Immunoregulatory method, nucleic acid composition for immunoregulation, and use thereof

Abstract

Provided is a polynucleotide from which a cell or extracellular vesicle capable of satisfactorily activating antigen-specific T cells can be produced.　Provided is a polynucleotide containing 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 on the outside of the membrane of a cell or 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 on the outside of the membrane of a cell or 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 on the outside of the membrane of a cell or 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 on the outside of the membrane of a cell or extracellular vesicle; and (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 on the outside of the membrane of a cell or extracellular vesicle.

Description

Immunoregulation method, immunoregulatory nucleic acid composition and use thereof

[0001] <Cross Reference> This application claims priority from a Japanese patent application (Patent Application No. 2021-142688) filed on September 1, 2021, the entire contents of which, including prior art documents, are incorporated herein by reference.

[0002] The present invention relates to a method for immunoregulation, a nucleic acid composition for immunoregulation, and uses thereof.

[0003] It is known that antigen-specific T cells (e.g., cytotoxic T cells, helper T cells, etc.) play a central role in immune responses such as the elimination of cancer cells and the like by the living body, and the regulation of responses to autoantigens, allergens, etc. Antigen-specific T cells recognize binding complexes 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, etc., that present antigens, and regulate responses to autoantigens, allergens, etc. Therefore, it is believed that activating, proliferating, differentiating, etc., antigen-specific T cells is particularly important in immune responses.

[0004] As a method for activating antigen-specific T cells, in addition to the already-practical method of expressing chimeric antigen receptors in T cells, other methods have also been developed. For example, Patent Document 1 discloses that nanoparticles containing MHC molecules and T cell costimulatory molecules on their surface proliferate antigen-specific T cells. Furthermore, Non-Patent Document 1 discloses that exosomes expressing IL-12 on their membranes via PTGFRN proliferate model antigen-specific CD8-positive T cells.

[0005] Special Publication No. 2016-520518

[0006] 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, 2021, 34th Annual Meeting of the Society for Immuno-therapy of CancerJournal of Extracellular Vesicles(2018);7:1535750ONCOIMMUNOLOGY 2020, VOL. 9, NO. 1, e1673125

[0007] The present inventors have attempted to use extracellular vesicles containing MHC molecules and T cell costimulatory molecules in their membranes as a new method for activating antigen-specific T cells, but when attempts were made to activate antigen-specific T cells using these extracellular vesicles, they found for the first time that they were unable to satisfactorily activate antigen-specific T cells.

[0008] Therefore, an object of the present invention is to provide a novel immunoregulatory method, a nucleic acid composition for immunoregulation, and uses thereof.

[0009] In view of the above problems, the present inventors conducted extensive research and unexpectedly found that T cells can be activated by using a polynucleotide capable of producing cells or extracellular vesicles containing an MHC molecule and a T cell-stimulating cytokine in the membrane, and thus completed the present invention.

[0010] Thus, the present invention includes the following: [0] A cell or extracellular vesicle that presents an antigen-presenting MHC molecule and a T cell-stimulating cytokine on the extracellular side. [1] An antigen-presenting cell or antigen-presenting extracellular vesicle comprising, in its membrane, the following: a protein (A) comprising an antigen-presenting MHC molecule, capable of presenting the antigen on the extracellular side; and a protein (B) comprising a T cell-stimulating cytokine or a subunit thereof, capable of presenting the T cell-stimulating cytokine on the extracellular side. [2] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1], wherein the protein (A) comprising an antigen-presenting MHC molecule, capable of presenting the antigen on the extracellular side, is a fusion protein or protein complex capable of presenting the antigen on the extracellular side, comprising an antigen-presenting MHC molecule and a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein that can bind to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof. [3] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1], wherein the protein (B) capable of extramembrane presentation of the T cell-stimulating cytokine, comprising the T cell-stimulating cytokine or a subunit thereof, is a fusion protein capable of extramembrane presentation of the T cell-stimulating cytokine, comprising the T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof.[4] The protein (A) capable of presenting an antigen extramembraneably, comprising the antigen-presenting MHC molecule, is: 1) a fusion protein or protein complex capable of presenting the antigen extramembraneably, comprising an antigen-presenting MHC molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a membrane-binding domain thereof; 2) a fusion protein comprising an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a membrane-binding domain thereof; 3) a fusion protein comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) an MHC class I α chain or β chain. ２ (A-4) an amino acid sequence of an MHC class II α chain or an MHC class II β chain, in this order; (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a β ２and a protein comprising the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain; 4) a fusion protein comprising an amino acid sequence comprising, from its N-terminus, (A-1) an MHC class I molecule-restricted antigenic peptide, (A-2) the amino acid sequence of an optionally present spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class I molecule, (A-4) the spacer sequence that may be present, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof; 5) a fusion protein comprising, from its N-terminus, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) the spacer sequence that may be present, (A-3) the amino acid sequence of an MHC class II β chain, (A-4) the spacer sequence that may be present, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. (A-6) a protein comprising the amino acid sequence of an MHC class II α chain; 6) a protein complex comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order, (A-1) an amino acid sequence of an MHC class II molecule-restricted antigen peptide, (A-2) an optional spacer sequence, (A-3) an amino acid sequence of an MHC class II β chain, (A-4) an optional spacer sequence, and (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain.

[0011] [5] The protein (B) comprising the T cell stimulating cytokine or a subunit thereof and capable of extramembrane display of the T cell stimulating cytokine is selected from the group consisting of: 1) a fusion protein comprising a T cell stimulating cytokine or a subunit thereof and a partial sequence of a tetraspanin, capable of extramembrane display of the T cell stimulating cytokine, wherein the partial sequence of the tetraspanin has at least two transmembrane domains and the T cell stimulating cytokine is disposed between the two transmembrane domains; 2) a fusion protein comprising a T cell stimulating cytokine or a subunit thereof and MFG-E8 or a domain thereof, capable of extramembrane display of the T cell stimulating cytokine; 3) a fusion protein comprising, from the N-terminus thereof: (B-1) a partial sequence of a tetraspanin comprising, from the N-terminus thereof, transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3; (B-2) an optional spacer sequence; (B-3) the amino acid sequence of a T cell stimulating cytokine; (B-4) an optional spacer sequence; (B-5) a partial sequence of a tetraspanin comprising transmembrane domain 4, in this order; or 4) a fusion protein capable of extramembrane presentation of a first T cell-stimulating cytokine, comprising an amino acid sequence comprising, from the N-terminus thereof, (B-3) the amino acid sequence of a T cell-stimulating cytokine, (B-4) an optional spacer sequence, and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof, in this order. [6] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1], wherein the T cell-stimulating cytokine or a subunit thereof is IL-2, IL-4, IL-6, IL-12, a subunit of IL-12, IL-15, or TGF-β.

[0012] [7] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1], further comprising in its membrane: a protein (C) comprising a T cell costimulatory molecule, capable of interacting with a T cell; [8] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [7], wherein the protein (C) comprising a T cell costimulatory molecule, capable of interacting with a T cell, is a fusion protein comprising a T cell costimulatory molecule and a membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle, or a protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle, capable of interacting with the T cell costimulatory molecule. [9] The antigen-presenting extracellular vesicle according to [7], wherein the protein (C) comprising a T cell costimulatory molecule and capable of interacting with a T cell comprises: 1) a fusion protein comprising a T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof, capable of interacting with the T cell; 2) a fusion protein comprising an amino acid sequence comprising, from the N-terminus, the following in this order: (C-1) the amino acid sequence of a T cell costimulatory molecule, (C-2) an optional spacer sequence, and (C-3) the amino acid sequence of a tetraspanin or a transmembrane domain thereof.

[10] The antigen-presenting cell according to [7], wherein the protein (C) comprising a T cell costimulatory molecule and capable of interacting with a T cell comprises: a fusion protein comprising a T cell costimulatory molecule comprising a transmembrane domain, capable of interacting with the T cell.

