into a pCAG-puro vector to prepare a vector that expresses the fusion protein. IL-12p35 A polynucleotide (SEQ ID NO: 98) encoding IL-12p35 (SEQ ID NO: 97), another subunit of IL-12, was inserted into a pCAG-puro or pMX vector to prepare a vector expressing IL-12p35.

[0243] CD81-IL-6 To express IL-6, one of the T cell-stimulating cytokines, on the membrane of extracellular vesicles, a polynucleotide encoding the full-length sequence of IL-6 excluding the signal peptide (SEQ ID NO: 99) (SEQ ID NO: 100) was introduced into a polynucleotide encoding the extracellular loop of the tetraspanin CD81. This polynucleotide encoding the CD81-IL-6 fusion protein (SEQ ID NO: 101) (SEQ ID NO: 102) was then inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein.

[0244] hCD80-hCD9 To express human CD80, one of the T cell costimulatory molecules, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 108) encoding a fusion protein (SEQ ID NO: 107) of human CD80 and the tetraspanin human CD9 was inserted into a pCAG-puro or pMX vector to prepare a vector that expresses the fusion protein. sc-Trimer-CD81―IL―2 To express IL-2, one of the T cell-stimulating cytokines, on the membrane of extracellular vesicles, a polynucleotide encoding the CD81-IL2 fusion peptide was prepared, similar to the CD81-IL-4 described above. This polynucleotide sequence was then combined with a nucleotide encoding sc-Trimer- to produce a polynucleotide encoding sc-Trimer-CD81-IL-2 (SEQ ID NO: 135) (SEQ ID NO: 136). This polynucleotide was then inserted into a pCAG-puro or pMX vector to prepare a vector for expressing the fusion protein.

[0245] hsc-Trimer-hCD81 Using the above-mentioned sc-Trimer-CD81 as a human gene sequence (HLA-A2402 was used as the MHC-I sequence), a polynucleotide encoding hsc-Trimer-hCD81 (sequence number 131) was constructed (sequence number 132), and this was inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein. SARS-CoV2sc-Trimer-hCD81 Using the SARS-CoV-2 peptide (amino acid sequence: SEQ ID NO: 141; polynucleotide sequence: SEQ ID NO: 142) as the antigen and HLA-A0201 as the MHC molecule, a polynucleotide (SEQ ID NO: 148) encoding the antigen-presenting MHC molecule (SARS-CoV2sc-Trimer; amino acid sequence: SEQ ID NO: 147) was constructed. This polynucleotide was then combined with a polynucleotide encoding hCD81 to produce a polynucleotide (SEQ ID NO: 150) encoding SARS-CoV2sc-Trimer-hCD81 (SEQ ID NO: 149). The constructed polynucleotides were inserted into pCAG-puro or pMX vectors to prepare vectors that express the fusion protein. hCD63-hIL-2 The above CD63-IL-2 was constructed using a human gene sequence. A polynucleotide encoding hCD63-hIL-2 (sequence number 115) (sequence number 116) was created and inserted into a pCAG-puro or pMX vector to prepare a vector for expressing the fusion protein.

[0246] CD63 - Akaluc As a negative control, CD63 and Akaluc luciferase were fused to create a polynucleotide (SEQ ID NO: 140) for localizing the AlkaLuc fusion protein (SEQ ID NO: 139) to extracellular vesicles. This polynucleotide was then inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein.

[0247] The sequences used in the examples are shown in Tables 1 to 13 below. The underlined portion in each sequence indicates the signal peptide.

[0248] [Table 1-1] [Table 1-2] [Table 1-3] Table 1-4 Table 1-5 Table 1-6

[0249] Table 2-1 Table 2-2 Table 2-3

[0250] Table 3-1 Table 3-2 Table 3-3

[0251] Table 4-1 Table 4-2 Table 4-3 Table 4-4

[0252] Table 5-1 Table 5-2 Table 5-3 Table 5-4

[0253] Table 6-1 Table 6-2

[0254] Table 7-1 Table 7-2 Table 7-3 Table 7-4

[0255] Table 8-1 Table 8-2 Table 8-3

[0256] Table 9-1 Table 9-2

[0257] Table 10-1 Table 10-2

[0258] Table 11-1 Table 11-2

[0259] Table 12-1 Table 12-2 Table 12-3 Table 12-4 Table 12-5

[0260] 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

[0261] Table 14-1 Table 14-2 Table 14-3 Table 14-4

[0262] Preparation of fetal bovine serum from which exosomes have been removed Fetal bovine serum from which exosomes have been removed was obtained by stirring 10 mL of inactivated FBS and 2 mL of 50% Poly(ethylene glycol) 10,000 solution (Sigma-Aldrich, #81280) at 4°C for 2 hours, followed by centrifugation at 1500 × g, 4°C, for 30 minutes to precipitate the exosomes, and collecting the supernatant.

[0263] [Example 1] Extracellular vesicle containing MHC class I molecules and T cell-stimulating cytokines in its membrane, which presents antigens. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were simultaneously transfected with two plasmids (pCAG vectors encoding sc-Trimer-CD81 and CD63-IL-2, respectively) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3 hours after transfection, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with exosome-removed 2% fetal bovine serum and penicillin / streptomycin. 72 hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and centrifuged at 300 g for 5 minutes. The supernatant was collected again 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 again and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicles of Example 1 (Figure 2A). The concentration of extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0264] [Example 2] Antigen-presenting extracellular vesicle containing MHC class I molecules, T cell costimulatory molecules, and T cell-stimulating cytokines in its membrane. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were simultaneously transfected with three plasmids prepared above (pCAG vectors encoding sc-Trimer-CD81, CD80-CD9, and CD63-IL-2, respectively) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. Three hours after transfection, the medium was changed, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. Seventy-two hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and centrifuged at 300 g for 5 minutes. The supernatant was collected again 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 again and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle for Example 2 (Figure 2B). The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0265] [Example 3] Extracellular antigen-presenting vesicle containing an MHC class II molecule, a T cell costimulatory molecule, and a T cell-stimulating cytokine in its membrane. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Four plasmids prepared above (pCAG vectors encoding sc-Dimer-CD81, MHC class IIα chain, CD80-CD9, and CD63-IL-2, respectively) were simultaneously transfected into cells at approximately 50% confluence using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3 hours after transfection, and 24 hours after transfection, the medium was changed again to Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. 72 hours after transfection, the supernatant was collected, filtered 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle in Example 3 (Figure 2C). The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0266] [Example 4] Extracellular antigen-presenting vesicle containing MHC class II molecules, T cell costimulatory molecules, and T cell-stimulating cytokines in its membrane. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Five plasmids (pCAG vectors encoding sc-Dimer-CD81, MHC class IIα chain, CD80-CD9, TGF-β-MFGE8, and CD63-IL-2, respectively) were simultaneously transfected into cells at approximately 50% confluence using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3 hours after transfection, and 24 hours after transfection, it was changed again to Dulbecco's Modified Eagle Medium supplemented with exosome-removed 2% fetal bovine serum and penicillin / streptomycin. 72 hours after transfection, the supernatant was collected, filtered through a 0.22 μm filter, and 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle in Example 4 (Figure 2D). The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0267] [Example 5] Extracellular antigen-presenting vesicle containing an MHC class II molecule, a T cell costimulatory molecule, and a T cell-stimulating cytokine in its membrane. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Four plasmids prepared above (pCAG vectors encoding sc-Dimer-CD81, MHC class IIα chain, CD80-CD9, and CD81-IL-4, respectively) were simultaneously transfected into cells at approximately 50% confluence using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3 hours after transfection, and 24 hours after transfection, the medium was changed again to Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. 72 hours after transfection, the supernatant was collected, filtered 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicles in Example 5 (Figure 2E). The concentration of the antigen-presenting extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0268] [Reference Example 1] Control extracellular vesicles HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. The medium was changed when the cells reached approximately 50% confluence, and after 24 hours, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin from which exosomes had been removed. 48 hours after changing to the exosome-removed medium, 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, and the mixture was centrifuged at 100,000 g for 2 hours. The supernatant was removed, and the pellet was suspended in 100 μL of PBS. This suspension was used as the extracellular vesicle for Reference Example 1. The concentration of antigen-presenting extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0269] [Example 2] Extracellular vesicles containing MHC class I molecules in their membrane HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with a plasmid (pCAG vector encoding sc-Trimer-CD81) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. Three hours after transfection, the medium was changed, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. Seventy-two hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and 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 removing the supernatant, the pellet was washed with PBS. PBS was added to the pellet, and it was centrifuged at 100,000 g for 2 hours. After removing the supernatant, the pellet was suspended in 100 μL of PBS and used as the extracellular vesicles for Reference Example 2. The concentration of extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0270] [Reference Example 3] Extracellular vesicles containing T cell costimulatory molecules in their membrane HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with a plasmid (a pCAG vector encoding CD80-CD9) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. Three hours after transfection, the medium was changed, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. Seventy-two hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and 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 removing the supernatant, the pellet was washed with PBS. PBS was added to the pellet, and it was centrifuged at 100,000 g for 2 hours. After removing the supernatant, the pellet was suspended in 100 μL of PBS and used as the extracellular vesicles for Reference Example 3. The concentration of the extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. [Reference Example 4] Extracellular vesicles containing T cell-stimulating cytokines in their membrane HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with a plasmid (pCAG vector encoding CD63-IL-2) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. Three hours after transfection, the medium was changed, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. Seventy-two hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and 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 removing the supernatant, the pellet was washed with PBS. PBS was added to the pellet, and it was centrifuged at 100,000 g for 2 hours. After removing the supernatant, the pellet was suspended in 100 μL of PBS and used as the extracellular vesicles for Reference Example 4. The concentration of the extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0271] [Reference Example 5] Extracellular vesicles containing MHC class I molecules and T cell costimulatory molecules in their membranes. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were simultaneously transfected with two plasmids (pCAG vectors encoding sc-Trimer-CD81 and CD80-CD9, respectively) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3 hours after transfection, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. 72 hours after transfection, the supernatant was collected, passed through a 0.22 μm filter, and centrifuged at 300 g for 5 minutes. The supernatant was collected again 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 again and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the extracellular vesicles in Reference Example 5. The concentration of extracellular vesicles was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0272] Test Example 1-1: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 2 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • APC conjugate anti-mouse H-2KbOVA complex antibody (25-D1.16 Biolegend) • PE conjugate anti-mouse CD80 antibody (16-10A1, Biolegend) • Brilliant Violet421 conjugate anti-mouse IL-2 antibody (JES6-5H4, manufactured by Biolegend) The results are shown in Figure 3A.

[0273] [result] The results from Test Example 1-1 show that the antigen-presenting extracellular vesicles of Example 2 contain MHC class I molecules, CD80, and IL-2 that present the OVA antigen on their membrane (Figure 3A).

[0274] Test Example 1-2: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 3 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows. After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • APC conjugate anti-mouse IL-2 antibody (JES6-5H4, manufactured by Biolegend) • PE conjugate anti-mouse CD80 antibody (16-10A1, Biolegend) • APC-Cy7 conjugate anti-mouse IA / IE antibody (M5 / 114.15.2 Biolegend) The results are shown in Figure 3B.

[0275] [result] The results from Test Examples 1-2 show that the antigen-presenting extracellular vesicles of Example 3 contain MHC class II molecules, CD80, and IL-2 that present the OVA antigen on their membrane (Figure 3B).

[0276] Test Examples 1-3: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 4 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows. After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • APC conjugate anti-mouse IL-2 antibody (JES6-5H4, manufactured by Biolegend) • PE conjugate anti-mouse CD80 antibody (16-10A1, Biolegend) • APC-Cy7 conjugate anti-mouse IA / IE antibody (M5 / 114.15.2 Biolegend) • APC conjugate anti-mouse LAP (TGF-β1) antibody (TW7-16B4, manufactured by Biolegend) The results are shown in Figure 3C.

[0277] [result] The results from Test Examples 1-3 show that the antigen-presenting extracellular vesicles of Example 4 contain MHC class II molecules, CD80, IL-2, and TGF-β1 that present the OVA antigen on their membrane (Figure 3C).