[11] The antigen-presenting extracellular vesicle according to [1], wherein the extracellular vesicle is an exosome.

[0013] [1A] An antigen-presenting cell or antigen-presenting extracellular vesicle, the membrane of which comprises: an antigen-presenting MHC molecule and a T cell-stimulating cytokine or a subunit thereof, and a fusion protein (D) capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine. [2A] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1A], wherein the fusion protein (D) comprises an antigen-presenting MHC molecule and a T cell-stimulating cytokine or a subunit thereof, and capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine comprises the antigen-presenting MHC molecule, the T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of localizing in the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof. [3A] The antigen-presenting extracellular vesicle according to [2A], wherein the membrane protein capable of being localized in 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] The antigen-presenting cell according to [2A], wherein the membrane protein capable of being localized in the membrane of the cell or the protein capable of binding to the membrane of the cell is CD8.[5A] The fusion protein (D) comprises: 1) an amino acid sequence encoding, from the N-terminus, (D-1) an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and the T cell-stimulating cytokine or a subunit thereof; 2) an amino acid sequence encoding, from the N-terminus, (D-1) a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and the T cell-stimulating cytokine or a subunit thereof, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) an MHC molecule-restricted antigen peptide, in this order; or 3) An antigen-presenting extracellular vesicle according to [3A], comprising, from its N-terminus, (1) at least one T cell-stimulating cytokine or a subunit thereof, (2) an optional spacer sequence, and (3) an amino acid sequence encoding MFG-E8 in this order. [6A] The antigen-presenting cell according to [4A], wherein the fusion protein (D) comprises: 1) an amino acid sequence encoding, from the N-terminus, (D-1) an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule comprising a transmembrane domain, (D-4) an optional spacer sequence, and (D-5) a fusion peptide comprising CD8 or a transmembrane domain thereof, and the T cell-stimulating cytokine or a subunit thereof, in this order; or 2) an amino acid sequence encoding, from the N-terminus, (D-1) a fusion peptide comprising CD8 or a transmembrane domain thereof, and the T cell-stimulating cytokine or a subunit thereof, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule comprising a transmembrane domain, (D-4) an optional spacer sequence, and (D-5) an MHC molecule-restricted antigen peptide, in this order.[8A] The antigen-presenting cell according to [6A], wherein the fusion peptide comprises an amino acid sequence encoding, from the N-terminus, (1) the T cell-stimulating cytokine or a subunit thereof, (2) an optional spacer sequence, and (3) CD8 or a transmembrane domain thereof. [9A] The antigen-presenting extracellular vesicle according to [5A], wherein the MHC molecule-restricted antigenic peptide is an MHC class I molecule-restricted antigenic peptide, and the single-chain MHC molecule comprises the extracellular region of an MHC class I α chain. [10A] The antigen-presenting extracellular vesicle according to [5A], wherein the MHC molecule-restricted antigenic peptide is an MHC class II molecule-restricted antigenic 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. [11A] The antigen-presenting cell according to [7A], wherein the MHC molecule-restricted antigenic peptide is an MHC class I molecule-restricted antigenic peptide, and the single-chain MHC molecule comprising the transmembrane domain comprises an MHC class I α chain. [12A] The antigen-presenting cell according to [7A], wherein the MHC molecule-restricted antigenic peptide is an MHC class II molecule-restricted antigenic peptide, and the single-chain MHC molecule comprising the transmembrane domain comprises an MHC class II α chain and / or an MHC class II β chain.

[0014] [13A] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1A], further comprising in its membrane a protein (C) comprising a T cell costimulatory molecule and capable of interacting with the T cell. [14A] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [13A], wherein the protein (C) comprises the T cell costimulatory molecule and a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof. [15A] The antigen-presenting extracellular vesicle according to [14A], wherein the protein (C) comprises the T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof. [16A] The antigen-presenting cell according to [14A], wherein the protein (C) comprises a T cell costimulatory molecule comprising a transmembrane domain. [17A] The antigen-presenting extracellular vesicle according to [1A], wherein the extracellular vesicle is an exosome.

[0015] [1B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [1], wherein the protein (A) and the protein (B) are fused to form a single protein. (C) [2B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [8], wherein the protein (A) and the protein (C) are fused to form a single protein. [3B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [8], wherein the protein (B) and the protein (C) are fused to form a single protein. [4B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [8], wherein the protein (A), the protein (B), and the protein (C) are fused to form a single protein. [5B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to [13A], wherein the protein (D) and the protein (C) are fused to form a single protein. [6B] The antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1B] to [5B], wherein the extracellular vesicle is an exosome.

[0016] [7B] A pharmaceutical composition comprising the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] and a pharmacologically acceptable carrier.

[0017] [1C] A pharmaceutical composition for treating or preventing cancer, comprising the antigen-presenting cell or antigen-presenting extracellular vesicle described in any one of [1] to [6B]; wherein the antigen peptide preferably comprises a cancer antigen peptide. [2C] A pharmaceutical composition for treating or preventing an autoimmune disease, comprising the antigen-presenting cell or antigen-presenting extracellular vesicle described in any one of [1] to [6B]; wherein the antigen peptide preferably comprises an autoantigen peptide. [3C] A pharmaceutical composition for treating or preventing an allergic disease, comprising the antigen-presenting cell or antigen-presenting extracellular vesicle described in any one of [1] to [6B]; wherein the antigen peptide preferably 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 cell or 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 the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] and a pharmacologically acceptable carrier; wherein the antigen peptide is preferably derived from an infectious pathogen that causes the infectious disease.

[0018] [1D] The antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] for use in the treatment or prevention of cancer; wherein preferably the antigenic peptide comprises a cancer antigen peptide. [2D] The antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] for use in the treatment or prevention of autoimmune disease; wherein preferably the antigenic peptide comprises an autoantigenic peptide. [3D] The antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] for use in the treatment or prevention of allergic disease; wherein preferably the antigenic peptide comprises an allergen. [4D] The antigen-presenting cell or antigen-presenting extracellular vesicle for use according to [1D], which is used together with an immune checkpoint inhibitor. [5D] The antigen-presenting cell or antigen-presenting extracellular vesicle for use according to [4D], wherein the immune checkpoint inhibitor is present on a membrane. [6D] The antigen-presenting cell or 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] The antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B], for use in treating or preventing an infectious disease; wherein the antigen peptide is preferably derived from an infectious pathogen that causes the infectious disease.

[0019] [1E] Use of the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] in the manufacture of a medicament for treating or preventing cancer; wherein the antigen peptide preferably comprises a cancer antigen peptide. [2E] Use of the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] in the manufacture of a medicament for treating or preventing an autoimmune disease; wherein the antigen peptide preferably comprises an autoantigen peptide. [3E] Use of the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] in the manufacture of a medicament for treating or preventing an allergic disease; wherein the antigen peptide preferably comprises an allergen. [4E] The use according to [1E], wherein the medicament is used together 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 cell or 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 the antigen-presenting cell or antigen-presenting extracellular vesicle according to any one of [1] to [6B] in the manufacture of a medicament for treating or preventing an infectious disease; wherein the antigen peptide is preferably derived from an infectious pathogen that causes the infectious disease.