[0278] Test Examples 1-4: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 5 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows. After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • Alexa Fluor 488 conjugate anti-mouse IL-4 antibody (manufactured by 11B11 Biolegend) • PE conjugate anti-mouse CD80 antibody (16-10A1, Biolegend) • APC-Cy7 conjugate anti-mouse IA / IE antibody (M5 / 114.15.2 Biolegend) The results are shown in Fig. 3D.

[0279] [Results] From the results of Test Examples 1-4, it can be seen that the antigen-presenting extracellular vesicles of Example 5 contain MHC class II molecules, CD80, and IL-4 that present the OVA antigen on their membranes (Fig. 3D).

[0280] Experiment Example 2: In vitro activation of OVA-specific CD8-positive T cells (OT-1 T cells) by antigen-presenting extracellular vesicles. In order to examine whether the antigen-presenting extracellular vesicles activate antigen-specific CD8-positive T cells, etc., the following test was carried out in vitro. Lymph nodes excised from OT-1 mice, which are OVA-reactive TCR transgenic mice, were disrupted on a 100-μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (manufactured by Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. 2 × 10 5 stained lymph node cells were suspended in 2,00 μL of RPMI1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin, and the antigen-presenting extracellular vesicles of Example 1 or 2 (final concentration 3 μg / mL), or a mixture of three types of extracellular vesicles of Reference Examples 2 to 4 (each final concentration of the three types of extracellular vesicles 3 μg / mL), or the extracellular vesicles of Reference Example 1, 2, or 5 (final concentration 3 μg / mL) were added, and after culturing in a 96-well round-bottom plate for 3 days, immunostaining was performed. The antibodies used for staining were as follows (staining time: 15 minutes, temperature: 4°C). After staining, the fluorescence intensity of CellTrace Violet, a cell proliferation assay reagent in OT-1 T cells, was detected using a flow cytometer FACSCantoII (manufactured by BD Biosciences). · APC-conjugated anti-mouse CD8 antibody (manufactured by 53-6.7 Biolegend) · PE-conjugated anti-mouse TCR Vb5.1,5.2 antibody (MR9-4 Biolegend) The results are shown in Fig. 4.

[0281] [result] The results of Test Example 2 showed that the antigen-presenting extracellular vesicles of Examples 1 and 2 significantly differentiated and / or proliferated antigen-specific CD8-positive T cells compared to the mixture of the three types of extracellular vesicles of Reference Examples 2-4, or the extracellular vesicles of Reference Examples 1, 2, and 5 (Figure 4).

[0282] Experiment Example 3: In vivo activation of OVA-specific CD8-positive T cells (OT-1 T cells) by antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles activate antigen-specific CD8-positive T cells, the following tests were performed in vivo. Lymph nodes were excised from OT-1 mice, which are OVA-responsive TCR transgenic mice, and lymphocyte suspensions were prepared in the same manner as in Test Example 2. Lymph nodes were also excised from CD45.1 congenic mice in the same manner, and lymphocyte suspensions were prepared. The respective lymphocyte suspensions were mixed in a 1:1 ratio and stained using CellTrace Violet, a cell proliferation assay reagent. 1 × 10⁶ cells were suspended in PBS. 7 CellTrace Violet-stained mixed lymphocyte suspensions were transferred into CD45.1 / CD45.2 congenic mice via the tail vein. The following day, 50 μg of antigen-presenting extracellular vesicles from Example 2 or Reference Example 1, or a mixture of 1.5 μg of IL-2 (Biolegend) and 50 μg of anti-mouse IL-2 antibody (S4B6-1 Bio X Cell) (IL-2 / anti-IL-2 antibody conjugate) was transferred into CD45.1 / CD45.2 congenic mice via the tail vein. Four days after cell transfer, lymph nodes were excised from recipient mice, lymphocyte suspensions were prepared, and immunostaining was performed. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After staining, the luminescence intensity of CellTrace Violet, a cell proliferation assay reagent, was detected in the transferred OT-1 T cells and wild-type CD8 T cells using a flow cytometer FACSCantoII (BD Biosciences). • PE-Cy7 conjugate anti-mouse CD8 antibody (53-6.7 Biolegend) • PE conjugate anti-mouse TCR Vb5.1,5.2 antibody (MR9-4, Biolegend) • FITC conjugate anti-mouse CD45.1 antibody (A20 Biolegend) • APC conjugate anti-mouse CD45.2 antibody (manufactured by 104 Biolegend) The results are shown in Figure 5.

[0283] [result] The results of Test Example 3 show that, compared to the extracellular vesicles of Reference Example 1, the antigen-presenting extracellular vesicles of Example 2 significantly differentiated and / or proliferated antigen-specific CD8-positive T cells in vivo without activating other CD8-positive T cells (antigen-nonspecific CD8-positive T cells) (Figure 5). Furthermore, since the antigen-presenting extracellular vesicles of Example 2 hardly activated other CD8-positive T cells (antigen-nonspecific CD8-positive T cells) compared to the IL-2 / anti-IL-2 antibody complex, it is possible that they have a lower risk of serious side effects such as cytokine storms (Figure 5).

[0284] Experiment Example 4: In vitro activation of OVA-specific CD4-positive T cells (OT-2 T cells) by antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles activate antigen-specific CD4-positive T cells, the following tests were performed in vitro. Lymph nodes excised from OT-2 mice, which are OVA-reactive CD4TCR transgenic mice, were lysed on a 100 μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. Stained lymph node cells: 2 × 10⁶ 5The cells were suspended in 200 μL of RPMI1640 medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin. Extracellular vesicles from Example 3 or Reference Example 1 were added to achieve a final concentration of 10 μg / mL, and the cells were cultured in a 96-well round-bottom plate for 4 days. After 4 days, the cells were harvested and immunostained. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After staining, the luminescence intensity of CellTrace Violet, a cell proliferation assay reagent for OT-2 T cells, was detected using a flow cytometer FACSCanto II (BD Biosciences). • PE-Cy7 conjugate anti-mouse CD4 antibody (RM4-5, Biolegend) • APC conjugate anti-mouse TCR Vb5.1,5.2 antibody (MR9-4, Biolegend) The results are shown in Figure 6.

[0285] [result] The results of Test Example 4 showed that the antigen-presenting extracellular vesicles of Example 3 significantly differentiated and / or proliferated antigen-specific CD4 T cells compared to the extracellular vesicles of Reference Example 1 (Figure 6).

[0286] Experiment Example 5: In vitro differentiation induction of OVA-specific CD4-positive T cells (OT-2 T cells) into regulatory T cells by antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles induce antigen-specific CD4-positive T cells into regulatory T cells (Tregs), the following in vitro tests were performed. Lymph nodes excised from OT-2 mice, which are OVA-reactive CD4TCR transgenic mice, were lysed on a 100 μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. Stained lymph node cells: 2 × 10⁶ 5The cells were suspended in 200 μL of RPMI1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin. Antigen-presenting extracellular vesicles from Example 4 or Reference Example 1 were added to achieve a final concentration of 10 μg / mL, and the cells were cultured in a 96-well round-bottom plate for 4 days. After 4 days, the cells were harvested and extracellular immunostaining was performed. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After extracellular staining, intracellular immunostaining was performed using True-Nuclear Transcription Factor Buffer Set (Biolegend) and anti-mouse FOXP3 antibody according to the manufacturer's instructions. After intracellular staining, the expression of CD25 and FOXP3 molecules, which are regulatory T cell markers on OT-2 T cells, was detected using a flow cytometer FACSCanto II (BD Biosciences). • PE conjugate anti-mouse CD25 antibody (PC61, manufactured by Biolegend) • PE-Cy7 conjugate anti-mouse CD4 antibody (RM4-5, Biolegend) • APC conjugate anti-mouse TCR Vb5.1,5.2 antibody (MR9-4, Biolegend) • Alexa Fluor 488 conjugate anti-mouse FOXP3 antibody (MF-14 Biolegend) The results are shown in Figure 7.

[0287] [result] The results of Test Example 5 show that the antigen-presenting extracellular vesicles of Example 4 induced differentiation of antigen-specific CD4-positive T cells into regulatory T cells (preferably regulatory T cells expressing Foxp3) compared to the extracellular vesicles of Reference Example 1 (Figure 7).

[0288] Experiment Example 6: In vitro differentiation induction of OVA-specific CD4-positive T cells (OT-2 T cells) into Th2 T cells by antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles induce differentiation of antigen-specific CD4-positive T cells into Th2 T cells, the following in vitro tests were performed. Lymph nodes excised from OT-2 mice, which are OVA-reactive CD4TCR transgenic mice, were lysed on a 100 μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. Stained lymph node cells: 2 × 10⁶ 5 The cells were suspended in 200 μL of RPMI1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin. Extracellular vesicles presenting antigens from Example 3 or 5, or from Reference Example 1, were added to achieve a final concentration of 10 μg / mL, and the cells were cultured in a 96-well round-bottom plate for 4 days. After 4 days, the cells were harvested and extracellular immunostaining was performed. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After extracellular staining, intracellular immunostaining was performed using True-Nuclear Transcription Factor Buffer Set (Biolegend) and anti-GATA3 antibody according to the manufacturer's instructions. After intracellular staining, the luminescence intensity of CellTrace Violet, an OT-2 T cell proliferation assay reagent, and the expression of GATA3, a Th2 T cell marker, were detected using a flow cytometer FACSCanto II (BD Biosciences). • PE conjugate anti-mouse TCR Vb5.1,5.2 antibody (MR9-4, Biolegend) • PE-Cy7 conjugate anti-mouse CD4 antibody (RM4-5, Biolegend) • APC conjugate anti-GATA3 antibody (16E10A23, Biolegend) The results are shown in Figure 8.

[0289] [result] The results of Test Example 6 show that the antigen-presenting extracellular vesicles of Examples 3 and 5, compared to the extracellular vesicles of Reference Example 1, induced the differentiation of antigen-specific CD4-positive T cells into Th2 cells in vitro (Figure 8). Th2 cells secrete cytokines such as IL-4 and IL-5, which differentiate and activate naive B cells that recognize the same antigen, promoting the induction of antigen-specific IgE production (i.e., activation of humoral immunity).

[0290] [Example 6] Extracellular antigen-presenting vesicle containing MHC class II molecules, T cell costimulatory molecules, and T cell-stimulating cytokines in its membrane 4 HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Four plasmids prepared above (pCAG vectors encoding sc-Dimer-CD81-IL-12p40, MHC class IIα chain, CD80-CD9, and IL-12p35, respectively) were simultaneously transfected into cells at approximately 50% confluence using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3-12 hours after transfection, and 24 hours after transfection, the medium was changed to Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin, from which exosomes had been removed. 72 hours after transfection, the supernatant was collected, filtered 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle in Example 6. The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0291] Test Examples 1-5: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 6 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows. After staining, the expression of each fusion protein was detected using a FACSCanto II flow cytometer (manufactured by BD Biosciences). • Alexa Fluor 488 conjugate anti-mouse IA / IE antibody (M5 / 114.15.2 Biolegend) • PE conjugate anti-mouse IL-12 antibody (C15.6 Biolegend) • Results for APC conjugate anti-mouse CD80 antibody (16-10A1, Biolegend) are shown in Figure 3E.

[0292] [result] The results from Test Examples 1-5 show that the antigen-presenting extracellular vesicles of Example 6 contain MHC class II molecules that present the OVA antigen, CD80, and functional IL-12 in their membrane (Figure 3E).