[0020] [1F] A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of the antigen-presenting cells or antigen-presenting extracellular vesicles described in any one of [1] to [6B] to activate and / or proliferate T cells in the subject that recognize a cancer antigen, and causing the activated and / or proliferated T cells to attack cancer cells, thereby treating or preventing cancer; wherein preferably the activated and / or proliferated T cells are CD8-positive cytotoxic T cells, and preferably the antigen peptide comprises a cancer antigen peptide. [2F] A method for treating or preventing autoimmune disease in a subject, comprising administering to the subject an effective amount of the antigen-presenting cells or antigen-presenting extracellular vesicles described in any one of [1] to [6B] to activate and / or proliferate T cells in the subject that recognize an autoantigen, thereby desensitizing the immune response to the autoantigen in the subject, thereby treating or preventing autoimmune disease; wherein preferably the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably the antigen peptide comprises an autoantigen peptide. [3F] A method for treating or preventing an allergic disease in a subject, comprising administering to the subject an effective amount of the antigen-presenting cells or antigen-presenting extracellular vesicles described in any one of [1] to [6B] to activate and / or proliferate T cells in the subject that recognize an allergen, thereby 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 (Tregs), and preferably the antigen peptide comprises an allergen. [4F] The method of [1F], wherein the antigen-presenting cells or antigen-presenting extracellular vesicles are administered together with an immune checkpoint inhibitor. [5F] The method of [4F], wherein the immune checkpoint inhibitor is present on the membrane of the antigen-presenting cells or antigen-presenting extracellular vesicles. [6F] The method of [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 to the subject an effective amount of the antigen-presenting cells or antigen-presenting extracellular vesicles described in any one of [1] to [6B], to 1) cause the secretion of inflammatory cytokines, thereby activating the innate immunity of the subject, and / or 2) endowing the subject with acquired immunity against the infectious pathogen that causes the infectious disease, thereby eliminating the infectious pathogen that causes the infectious disease in the body and / or suppressing its proliferation.

[0021] [1G] A method for activating and / or proliferating T cells against a specific antigen, the method comprising contacting T cells with an effective amount of the antigen-presenting cells or antigen-presenting extracellular vesicles described in any one of [1] to [6B] in vitro or ex vivo.

[0022] [1H] A fusion protein (A) comprising an antigen-presenting MHC molecule and capable of presenting the antigen-presenting MHC molecule on the extracellular membrane of a cell or extracellular vesicle. [2H] A fusion protein (B) comprising at least one T cell stimulatory cytokine or subunit thereof and capable of presenting the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle. [3H] A fusion protein (C) comprising a T cell costimulatory molecule and capable of presenting the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle. [4H] A fusion protein (D) comprising an antigen-presenting MHC molecule and at least one T cell stimulatory cytokine or subunit thereof and capable of presenting the antigen and the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle. [5H] A fusion protein (E) comprising an antigen-presenting MHC molecule and at least one T cell stimulatory cytokine or subunit thereof and a T cell costimulatory molecule and capable of presenting the antigen, the T cell stimulatory cytokine, and the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle.

[0023] [1I] (a) a sequence encoding a fusion protein (A) comprising an antigen-presenting MHC molecule and capable of presenting the antigen-presenting MHC molecule on the extracellular membrane of a cell or extracellular vesicle; (b) a sequence encoding a fusion protein (B) comprising at least one T cell stimulatory cytokine or a subunit thereof and capable of presenting the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle; (c) a sequence encoding a fusion protein (C) comprising a T cell costimulatory molecule and capable of presenting the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle; (d) a sequence encoding a fusion protein (D) comprising an antigen-presenting MHC molecule and at least one T cell stimulatory cytokine or a subunit thereof and capable of presenting the antigen and the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle; and (e) a sequence encoding a fusion protein (E) that comprises an antigen-presenting MHC molecule, at least one T cell stimulatory cytokine or a subunit thereof, and a T cell costimulatory molecule, and that is capable of presenting the antigen, the T cell stimulatory cytokine, and the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle;

[0024] [2I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an antigen-presenting MHC molecule and a membrane protein capable of being expressed on the membrane of a cell or an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or an extracellular vesicle or a membrane-binding domain thereof. [3I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an antigen-presenting MHC molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a membrane-binding domain thereof. [4I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an antigen-presenting MHC molecule comprising a transmembrane domain. [5I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. [6I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (A-2) an optional spacer sequence, and (A-3) an MHC class I α chain, β chain, or a tetraspanin. ２ The polynucleotide according to [1I], further comprising: (A-6) an amino acid sequence of a β-microglobulin, an MHC class II α chain, or an MHC class II β chain; (A-4) an optionally present spacer sequence; and (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order. ２The polynucleotide according to [6I], comprising the amino acid sequence of microglobulin, MHC class I α chain, MHC class II β chain, or MHC class II α chain. [8I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, from the N-terminus thereof, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class I molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. [9I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, in this order from the N-terminus, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of an MHC class II β chain, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. [10I] The polynucleotide according to [9I], further comprising (A-6) a sequence encoding the amino acid sequence of an MHC class II α chain. [11I] The polynucleotide according to [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, in this order from the N-terminus, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC molecule comprising a transmembrane domain. [12I] The fusion protein defined in (A) above comprises, from its N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (A-2) an optional spacer sequence, and (A-3) an MHC class I α chain, β chain, or both. ２ The polynucleotide according to [1I], further comprising an amino acid sequence of a fusion protein of a microglobulin and an MHC class I α chain, an MHC class II α chain, or an MHC class II β chain, in this order. ２The polynucleotide according to [12I], comprising a sequence encoding the amino acid sequence of a microglobulin, an MHC class II beta chain, or an MHC class II alpha chain. [14I] The polynucleotide of [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC class I molecule comprising a transmembrane domain. [15I] The polynucleotide of [1I], wherein the fusion protein defined in (A) comprises an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigen peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of an MHC class II β chain. [16I] The polynucleotide of [15I], further comprising a sequence encoding the amino acid sequence of an MHC class II α chain.

[0025] [17I] The polynucleotide of [1I], wherein the fusion protein defined in (B) comprises at least one T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of being expressed on the membrane of a cell or an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or an extracellular vesicle or a domain thereof. [18I] The polynucleotide of [1I], wherein the fusion protein defined in (B) comprises at least one T cell-stimulating cytokine or a subunit thereof, and a partial sequence of a tetraspanin, the partial sequence of the tetraspanin having at least two transmembrane domains, and the at least one T cell-stimulating cytokine being disposed between the two transmembrane domains. [19I] The polynucleotide of [1I], wherein the fusion protein defined in (B) comprises at least one T cell-stimulating cytokine or a subunit thereof, and MFG-E8 or a membrane-binding domain thereof. [20I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) comprises an amino acid sequence comprising, from its N-terminus, (B-1) a partial sequence of a tetraspanin comprising, from the N-terminus, transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (B-2) an optional spacer sequence, (B-3) the amino acid sequence of a first T cell-stimulating cytokine, (B-4) an optional spacer sequence, and (B-5) a partial sequence of a tetraspanin comprising transmembrane domain 4. [21I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) comprises an amino acid sequence comprising, from its N-terminus, (B-3) the amino acid sequence of a first T cell-stimulating cytokine, (B-4) an optional spacer sequence, and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof, in this order. [22I] The polynucleotide of claim 1, wherein the fusion protein defined in (B) comprises at least one T cell stimulating cytokine or a subunit thereof and CD8 or a transmembrane domain thereof.[23I] The polynucleotide according to [1I], wherein the fusion protein defined in (B) comprises an amino acid sequence comprising, from the N-terminus thereof, (B-3) the amino acid sequence of a first T cell-stimulating cytokine, (B-4) an optional spacer sequence, and (B-5) the amino acid sequence of CD8 or a transmembrane domain thereof. [24I] 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, IL-15, or TGF-β.

[0026] [25I] The polynucleotide of [1I], wherein the fusion protein defined in (C) comprises a T cell costimulatory molecule and a membrane protein capable of being expressed on the membrane of a cell or an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or an extracellular vesicle or a membrane-binding domain thereof. [26I] The polynucleotide of [1I], wherein the fusion protein defined in (C) comprises a T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a membrane-binding domain thereof. [27I] The polynucleotide of [1I], wherein the fusion protein defined in (C) comprises a T cell costimulatory molecule comprising a transmembrane domain. [28I] The polynucleotide according to [1I], wherein the fusion protein defined in (C) comprises an amino acid sequence comprising, from the N-terminus, (C-1) the amino acid sequence of a T cell costimulatory molecule, (C-2) an optional spacer sequence, and (C-3) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order.