[0293] [Example 7] Extracellular vesicle 5 containing an MHC class II molecule, a T cell costimulatory molecule, and a T cell stimulating cytokine in its membrane. HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were simultaneously transfected with five plasmids prepared above (pCAG vectors encoding sc-Dimer-CD81, MHC class IIα chain, CD80-CD9, CD81-IL-6, and TGF-β-MFGE8, respectively) using Polyethylenimine "Max" (Polysciences), according to the manufacturer's instructions. The medium was changed 3–12 hours after transfection, and 24 hours after transfection, the medium was changed again with Dulbecco's Modified Eagle Medium supplemented with exosome-removed 2% fetal bovine serum and penicillin / streptomycin. 72 hours after transfection, the supernatant was collected, filtered through a 0.22 μm filter, and 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle in Example 7. The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0294] Test Examples 1-6: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 7 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows. After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • FITC conjugate anti-mouse CD80 antibody (16-10A1, manufactured by Biolegend) • PE conjugate anti-mouse IL-6 antibody (MP5-20F3, manufactured by Biolegend) • APC-Cy7 conjugate anti-mouse IA / IE antibody (M5 / 114.15.2 Biolegend) • APC conjugate anti-mouse LAP (TGF-β1) antibody (TW7-16B4, manufactured by Biolegend) The results are shown in Figure 3F.

[0295] [result] The results from Test Examples 1-6 show that the antigen-presenting extracellular vesicles of Example 7 contain MHC class II molecules, CD80, IL-6, and TGFb that present the OVA antigen on their membrane (Figure 3F).

[0296] [Example 8] Establishment of cell lines stably expressing MHC class I molecules, T cell costimulatory molecules, and T cell-stimulating cytokines, and preparation of antigen-presenting extracellular vesicles PLAT-A cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were transfected with pMX vectors encoding CD80-CD9 or sc-Trimer-CD81-IL-2 using Polyethylenimine "Max" (Polysciences), according to the manufacturer's instructions. The medium was changed 12 hours after transfection, and the supernatant was collected 60 hours after transfection and centrifuged at 300 g for 5 minutes. The collected supernatant was used as viral particles. HEK293 cells were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. To cells with approximately 50% confluence, DOTAP transfection reagent (Roche) was added to virus particles incorporating the CD80-CD9 prepared above, according to the manufacturer's instructions, and then added to HEK293 cells. Cells with added virus particles were centrifuged at 2500 rpm for 3 hours. 24 hours after infection, the medium was changed, and after 1 week, CD80-positive cells were sorted using FACSMelody (BD Biosciences). After culturing the sorted CD80-positive cells for 1 week, they were seeded in dishes and cultured in Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. To cells with approximately 50% confluence, DOTAP transfection reagent was added to virus particles incorporating the sc-Trimer-CD81-IL-2 prepared above, according to the manufacturer's instructions, and then added to CD80-positive HEK293 cells. Cells with added virus particles were centrifuged at 2500 rpm for 3 hours. 24 hours after infection, the culture medium was changed, and one week later, CD80-positive, MHCI-positive cells were sorted using FACSMelody (BD Biosciences). The sorted cells were used as stable expression cells. The stable expression cells were seeded in dishes and cultured in Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum and penicillin / streptomycin.The supernatant of cells with approximately 50% confluence was replaced with Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum from which exosomes had been removed and penicillin / streptomycin. 72 hours after the medium change, 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 and centrifuged at 100,000 g for 2 hours, after which the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the antigen-presenting extracellular vesicle in Example 8.

[0297] Test Examples 1-7: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 8 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • PE conjugate anti-mouse H-2KbOVA complex antibody (25-D1.16 Biolegend) • FITC conjugate anti-mouse CD80 antibody (16-10A1, manufactured by Biolegend) • APC conjugate anti-mouse IL-2 antibody (JES6-5H4, manufactured by Biolegend) The results are shown in Figure 3G.

[0298] [result] The results from Test Examples 1-7 show that the antigen-presenting extracellular vesicles of Example 8 contain MHC class I molecules, CD80, and IL-2 that present the OVA antigen on their membrane (Figure 3G).

[0299] [Example 9] Antigen-presenting extracellular vesicle containing HLA class I molecule, human T cell costimulatory molecule, and human T cell stimulating cytokine in its membrane. HEK293T cells lacking B2m were seeded in cell culture dishes and cultured in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin. Cells at approximately 50% confluence were simultaneously transfected with two plasmids prepared above (pCAG vectors encoding HLAsc-Trimer-human CD81, human CD80-human CD9, and human CD63-IL2, respectively) using Polyethylenimine "Max" (Polysciences) according to the manufacturer's instructions. The medium was changed 3–12 hours after transfection, and 24 hours after transfection, the medium was changed to Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and penicillin / streptomycin from which exosomes had been removed. 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, then the supernatant was removed and the pellet was washed with PBS. PBS was added to the pellet and centrifuged at 100,000 g for 2 hours, then the supernatant was removed and the pellet was suspended in 100 μL of PBS, which was used as the humanized antigen-presenting extracellular vesicle in Example 9. The concentration of the antigen-presenting extracellular vesicle was measured using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. By using SARS-CoV2sc-Trimer-hCD81 instead of hsc-Trimer-hCD81, it is possible to create antigen-presenting MHC molecules that present the SARS-CoV2 peptide as an antigen, and humanized antigen-presenting extracellular vesicles that present hCD80 and hIL-2 on their surface.

[0300] Test Examples 1-8: Flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles The antigen-presenting extracellular vesicles from Example 9 were immunostained using the PS Capture® exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After staining, the expression of each fusion protein was detected using a FACSCantoII flow cytometer (manufactured by BD Biosciences). • APC conjugate anti-human IL-2 antibody (MQ1-17H12, manufactured by Biolegend) • PE conjugate anti-human CD80 antibody (2D10, manufactured by Biolegend) • APC conjugate anti-human β2m antibody (manufactured by 2M2 Biolegend) The results are shown in Figure 3H.

[0301] [result] The results from Test Examples 1-8 show that the antigen-presenting extracellular vesicles of Example 9 contain MHC class I molecules that present the WT1 antigen, hCD80, and hIL-2 on their membrane (Figure 3H).

[0302] Experiment Example 7: In vitro differentiation induction of OVA-specific CD4-positive T cells (OT-2 T cells) into Th1 T cells by antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles induce differentiation of antigen-specific CD4-positive T cells into Th1 T cells, the following in vitro tests were performed. Lymph nodes excised from OT-2 mice, which are OVA-reactive CD4TCR transgenic mice, were lysed on a 100 μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. Stained lymph node cells: 2 × 10⁶ 5The cells were suspended in 200 μL of RPMI1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin. Extracellular vesicles presenting antigens from Example 3 or 6, or from Reference Example 1, were added to achieve a final concentration of 10 μg / mL, and the cells were cultured in a 96-well round-bottom plate for 4 days. After 4 days, the cells were harvested and extracellular immunostaining was performed. The antibodies used for staining are as follows (staining time: 15 minutes, temperature: 4°C). After extracellular staining, intracellular immunostaining was performed using True-Nuclear Transcription Factor Buffer Set (Biolegend) and anti-T-bet antibody according to the manufacturer's instructions. After intracellular staining, the luminescence intensity of CellTrace Violet, an OT-2 T cell proliferation assay reagent, and the expression of T-bet, a Th1 T cell marker, were detected using a flow cytometer FACSCanto II (BD Biosciences). • PerCP / Cy5.5 conjugate anti-mouse TRVA2 antibody (B20.1 Biolegend) • APC-Cy7 conjugate anti-mouse CD4 antibody (RM4-5, Biolegend) • PE conjugate anti-T-bet antibody (4B10, manufactured by Biolegend) The results are shown in Figure 9.

[0303] [result] The results of Test Example 7 showed that the antigen-presenting extracellular vesicles of Example 6, compared to the extracellular vesicles of Reference Example 1, induced the differentiation of antigen-specific CD4-positive T cells into Th1 cells in vitro (Figure 9). Th1 cells produce IFN-γ and IL-2, etc., and promote the activation of macrophages and cytotoxic T cells that destroy pathogen cells, virus-infected cells, cancer cells, etc. (i.e., activation of cellular immunity).

[0304] Experiment Example 8: In vitro differentiation induction of OVA-specific CD4-positive T cells (OT-2 T cells) into Th17 T cells using antigen-presenting extracellular vesicles. To investigate whether antigen-presenting extracellular vesicles induce differentiation of antigen-specific CD4-positive T cells into Th17 T cells, the following in vitro tests were performed. Lymph nodes extracted from mice crossed with RORrt-GFP mice and OVA-reactive CD4TCR transgenic mice were lysed on a 100 μm filter to obtain a lymph node cell suspension. The cell suspension was stained using CellTrace Violet (Thermo Fisher Scientific), a cell proliferation assay reagent, according to the manufacturer's instructions. Stained lymph node cells: 2 × 10⁶ 5 The cells were suspended in 200 μL of RPMI1640 medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and penicillin / streptomycin. Antigen-presenting vesicles from Example 7 or extracellular vesicles from Reference Example 1 were added to achieve a final concentration of 10 μg / mL, and the cells were cultured in a 96-well round-bottom plate for 4 days. After 4...

Claims

1. (a) A polynucleotide comprising a sequence encoding a fusion protein (A) capable of presenting the antigen-presenting MHC molecule outside the membrane of an extracellular vesicle, the fusion protein comprising an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; (b) A polynucleotide comprising a sequence encoding a fusion protein (B) capable of presenting the T cell-stimulating cytokine outside the extracellular vesicle membrane, comprising a T cell-stimulating cytokine or a subunit thereof, and a membrane protein or its transmembrane domain capable of being expressed on the membrane of an extracellular vesicle, or a protein or its domain capable of binding to the membrane of an extracellular vesicle; and (c) A polynucleotide comprising a sequence encoding a fusion protein (C) capable of presenting the T cell costimulatory molecule outside the extracellular vesicle membrane, the fusion protein comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle, or a protein or its domain that can bind to the membrane of the extracellular vesicle. It is a combination of, The antigen-presenting MHC molecule is an MHC class I molecule or an MHC class II molecule. combination.

2. (d) A polynucleotide comprising a sequence encoding a fusion protein (D) capable of presenting the antigen-presenting MHC molecule and the T cell-stimulating cytokine outside the membrane of an extracellular vesicle, the fusion protein comprising an antigen-presenting MHC molecule, a T cell-stimulating cytokine or a subunit thereof, and a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle, The antigen-presenting MHC molecule is an MHC class I molecule or an MHC class II molecule. Polynucleotide.

3. The polynucleotide according to Claim 2; and (c) A polynucleotide comprising a sequence encoding a fusion protein (C) capable of presenting the T cell costimulatory molecule outside the extracellular vesicle membrane, the fusion protein comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle, or a protein or its domain that can bind to the membrane of the extracellular vesicle. A combination.

4. The polynucleotide combination according to claim 1, wherein the fusion protein defined in (A) above comprises an antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle.

5. The polynucleotide combination according to claim 1, wherein the fusion protein defined in (B) above comprises a T cell stimulating cytokine or a subunit thereof, and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle.

6. The polynucleotide combination according to claim 1, wherein the fusion protein defined in (A) above comprises an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain.

7. The polynucleotide combination according to claim 1, wherein the fusion protein defined in (B) above comprises a T cell stimulating cytokine or a subunit thereof and a partial sequence of tetraspanin, the partial sequence of tetraspanin having at least two transmembrane domains, and the T cell stimulating cytokine is positioned between the two transmembrane domains.

8. The polynucleotide combination according to claim 1, wherein the fusion protein defined in (B) above comprises a T cell stimulating cytokine or a subunit thereof and MFG-E8 or a domain thereof.

9. The fusion protein defined in (A) above is: From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) single chain MHC molecule, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the above.

10. The fusion protein defined in (A) above From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β ２ Microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the above.

11. Furthermore, β ２ A combination of polynucleotides according to claim 10, comprising a polynucleotide containing microglobulin, an MHC class Iα chain, an MHC class IIβ chain, or a sequence encoding an amino acid sequence of an MHC class IIα chain.

12. The fusion protein defined in (B) above From the N-terminus, (B-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, from the N-terminus. (B-2) A spacer arrangement that may exist, (B-3) T cell stimulating cytokines, (B-4) A spacer arrangement that may be present, and (B-5) Partial sequence of a tetraspanin containing transmembrane domain 4 A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the above.

13. The fusion protein defined in (B) above From the N-terminus, (B-3) T cell stimulating cytokines, (B-4) A spacer arrangement that may be present, and (B-5) MFG-E8 A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the above.

14. The fusion protein defined in (A) above is From the N-terminus, (A-1) MHC class I molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the following.