[0027] [29I] The polynucleotide according to [1I], wherein the fusion protein defined in (D) comprises: the antigen-presenting MHC molecule; the at least one T cell-stimulating cytokine or a subunit thereof; and a membrane protein capable of localizing in the membrane of a cell or an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or an extracellular vesicle or a membrane-binding domain thereof. [30I] The polynucleotide according to [29I], wherein the membrane protein capable of localizing in the membrane of an extracellular vesicle or the protein capable of binding to the membrane of an extracellular vesicle is tetraspanin or MFG-E8. [31I] The polynucleotide according to [29I], wherein the membrane protein capable of localizing in the membrane of a cell or the protein capable of binding to the membrane of a cell is CD8. [32I] The polynucleotide according to [1I], wherein the fusion protein defined in (D) comprises an amino acid sequence comprising, from the N-terminus, (D-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) the amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) the amino acid sequence of a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a membrane-binding domain thereof, and at least one T cell-stimulating cytokine or a subunit thereof. [33I] The polynucleotide according to [1I], wherein the fusion protein defined in (D) comprises an amino acid sequence comprising, from the N-terminus, (D-1) an amino acid sequence of a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a membrane-binding domain thereof, and at least one T cell-stimulating cytokine or a subunit thereof; (D-2) an optionally present spacer sequence; (D-3) an amino acid sequence of a single-chain MHC molecule; (D-4) an optionally present spacer sequence; and (D-5) an amino acid sequence of an MHC molecule-restricted antigen peptide, in this order.[34I] The polynucleotide according to [32I] or [33I], wherein the fusion peptide comprising the tetraspanin or a transmembrane domain thereof or MFG-E8 or a membrane-binding domain thereof and the at least one T cell-stimulating cytokine or a subunit thereof comprises an amino acid sequence comprising, in this order from the N-terminus: (1) a partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3; (2) an optional spacer sequence; (3) the amino acid sequence of the at least one T cell-stimulating cytokine or a subunit thereof; (4) an optional spacer sequence; and (5) a partial sequence of a tetraspanin comprising transmembrane domain 4. [35I] The polynucleotide according to [32I] or [33I], wherein the fusion peptide comprising a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a membrane-binding domain thereof, and the at least one T cell-stimulating cytokine or a subunit thereof, comprises an amino acid sequence comprising, from the N-terminus, (1) the amino acid sequence of the at least one T cell-stimulating cytokine or a subunit thereof, (2) an optional spacer sequence, and (3) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof. [36I] The polynucleotide according to [1I], wherein the fusion protein defined in (D) comprises the antigen-presenting MHC molecule, the at least one T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of localizing in a cell membrane or a transmembrane domain thereof, or a protein capable of binding to a cell membrane or a membrane-binding domain thereof. [37I] The polynucleotide according to [32I] or [33I], wherein the MHC molecule-restricted antigenic peptide is an MHC class I molecule-restricted antigenic peptide and the single-chain MHC molecule comprises the extracellular domain of the MHC class I α chain. [38I] The polynucleotide according to [32I] or [33I], wherein the MHC molecule-restricted antigenic peptide is an MHC class II molecule-restricted antigenic peptide and the single-chain MHC molecule comprises the extracellular domain of the MHC class II α chain and / or the extracellular domain of the MHC class II β chain.

[0028] [1J] The polynucleotide according to [1I], comprising the sequence defined in (a) and the sequence defined in (b). [2J] The polynucleotide according to [1J], encoding an amino acid sequence in which the fusion protein (A) and the fusion protein (B) are fused together. [3J] The polynucleotide according to [2J], encoding an amino acid sequence in which the fusion protein (A) and the fusion protein (B) are fused together via at least one 2A peptide. [4J] The polynucleotide according to [1J], further comprising the sequence defined in (c). [5J] The polynucleotide according to [4J], encoding an amino acid sequence in which the fusion protein (A), the fusion protein (B), and the fusion protein (C) are fused together. [6J] The polynucleotide according to [5J], encoding an amino acid sequence in which the fusion protein (A), the fusion protein (B), and the fusion protein (C) are fused together via at least one independent 2A peptide. [7J] The polynucleotide according to [6J], comprising, from the 5' end, in this order: a sequence defined in (a); a sequence encoding at least one first 2A peptide; a sequence defined in (b); a sequence encoding at least one second 2A peptide; and a sequence defined in (c).

[0029] [8J] The polynucleotide according to [1I], comprising the sequence defined in (d). [9J] The polynucleotide according to [8J], further comprising the sequence defined in (c). [10J] The polynucleotide according to [9J], encoding an amino acid sequence in which the fusion protein (D) and the fusion protein (C) are fused together. [11J] The polynucleotide according to [10J], encoding an amino acid sequence in which the fusion protein (D) and the fusion protein (C) are fused together via at least one 2A peptide. [12J] The polynucleotide according to [1I], comprising the sequence defined in (e). [13J] A vector comprising the polynucleotide according to any one of [1I] to [12J]. [14J] A pharmaceutical composition comprising the polynucleotide according to any one of [1I] to [12J] or the vector according to [13J], and a pharmacologically acceptable carrier.

[0030] [1K] A cell transformed with the polynucleotide described in any one of [1I] to [12J] or the vector described in [13J]. [2K] A culture supernatant obtained by culturing the cell described in [1K]. [3K] Antigen-presenting extracellular vesicles obtained from the culture supernatant described in [2K]. [4K] A method for producing the antigen-presenting extracellular vesicles described in [1], comprising a step of recovering the culture supernatant obtained by culturing the cell described in [1K]. [5K] A pharmaceutical composition comprising the culture supernatant described in [2K].

[0031] [1L] A pharmaceutical composition for treating or preventing cancer, comprising the polynucleotide of any one of [1I] to [12J] or the vector of [13J]. [2L] A pharmaceutical composition for treating or preventing an autoimmune disease, comprising the polynucleotide of any one of [1I] to [12J] or the vector of [13J]; wherein preferably the antigenic peptide comprises an autoantigenic peptide. [3L] A pharmaceutical composition for treating or preventing an allergic disease, comprising the polynucleotide of any one of [1I] to [12J] or the vector of [13J]; wherein preferably the antigenic peptide comprises an allergen. [4L] A pharmaceutical composition for treating or preventing an infectious disease, comprising the polynucleotide of any one of [1I] to [12J] or the vector of [13J] and a pharmacologically acceptable carrier; wherein preferably the antigenic peptide is derived from an infectious pathogen that causes the infectious disease.

[0032] [1M] The polynucleotide of any one of [1I] to [12J] or the vector of [13J] for use in the treatment or prevention of cancer; wherein preferably the antigenic peptide comprises a cancer antigen peptide. [2M] The polynucleotide of any one of [1I] to [12J] or the vector of [13J] for use in the treatment or prevention of autoimmune disease; wherein preferably the antigenic peptide comprises an autoantigenic peptide. [3M] The polynucleotide of any one of [1I] to [12J] or the vector of [13J] for use in the treatment or prevention of allergic disease; wherein preferably the antigenic peptide comprises an allergen. [4M] The polynucleotide of any one of [1I] to [12J] or the vector of [13J] for use in the treatment or prevention of infectious disease; wherein preferably the antigenic peptide is derived from an infectious pathogen that causes the infectious disease.

[0033] [1N] Use of the polynucleotide of any one of [1I] to [12J] or the vector of [13J] in the manufacture of a medicament for treating or preventing cancer; wherein preferably the antigenic peptide comprises a cancer antigen peptide. [2N] Use of the polynucleotide of any one of [1I] to [12J] or the vector of [13J] in the manufacture of a medicament for treating or preventing an autoimmune disease; wherein preferably the antigenic peptide comprises an autoantigenic peptide. [3N] Use of the polynucleotide of any one of [1I] to [12J] or the vector of [13J] in the manufacture of a medicament for treating or preventing an allergic disease; wherein preferably the antigenic peptide comprises an allergen. [4N] Use of the polynucleotide of any one of [1I] to [12J] or the vector of [13J] in the manufacture of a medicament for treating or preventing an infectious disease; wherein preferably the antigenic peptide is derived from an infectious pathogen that causes the infectious disease.