15. The fusion protein defined in (A) above is From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A combination of polynucleotides according to claim 1, comprising an amino acid sequence consisting of the above.

16. The polynucleotide combination according to claim 15, further comprising (A-6) a polynucleotide containing a sequence encoding the amino acid sequence of an MHC class IIα chain.

17. The fusion protein defined in (C) above is (C) A combination of polynucleotides according to claim 1 or 3, comprising a T cell costimulatory molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain.

18. The fusion protein defined in (C) above is From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanin A combination of polynucleotides according to claim 1 or 3, comprising an amino acid sequence consisting of the above.

19. The fusion protein defined in (D) above is From the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) single chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, the T cell stimulating cytokine or its subunit, The polynucleotide according to claim 2, comprising an amino acid sequence encoding in this order.

20. The fusion protein defined in (D) above is From the N-terminus, (D-1) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and the T cell stimulating cytokine or its subunit. (D-2) A spacer arrangement that may exist, (D-3) single chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, The polynucleotide according to claim 2, comprising an amino acid sequence encoding in this order.

21. A fusion peptide comprising the tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, From the N-terminus, (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) A spacer arrangement that may exist, (3) The T cell stimulating cytokine or its subunit, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 The polynucleotide according to claim 19 or 20, comprising an amino acid sequence encoding in this order.

22. A fusion peptide comprising the tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, From the N-terminus, (1) The T cell stimulating cytokine or its subunit, (2) A spacer arrangement which may exist, and (3) MFG-E8 The polynucleotide according to claim 19 or 20, comprising an amino acid sequence encoding in this order.

23. The MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular domain of an MHC class I α chain. or The MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular domain of the MHC class IIα chain and / or the extracellular domain of the MHC class IIβ chain. The polynucleotide according to claim 19 or 20.

24. The polynucleotide or combination of polynucleotides according to any one of claims 1 to 23, wherein the T cell stimulating cytokine is IL-2, IL-4, IL-6, IL-12, a subunit of IL-12, or TGF-β.

25. The combination of polynucleotides or polynucleotides according to any one of claims 1 to 24 A vector containing; or A combination of vectors comprising each polynucleotide of the polynucleotide combination according to any one of claims 1, 3 to 18, or 24.

26. A combination of polynucleotides or polynucleotides according to any one of claims 1 to 24; or The vector or combination of vectors described in claim 25, A pharmaceutical composition comprising a pharmacologically acceptable carrier.

27. For the treatment or prevention of infectious diseases, cancer, autoimmune diseases, or allergic diseases, The pharmaceutical composition according to claim 26.

28. A method for activating and / or proliferating T cells in response to a specific antigen, A combination of polynucleotides or polynucleotides according to any one of claims 1 to 24; or The vector or combination of vectors described in claim 25 A method comprising introducing into cells in vitro or ex vivo to generate antigen-presenting cells and / or antigen-presenting extracellular vesicles, and contacting the generated antigen-presenting cells and / or antigen-presenting extracellular vesicles with T cells in vitro or ex vivo.

29. An antigen-presenting extracellular vesicle that presents antigen-presenting MHC molecules and T cell-stimulating cytokines outside the membrane, The following is on the membrane: (A) A fusion protein or protein complex capable of presenting the antigen extracellularly, comprising the antigen-presenting MHC molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; (B) A fusion protein capable of presenting the T cell-stimulating cytokine extracellularly, comprising the T cell-stimulating cytokine or its subunit and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle; and (C) A fusion protein that allows interaction between the T cell costimulatory molecule and T cells, comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of an extracellular vesicle, or a protein or its domain that can bind to the membrane of an extracellular vesicle. Includes, The antigen-presenting MHC molecule is an MHC class I molecule or an MHC class II molecule. Antigen-presenting extracellular vesicles.

30. An antigen-presenting extracellular vesicle that presents antigen-presenting MHC molecules and T cell-stimulating cytokines outside the membrane, The following is on the membrane: (D) the antigen-presenting MHC molecule; The T cell stimulating cytokine or its subunit and A fusion protein comprising a membrane protein or its transmembrane domain capable of localizing to the membrane of an extracellular vesicle, or a protein or its membrane-binding domain capable of binding to the membrane of an extracellular vesicle. Includes, The antigen-presenting MHC molecule is an MHC class I molecule or an MHC class II molecule. Antigen-presenting extracellular vesicles.

31. (C) An antigen-presenting extracellular vesicle according to claim 30, further comprising in its membrane a fusion protein that can interact with a T cell, comprising a T cell costimulatory molecule and a membrane protein or its transmembrane domain that can be expressed on the membrane of the extracellular vesicle, or a protein or its domain that can bind to the membrane of the extracellular vesicle.

32. An antigen-presenting extracellular vesicle according to claim 29, wherein its membrane has the following: (A) A fusion protein or protein complex capable of presenting the antigen extramembrane, comprising an antigen-presenting MHC molecule and a tetraspanin or its transmembrane domain or MFG-E8 or its domain; and (B) A fusion protein capable of presenting the T cell-stimulating cytokine extramembrane, comprising a T cell-stimulating cytokine or a subunit thereof and a partial sequence of tetraspanin, wherein the partial sequence of tetraspanin has at least two transmembrane domains, and the T cell-stimulating cytokine is positioned between the two transmembrane domains, or (B) A fusion protein comprising a T cell-stimulating cytokine or a subunit thereof and MFG-E8 or its domain, capable of presenting the T cell-stimulating cytokine extramembrane; An extracellular vesicle containing an antigen presenting cell.