[0034] [1O] A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of the polynucleotide described in any one of [1I] to [12J] or the vector described in [13J], thereby activating and / or proliferating T cells in the subject that recognize a cancer antigen, and allowing the activated and / or proliferated T cells to attack cancer cells, thereby treating or preventing cancer; wherein preferably, the activated and / or proliferated T cells are CD8-positive cytotoxic T cells, and preferably, the antigen peptide comprises a cancer antigen peptide. [2O] A method for treating or preventing an autoimmune disease in a subject, comprising administering to the subject an effective amount of the polynucleotide described in any one of [1I] to [12J] or the vector described in [13J], thereby activating and / or proliferating T cells in the subject that recognize an autoantigen and desensitizing the immune response to the autoantigen in the subject, thereby treating or preventing the autoimmune disease; wherein preferably, the activated and / or proliferated T cells are CD4-positive regulatory T cells (Treg), and preferably, the antigen peptide comprises an autoantigen peptide. [3O] A method for treating or preventing an allergic disease in a subject, comprising administering to the subject an effective amount of the polynucleotide described in any one of [1I] to [12J] or the vector described in [13J], thereby activating and / or proliferating T cells in the subject that recognize an allergen 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 comprises an allergen. [4O] A method for treating or preventing an infectious disease in a subject, comprising administering to the subject an effective amount of a polynucleotide described in any one of [1I] to [12J] or a vector described in [13J], to 1) cause the secretion of inflammatory cytokines, thereby activating the subject's innate immunity, and / or 2) endowing the subject with acquired immunity against the infectious pathogen that causes the infectious disease, thereby eliminating the infectious pathogen that causes the infectious disease in the body and / or suppressing its proliferation.

[0035] [1P] A method for activating and / or proliferating T cells against a specific antigen, the method comprising introducing the polynucleotide described in any one of [1I] to [12J] or the vector described in [13J] 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.

[0036] According to the present invention, T cells can be activated, etc., by using polynucleotides for producing cells (antigen-presenting cells) and extracellular vesicles (antigen-presenting extracellular vesicles) that contain antigen-presenting MHC molecules and T cell-stimulating cytokines in their membranes.

[0037] A model diagram of an antigen peptide-single-chain MHC class I molecule (sc-Trimer)-CD81 fusion protein is shown. The amino acid sequence of the antigen peptide-single-chain MHC class I molecule (sc-Trimer)-CD81 fusion protein is shown. A model diagram of a CD80-CD9 fusion protein is shown. The amino acid sequence of the CD80-CD9 fusion protein is shown. A model diagram of a CD63-IL-2 fusion protein is shown. The amino acid sequence of the CD63-IL-2 fusion protein is shown. A model diagram of an antigen peptide-MHC class II β chain (sc-Dimer)-CD81 fusion protein is shown. The amino acid sequence of the antigen peptide-MHC class II β chain (sc-Dimer)-CD81 fusion protein is shown. The amino acid sequence of the MHC class II α chain is shown. A model diagram of a TGF-β-MFG-E8 fusion protein is shown. The amino acid sequence of the TGF-β-MFG-E8 fusion protein is shown.

[0033] Figure 1 shows a model of a CD81-IL-4 fusion protein.

[0034] Figure 2 shows the amino acid sequence of a CD81-IL-4 fusion protein.

[0035] Figure 3 shows the nucleic acid sequence of a sc-Trimer-CD81-IL-2 fusion protein.

[0036] Figure 4 shows the nucleic acid sequence of a CD63-AkaLuc fusion protein.

[0037] Figure 5 shows the nucleic acid sequence encoding sc-Trimer-T2A-IL-2-CD8-P2A-CD80.

[0038] Figure 6 shows a model of antigen-presenting extracellular vesicles of Example 1.

[0039] Figure 7 shows a model of antigen-presenting extracellular vesicles of Example 2.

[0039] Figure 8 shows a model of antigen-presenting extracellular vesicles of Example 3.

[0039] Figure 9 shows a model of antigen-presenting extracellular vesicles of Example 5.

[0039] Figure 6 shows a model of antigen-presenting extracellular vesicles of Example 6.

[0039] Figure 7 shows a model of antigen-presenting extracellular vesicles of Example 7.

[0039] Figure 8 shows a model of antigen-presenting extracellular vesicles of Example 8.

[0039] Figure 9 shows a model of antigen-presenting extracellular vesicles of Example 9. A model diagram of antigen-presenting extracellular vesicles of Example 11 is shown. A model diagram of antigen-presenting extracellular vesicles of other embodiments is shown. In Test Example 1-1, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 2 are shown. In Test Example 1-2, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 3 are shown. In Test Example 1-3, the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 4 are shown.Test Example 1-4 shows the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 5. Test Example 1-5 shows the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 6. Test Example 1-6 shows the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 7. Test Example 1-7 shows the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 8. Test Example 1-8 shows the results of flow cytometry analysis of the fusion protein contained in the membrane of the antigen-presenting extracellular vesicles of Example 9. Test Example 2 shows the results of 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). Test Example 3 shows 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). Test Example 4 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 3 activate antigen-specific CD4-positive T cells. Test Example 5 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 4 induce the differentiation of antigen-specific CD4-positive T cells (OT-2 T cells) into regulatory T cells. Test Example 6 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Examples 3 and 5 induce the differentiation of antigen-specific CD4-positive T cells (OT-2 T cells) into Th2 T cells. Test Example 7 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 6 induce the differentiation of antigen-specific CD4-positive T cells into Th1 cells. Test Example 8 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 7 induce the differentiation of antigen-specific CD4-positive T cells into Th17 cells. Test Example 9 shows that the antigen-presenting extracellular vesicles of Examples 1 and 8 significantly proliferated antigen-specific CD8-positive T cells. Test Example 10 shows that the antigen-presenting extracellular vesicles of Example 8 significantly suppressed the proliferation of B16 melanoma cells.Test Example 11 shows the results of an in vivo evaluation of whether the mRNA of Example 10 activates antigen-specific CD8-positive T cells (OT-1). Test Example 12 shows the results of an in vivo evaluation of whether the mRNA of Example 10 activates endogenous antigen-specific CD8-positive T cells. Test Example 13 shows the results of an in vivo evaluation of whether the extracellular vesicles of Example 6 differentiate antigen-specific CD4-positive T cells into Th1 cells. Test Example 14 shows the results of an in vivo evaluation of whether the antigen-presenting extracellular vesicles of Example 6 suppress the proliferation of melanoma cells. Test Example 15 shows the results of flow cytometry of the antigen-presenting extracellular vesicles of Example 11. Test Example 16 shows the results of an in vitro evaluation of whether the antigen-presenting extracellular vesicles of Example 11 differentiate antigen-specific CD4-positive T cells into Th1 cells. Test Example 17 shows the results of an in vivo evaluation of whether the antigen-presenting extracellular vesicles of Example 12 suppress the proliferation of T lymphoma cells. Test Example 1A shows the results of an in vitro evaluation of whether the mRNA of Example 1A expresses antigen-MHCI complexes, CD80, and IL-2 on cells. Test Example 2A shows the results of an in vitro evaluation of whether antigen-presenting cells induced by the mRNA of Example 1A proliferate antigen-specific CD8-positive T cells. Test Example 3A shows the results of an in vivo evaluation of whether the mRNA of Example 1A expresses antigen-MHCI complexes, CD80, and IL-2 on cells. Test Example 4A shows the results of an in vivo evaluation of whether the mRNA of Example 1A proliferates endogenous OVA-reactive CD8 T cells. (a) Nucleic acid sequence of sc-Trimer-T2A-IL-15sa-P2A-CD80 fusion protein. (b) Nucleic acid sequence of neoantigen-presenting sc-Trimer-T2A-IL-2-CD8-P2A-CD80 fusion protein. (c) Nucleic acid sequence of OVAp-MHCIIβ-P2A-MHCIIα-T2A-IL-12sc-CD8-P2A-CD80 fusion protein. (d) Nucleic acid sequence of neoantigen-presenting sc-Trimer-CD81-IL-2 fusion protein.Test Example 5A shows the results of in vivo evaluation of whether the mRNA of Example 2A expresses antigen-MHCI complexes, CD80, and IL-15sa on cells. Test Example 6A shows the results of in vivo evaluation of whether the mRNA of Example 2A expands endogenous OVA-reactive CD8 T cells. Test Example 7A shows the results of in vivo evaluation of whether the mRNA of Example 3A expresses antigen-MHCI complexes, CD80, and IL-2 on cells. Test Example 8A shows the results of in vivo evaluation of whether the mRNA of Example 3A expands endogenous Gtf2i-reactive CD8 T cells. Test Example 9A shows the results of in vivo evaluation of whether the mRNA of Example 4A expresses antigen-MHCII complexes, CD80, and IL-12 on cells. Test Example 10A shows the results of in vivo evaluation of whether endogenous OVA-reactive CD8 T cells are expanded by the mRNA of Example 4A. Test Example 11A shows the results of in vivo evaluation of whether endogenous RPL18-reactive CD8 T cells are expanded by the mRNA of Example 5A.

[0038] definition

[0039] Comprising: As used herein, "comprising" encompasses "substantially comprising," "essentially comprising," "consisting essentially of," and "consisting of."

[0040] Extracellular Vesicles As used herein, the term "extracellular vesicles" is not particularly limited as long as they are vesicles secreted from cells, and examples thereof include exosomes, microvesicles (MVs), apoptotic bodies, and the like.

[0041] 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 endocytic pathway. Examples of exosome components include proteins, nucleic acids (mRNA, miRNA, non-coating RNA), and the like. Exosomes may have the function of mediating intercellular communication. Examples of exosome marker molecules include Alix, Tsg101, tetraspanins, flotillin, and phosphatidylserine.

[0042] As used herein, "microvesicles" refer to vesicles of about 50 to about 1000 nm that are derived from the cytoplasmic membrane. Examples of components of microvesicles include proteins, nucleic acids (mRNA, miRNA, non-coating RNA, etc.). Microvesicles may have functions such as mediating intercellular communication. Examples of marker molecules for microvesicles include integrins, selectins, CD40, CD154, etc.

[0043] As used herein, "apoptotic body" refers to a vesicle of about 500 to about 2000 nm derived from the cytoplasmic membrane. Constituent components of apoptotic bodies include, for example, fragmented nuclei and organelles. Apoptotic bodies may have functions such as inducing phagocytosis. Marker molecules for apoptotic bodies include, for example, Annexin V and phosphatidylserine.

[0044] As used herein, "antigen-presenting extracellular vesicles" refer to extracellular vesicles that present antigens on the outside of their membranes.

[0045] Antigen-presenting cells. As used herein, the term "antigen-presenting cells" refers to cells that artificially present one or more antigens on the outside of their membrane. It is preferable that antigen-presenting cells present one or more cytokines (e.g., T cell-stimulating cytokines, etc., as defined below) on the outside of their membrane. It is preferable that antigens are presented on the outside of their membrane by being fixed on the outside of their membrane, more preferably in the form of a fusion molecule fused with a major histocompatibility complex molecule as defined below (i.e., the antigens are not temporarily attached to the outer membrane). It is preferable that antigen-presenting cells transiently express antigenic peptides and cytokines and simultaneously present them on the outside of their membrane by introducing a polynucleotide containing a sequence encoding one or more antigenic peptides and a sequence encoding one or more cytokines. Furthermore, it is preferable that antigen-presenting cells present any auxiliary signal (e.g., a T cell costimulatory molecule as defined below) on the outside of their membrane.

[0046] Major Histocompatibility Complex Molecules As used herein, the term "major histocompatibility complex (hereinafter also referred to as "MHC") molecule" is not particularly limited as long as it contains an antigen-binding cleft and is capable of binding to an antigen to be 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, examples include human leukocyte antigens (HLA), and in mice, examples include the H2 system.

[0047] HLA, which corresponds to MHC class I molecules, may 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. Examples of HLA-Cw polymorphisms include HLA-Cw0301, HLA-Cw0401, and HLA-Cw0602.

[0048] HLA, which corresponds to MHC class II molecules, may be classified into subtypes such as HLA-DR, HLA-DQ, and HLA-DP.

[0049] The MHC molecules described herein may be any molecules having a wild-type amino acid sequence (e.g., in the case of an MHC class I molecule, for example, an MHC class I α chain such as SEQ ID NO: 9, a β chain such as SEQ ID NO: 7) as long as they are capable of exerting their functions. ２ In the case of an MHC class II molecule, for example, an MHC class II α chain such as SEQ ID NO: 71, an MHC class II β chain such as SEQ ID NO: 37, a single-chain MHC class II molecule, etc., the amino acid sequence identity may be 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. Alternatively, the MHC molecules described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions, etc., of the wild-type amino acid sequence, so long as they are capable of exerting their functions.

[0050] 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, and examples thereof include antigen-presenting MHC class I molecules, antigen-presenting MHC class II molecules, etc. Examples of "antigen-presenting MHC class I molecules" include those that combine an antigen with an MHC class I α chain or its extracellular domain and a β ２a complex of an antigen and a single-chain MHC class I molecule; a fusion protein in which an antigen and a single-chain MHC class I molecule are bound; a complex of an antigen and a fusion protein of the extracellular domain of the MHC class I α-chain and another protein or a domain thereof or a fragment thereof (for example, a fusion protein of the extracellular domain of the MHC class I α-chain and the Fc portion of an antibody; a fusion protein of the extracellular domain of the MHC class I α-chain and the transmembrane domain of another membrane protein); and the like. Examples of "antigen-presenting MHC class II molecules" include complexes of an antigen, an MHC class II α chain or its extracellular domain, and an MHC class II β chain or its extracellular domain; a complex of an antigen and a single-chain MHC class II molecule; a complex of an MHC class II α chain and a fusion protein in which an antigen and an MHC class II β chain are bound; a fusion protein of an antigen, an MHC class II α chain and another protein or a domain thereof or a fragment thereof (e.g., a fusion protein of an MHC class II α chain extracellular domain and an Fc portion of an antibody; a fusion protein of an MHC class II α chain extracellular domain and a transmembrane domain of another membrane protein); and a fusion protein of an MHC class II β chain extracellular domain and another protein or a domain thereof or a fragment thereof (e.g., a fusion protein of an MHC class II β chain extracellular domain and an Fc portion of an antibody; a fusion protein of an MHC class II β chain extracellular domain and an amino acid sequence containing a transmembrane domain of another membrane protein).

[0051] As used herein, a "single-chain MHC molecule," "single-chain MHC class I molecule," or "single-chain MHC class II molecule" refers to a molecule that comprises an α chain or an extracellular domain thereof and a β chain or an extracellular domain thereof or a β chain of an MHC molecule (or an MHC class I molecule or an MHC class II molecule). ２ The term "single-chain MHC class I molecule" refers to a fusion protein in which an MHC class I α chain and a β ２Examples of "single-chain MHC class II molecules" include fusion proteins in which an MHC class II α chain and an MHC class II β chain are linked, optionally via a spacer sequence.

[0052] As used herein, the term "single-chain MHC molecule comprising a transmembrane domain" refers to a "single-chain MHC molecule" that comprises the original transmembrane domain of an MHC molecule (the transmembrane domain of an MHC class I α chain, an MHC class II α chain, or an MHC class II β chain).