33. An antigen-presenting extracellular vesicle according to claim 29, wherein its membrane has the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminal side, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) single chain MHC molecule, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A fusion protein comprising an amino acid sequence consisting of, (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC molecule-restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class Iα chain, β2 microglobulin, MHC class IIα chain, or MHC class IIβ chain (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) β2 microglobulin, a protein containing the amino acid sequence of an MHC class Iα chain, an MHC class IIβ chain, or an MHC class IIα chain Protein complexes including; and (B) From the N-terminus, (B-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, from the N-terminus. (B-2) A spacer arrangement that may exist, (B-3) T cell stimulating cytokines or their subunits, (B-4) A spacer arrangement that may be present, and (B-5) Partial sequence of a tetraspanin containing transmembrane domain 4 A fusion protein comprising an amino acid sequence consisting of the above, capable of presenting the T cell-stimulating cytokine extramembrane, or (B) From the N-terminus, (B-3) T cell stimulating cytokines or their subunits, (B-4) A spacer arrangement that may be present, and (B-5) MFG-E8 A fusion protein comprising an amino acid sequence comprising the above, capable of presenting the T cell-stimulating cytokine extramembrane; An extracellular vesicle containing an antigen presenting cell.

34. An antigen-presenting extracellular vesicle according to claim 29, wherein the membrane comprises the following: (A) A fusion protein capable of presenting an antigen peptide outside the membrane, wherein from the N-terminal side, (A-1) MHC class I molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) Single-chain MHC class I molecule, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of the following: An extracellular vesicle containing an antigen presenting cell.

35. An antigen-presenting extracellular vesicle according to claim 29, wherein the membrane comprises the following: (A) A protein complex capable of presenting an antigen peptide outside the membrane, From the N-terminus, (A-1) MHC class II molecularly restricted antigen peptide, (A-2) A spacer arrangement that may exist, (A-3) MHC class IIβ chain, (A-4) A spacer arrangement that may be present, and (A-5) Tetraspanin A fusion protein containing an amino acid sequence consisting of, (A-6) Proteins containing the amino acid sequence of the MHC class IIα chain protein complex An extracellular vesicle containing an antigen presenting cell.

36. An extracellular vesicle presenting an antigen according to claim 30, An antigen-presenting extracellular vesicle in which 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 is tetraspanin or MFG-E8.

37. An antigen-presenting extracellular vesicle according to claim 30, The aforementioned fusion protein, from the N-terminus, (D-1) MHC molecule-restricted antigen peptide, (D-2) A spacer arrangement that may exist, (D-3) single chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, the T cell stimulating cytokine or its subunit, An extracellular vesicle that presents an antigen and contains an amino acid sequence that codes for this in this order.

38. An extracellular antigen-presenting vesicle according to claim 30, The aforementioned fusion protein, from the N-terminus, (D-1) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain, and the T cell stimulating cytokine or its subunit. (D-2) A spacer arrangement that may exist, (D-3) single chain MHC molecule, (D-4) A spacer arrangement that may be present, and (D-5) MHC molecule-restricted antigen peptide, An extracellular vesicle that presents an antigen and contains an amino acid sequence that codes in this order.

39. The fusion peptide is, from the N-terminus, (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) A spacer arrangement that may exist, (3) The T cell stimulating cytokine or its subunit, (4) A spacer arrangement which may exist, and (5) Partial sequence of tetraspanin containing transmembrane domain 4 An antigen-presenting extracellular vesicle according to claim 37 or 38, comprising an amino acid sequence encoding in this order.

40. The fusion peptide is, from the N-terminus, (1) The T cell stimulating cytokine or its subunit, (2) A spacer arrangement which may exist, and (3) MFG-E8 An antigen-presenting extracellular vesicle according to claim 37 or 38, comprising an amino acid sequence encoding in this order.

41. The MHC molecule-restricted antigen peptide is an MHC class I molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular domain of an MHC class I α chain; Or, The MHC molecule-restricted antigen peptide is an MHC class II molecule-restricted antigen peptide, and the single-chain MHC molecule includes the extracellular domain of the MHC class IIα chain and / or the extracellular domain of the MHC class IIβ chain. An antigen-presenting extracellular vesicle according to claim 37 or 38.

42. The antigen-presenting extracellular vesicle according to any one of claims 29 to 41, wherein the T cell-stimulating cytokine is IL-2, IL-4, IL-6, IL-12, or TGF-β, or a subunit thereof.

43. An antigen-presenting extracellular vesicle according to claim 29 or 31, wherein its membrane comprises the following: (C) A fusion protein comprising a T cell costimulatory molecule and a tetraspanin or its transmembrane domain, or MFG-E8 or its domain, wherein the T cell costimulatory molecule and T cells can interact; An extracellular vesicle containing an antigen presenting cell.

44. An antigen-presenting extracellular vesicle according to claim 43, wherein its membrane has the following: (C) From the N-terminus, (C-1) T cell costimulatory molecule, (C-2) A spacer arrangement that may be present, and (C-3) Tetraspanin A fusion protein comprising an amino acid sequence comprising the above, which is capable of interacting with the T cell costimulatory molecule and T cells; An extracellular vesicle containing an antigen presenting cell.

45. A protein or protein complex defined in (A) is fused with a protein or protein complex defined in (B); A protein or protein complex as defined in (A) is fused with a protein or protein complex as defined in (C); A protein or protein complex as defined in (B) is fused with a protein or protein complex as defined in (C); or A protein or protein complex as defined in (A) is fused with a protein or protein complex as defined in (B) and a protein or protein complex as defined in (C). The antigen-presenting extracellular vesicle according to claim 29.

46. The antigen-presenting extracellular vesicle according to claim 31, wherein the (D) fusion protein and the (C) protein capable of interacting with T cells are fused.

47. The antigen-presenting extracellular vesicle according to any one of claims 29 to 46, wherein the extracellular vesicle is an exosome.

48. A pharmaceutical composition comprising an antigen-presenting extracellular vesicle according to any one of claims 29 to 47.

49. For treating or preventing infectious diseases, cancer, autoimmune diseases or allergic diseases, The pharmaceutical composition according to claim 48.

50. The pharmaceutical composition according to claim 49 for treating or preventing cancer, further comprising an immune checkpoint inhibitor.

51. The pharmaceutical composition according to claim 50, wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting extracellular vesicle.

52. The pharmaceutical composition according to claim 50 or 51, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or an active fragment thereof; an anti-CTLA-4 antibody or an active fragment thereof; and a PD-L1 antibody or an active fragment thereof.

53. A method for activating and / or proliferating T cells in response to a specific antigen, comprising contacting an antigen-presenting extracellular vesicle according to any one of claims 29 to 47 with T cells in vitro or ex vivo.

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