[0053] As used herein, "a protein (or fusion protein, protein complex, etc.) comprising an antigen-presenting MHC molecule and capable of presenting the antigen (or antigenic peptide) extramembrane" means a protein (or fusion protein, protein complex, etc.) that comprises at least an antigen-presenting MHC molecule and is capable of presenting the antigen (or antigenic peptide) extramembrane, thereby presenting the antigen to T cells, etc. The "protein (or fusion protein, protein complex, etc.) comprising an antigen-presenting MHC molecule and capable of presenting an antigen (or antigenic peptide) extramembrane" may be one that is expressed in the form of a fusion protein, protein complex, etc. using a plasmid, etc., so that it is expressed on the membrane of extracellular vesicles. Alternatively, when a soluble antigen-presenting MHC molecule (including, but not limited to, a fusion protein comprising an MHC class I α chain and an immunoglobulin heavy chain as described in Patent Document 1; a soluble MHC class I molecule as described in Japanese Patent Laid-Open No. 2007-161719) is used as the "protein (or fusion protein, protein complex, etc.) capable of extramembrane presentation of an antigen (or antigen peptide)," the soluble antigen-presenting MHC molecule and the extracellular vesicle may be bound to the membrane of the extracellular vesicle, if necessary, via a lipid linker, a peptide linker, or the like (for example, the method described in Japanese Patent Laid-Open No. 2018-104341 may be used as reference). Alternatively, a protein comprising a soluble antigen-presenting MHC molecule to which a desired tag (e.g., His tag, FLAG tag, PNE tag (SEQ ID NO: 79: NYHLENEVARLKKL) or the like) has been added at the N-terminus or C-terminus (the tag may be expressed, for example, as a fusion protein together with other components, or may be attached to a separately prepared soluble antigen-presenting MHC molecule via a linker or the like as necessary) and an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) or the like (for example, a method using a zPNE tag and an antibody against the tag or the like may be used as reference).

[0054] Antigen The term "antigen" as used herein is not particularly limited as long as it is capable of possessing antigenicity, and includes not only peptide antigens but also non-peptide antigens such as phospholipids and complex carbohydrates (e.g., bacterial membrane components such as mycolic acid and lipoarabinoanan).

[0055] The term "antigenic peptide" as used herein is not particularly limited as long as it is a peptide that can serve as an antigen, and may be naturally occurring, synthetically derived, or commercially available. Examples of antigen peptides include, but are not limited to, WT-1, α-fetoprotein, MAGE-1, MAGE-3, placental alkaline phosphatase sialyl-Lewis X, CA-125, CA-19, TAG-72, epithelial glycoprotein 2, 5T4, α-fetoprotein receptor, M2A, tyrosinase, Ras, p53, Her-2 / neu, EGF-R, estrogen receptor, progesterone receptor, myc, BCR-ABL, HPV type 16, melanotransferrin, MUC1, CD10, CD19, CD20, CD37, CD45R, IL-2 receptor α chain, T cell receptor, prostatic acid phosphatase, GP100, MelanA / Mart-1, gp75 / Brown, BAGE, S-100, itokeratin, CYFRA21-1, Tumor-associated antigen peptides such as Ep-CAM, Gtf2i, and RPL18; autoantigenic peptides such as insulin, glutamic acid decarboxylase, ICA512 / IA-2 protein tyrosine phosphatase, ICA12, ICA69, preproinsulin, HSP60, carboxypeptidase H, peripherin, GM1-2, vitronectin, beta-crystallin, calreticulin, serotransferrin, keratin, pyruvate carboxylase, C1, villin 2, nucleosomes, ribonucleoproteins, myelin oligodendrocyte glycoprotein, myelin-associated glycoprotein, myelin / oligodendrocyte basic protein, oligodendrocyte-specific protein, myelin basic protein, and proteolipid apoprotein;Examples of antigenic peptides include antigenic peptides derived from infectious pathogens such as protozoa (e.g., Plasmodium, Leishmania species, Trypanosoma species), bacteria (e.g., gram-positive cocci, gram-positive bacilli, gram-negative bacteria, and anaerobic bacteria), fungi (e.g., Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, and Sporothrix), viruses (e.g., adenovirus, herpes simplex virus, papillomavirus, respiratory syncytial virus, poxvirus, HIV, influenza virus, and coronaviruses such as SARS-CoV and SARS-CoV2), intracellular parasites (e.g., Chlamydiaceae, Mycoplasmataceae, Acholeplasmataceae, and Rickettsiaceae), and helminths (e.g., nematodes, trematodes, and cestodes); and other antigenic peptides such as prions. Antigenic peptides may include allergens that cause allergic symptoms. Examples of allergens include peptides derived from the above-mentioned protozoa, bacteria, fungi, intracellular parasites, and helminths, as well as exogenous peptides, such as peptides derived from house dust, mites, animals (e.g., companion animals such as cats and dogs), and pollen (e.g., cedar and cypress). More specifically, proteins contained in cedar pollen, such as Cryj1, are exemplified. Alternatively, allergens that cause allergic symptoms may be derived from food. Examples of allergens that cause allergic symptoms beyond those caused by food include peptides derived from chicken eggs, milk, wheat, buckwheat, crab, shrimp, and peanuts.

[0056] As used herein, "MHC molecule-restricted antigenic peptide" refers to an antigenic peptide that can bind to an MHC molecule in vitro, in vivo, and / or ex vivo, etc. The number of amino acid residues in an "MHC molecule-restricted antigenic peptide" is usually about 7 to about 30. Examples of "MHC molecule-restricted antigenic peptides" include MHC class I molecule-restricted antigenic peptides, MHC class II molecule-restricted antigenic peptides, etc.

[0057] As used herein, the term "MHC class I molecule-restricted antigenic peptide" refers to an antigenic peptide that can bind to an MHC class I molecule in vitro, in vivo, and / or ex vivo. When an MHC class I molecule-restricted antigenic peptide is presented on the extracellular membrane of an extracellular vesicle, the antigenic peptide can be recognized by precursor T cells and induce cytotoxic T cells. The number of amino acid residues in an "MHC class I molecule-restricted antigenic peptide" is typically 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 still more preferably about 7 to about 12.

[0058] As used herein, the term "MHC class II molecule-restricted antigenic peptide" refers to an antigenic peptide that can bind to an MHC class II molecule in vitro, in vivo, and / or ex vivo. When an MHC class II molecule-restricted antigenic peptide is presented on the extracellular membrane of an extracellular vesicle, the antigenic peptide can be recognized by precursor T cells and the like, and can induce helper T cells and the like. The number of amino acid residues in an "MHC class II molecule-restricted antigenic peptide" is typically about 7 to about 30, preferably about 10 to about 25, and more preferably about 12 to about 24.

[0059] The "MHC molecule-restricted antigenic peptide," "MHC class I molecule-restricted antigenic peptide," or "MHC class II molecule-restricted antigenic peptide" is not particularly limited as long as it is an antigenic peptide that can bind to an MHC molecule, an MHC class I molecule, or an MHC class II molecule.

[0060] T Cell-Stimulating Cytokines As used herein, the term "T cell-stimulating cytokine" is not particularly limited as long as it is a cytokine capable of stimulating (e.g., activating, suppressing, etc.) T cells via a receptor expressed on the membrane of the T cell or the like. Examples of T cell-stimulating cytokines include, but are not limited to, IL-2, IL-4, IL-6, IL-12, IL-15, TGF-β, IFN-α, IFN-γ, etc. Among these, those capable of forming homo- or hetero-subunit multimers (e.g., IL-12, TGF-β, etc.) may have a continuous amino acid sequence, optionally linked via a peptide linker, etc., as long as they are functional (i.e., as long as they have the desired pharmacological activity). As long as they maintain the ability to stimulate T cells, they may be bound to or fused with other full-length proteins or partial sequence peptides thereof (e.g., the Sushi domain of the IL-15 receptor).

[0061] The T cell stimulating cytokines described herein may be derived from any animal species, including 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 primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, 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.

[0062] 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 to the wild-type amino acid sequence (e.g., in the case of IL-2, for example, SEQ ID NO: 25; in the case of IL-4, for example, SEQ ID NO: 53), so long as they are able to exert their functions. Alternatively, the T cell-stimulating cytokines described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions, etc., of the wild-type amino acid sequence, so long as they are able to exert their functions.

[0063] As used herein, "a protein comprising a (first or second) T cell-stimulating cytokine and capable of displaying the (first or second) T cell-stimulating cytokine on the extracellular membrane" means a protein that contains at least a T cell-stimulating cytokine and is capable of displaying the T cell-stimulating cytokine on the extracellular membrane of a cell or extracellular vesicle. The "protein comprising a (first or second) T cell-stimulating cytokine and capable of displaying the (first or second) T cell-stimulating cytokine on the extracellular membrane" may be expressed as a fusion protein comprising a T cell-stimulating cytokine and a membrane protein or a fragment comprising its transmembrane domain, using a plasmid or the like, so that the fusion protein is expressed on the membrane of a cell or extracellular vesicle. Alternatively, when a soluble T cell stimulatory cytokine (including a (first or second) T cell stimulatory cytokine and capable of extramembrane presentation of the (first or second) T cell stimulatory cytokine) is used (including, but not limited to, a T cell stimulatory cytokine itself; a fusion protein of a T cell stimulatory cytokine with an Fc portion of an antibody; a complex of a T cell stimulatory cytokine with an antibody that recognizes the T cell stimulatory cytokine or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody)), the soluble T cell stimulatory cytokine may be bound to the membrane of a cell or extracellular vesicle via a lipid linker, peptide linker, or the like, as necessary (for example, a method described in JP 2018-104341 A or the like may be referenced).Alternatively, a soluble T cell-stimulating cytokine to which a desired tag (e.g., a His tag, a FLAG tag, or a PNE tag) has been added at the N-terminus or C-terminus (the tag may be expressed, for example, as a fusion protein together with other components, or may be attached to a separately prepared soluble T cell-stimulating cytokine via a linker or the like, as necessary) may be mixed with cells or extracellular vesicles containing in their membranes a protein containing an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) or the like (e.g., an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) itself, bound to the membrane of a cell or extracellular vesicle via a linker or the like, as necessary; a fusion protein in which a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a nanobody against the tag is bound to the N-terminus or C-terminus of its transmembrane domain, etc.) under desired conditions (e.g., Raj D, et al., Gut., 2019 Jun;68(6):1052-1064, etc., may be used as a reference.) In the case of a T cell-stimulating cytokine formed by a multimer of subunits, if one of the subunits is a protein that can be presented on the extracellular membrane of a cell or extracellular vesicle, the remaining subunits do not need to be in a form that can be presented on the extracellular membrane. If one of the subunits is a protein that can be presented on the extracellular membrane of an extracellular vesicle, a functional T cell-stimulating cytokine can be constructed on the extracellular membrane of an extracellular vesicle by adding or co-expressing another subunit.

[0064] T Cell Costimulatory Molecule As used herein, the term "T cell costimulatory molecule" refers to a molecule that can contribute to T cell activation, etc. by interacting with a molecule present on the membrane of a T cell, such as CD28 or CD134. Examples of T cell costimulatory molecules include, but are not limited to, molecules such as CD80 and CD86, or their extracellular domains or functional fragments thereof; antibodies such as anti-CD28 antibodies and anti-CD134 antibodies, or antigen-binding fragments thereof (e.g., scFv, Fab, or nanobody); fusion proteins (or complexes or associations) of these with the transmembrane domain of another protein or the Fc portion of an antibody, etc.

[0065] As used herein, a "T cell costimulatory molecule comprising a transmembrane domain" refers to a "T cell costimulatory molecule" that also comprises a transmembrane domain derived from a T cell costimulatory molecule.

[0066] The T cell costimulatory molecules described herein may be derived from any animal species, including 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 primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, 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.

[0067] 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 to the wild-type amino acid sequence (e.g., in the case of CD80, for example, SEQ ID NO: 67), so long as they are able to exert the above-described functions. Alternatively, the T cell costimulatory molecules described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions, etc., of the wild-type amino acid sequence, so long as they are able to exert their functions.

[0068] As used herein, "a protein comprising a T cell costimulatory molecule and capable of interacting with a T cell" means a protein comprising at least a T cell costimulatory molecule and capable of interacting with a molecule present on the membrane of a T cell. In other words, it means that at least the portion of the T cell costimulatory molecule that is capable of interacting with a T cell is located outside the membrane of a cell or extracellular vesicle. A "protein comprising a T cell costimulatory molecule and capable of interacting with a T cell" may be expressed using a plasmid or the like so that it is expressed on the membrane of a cell or extracellular vesicle. Alternatively, when a soluble T cell costimulatory molecule (including a T cell costimulatory molecule and capable of interacting with the T cell) is used, the "protein capable of interacting with the T cell costimulatory molecule and the T cell" may be one in which the soluble T cell costimulatory molecule is bound to the membrane of a cell or extracellular vesicle via a lipid linker, spacer sequence, or the like, as necessary (for example, see the method described in JP 2018-104341 A).Alternatively, a soluble T cell costimulatory molecule to which a desired tag (e.g., a His tag, a FLAG tag, or a PNE tag) has been added at the N-terminus or C-terminus (the tag may be expressed, for example, as a fusion protein together with other components, or may be attached to a separately prepared soluble T cell costimulatory molecule via a linker or the like, as necessary) may be mixed with cells or extracellular vesicles containing, in their membranes, a protein containing an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) or the like (e.g., an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) itself, bound to the membrane of a cell or extracellular vesicle via a linker or the like, as necessary; a fusion protein in which a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a nanobody against the tag is bound to the N-terminus or C-terminus of its transmembrane domain, etc.) under desired conditions (e.g., Raj D, et al., Gut., 2019 Jun;68(6):1052-1064, etc., may be used as a reference).

[0069] As used herein, the term "membrane protein or transmembrane domain thereof capable of being expressed on the cell membrane" can refer to any membrane protein or transmembrane domain thereof, as long as it is capable of being expressed on the cell membrane. Although not limited thereto, preferred membrane proteins or transmembrane domains thereof include those that comprise part or all of CD8, CD4, CD28, transferrin receptor, etc., or part or all of the Fc region of membrane-bound immunoglobulin molecules such as IgG1, IgG2, and IgG4. As used herein, the term "protein or domain thereof capable of binding to the cell membrane" can refer to any protein or domain thereof, as long as it is capable of binding to the cell membrane.

[0070] As used herein, any membrane protein or transmembrane domain thereof that can be expressed in the membrane of an extracellular vesicle can be selected as the "membrane protein or transmembrane domain thereof that can be expressed in the membrane of an extracellular vesicle." The "membrane protein or transmembrane domain thereof that can be expressed in the membrane of an extracellular vesicle" is preferably a membrane protein known to be able to be expressed in extracellular vesicles (e.g., exosomes, etc.) (e.g., tetraspanin, CD58, ICAM-1, PTGFRN (see, for example, Non-Patent Document 1, WO 2019 / 183578, etc.)), or a transmembrane domain thereof.

[0071] As used herein, any protein or domain thereof capable of binding to the membrane of an extracellular vesicle can be selected as the "protein or domain thereof capable of binding to the membrane of an extracellular vesicle," as long as it is capable of binding to the membrane of an extracellular vesicle. Preferred "proteins or domains thereof capable of binding to the membrane of an extracellular vesicle" are those known to be capable of binding to the membrane of an extracellular vesicle (e.g., exosomes) (e.g., MFG-E8 or domains thereof (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)).

[0072] The "membrane protein or transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle" or "protein or domain thereof that can bind to the membrane of a cell or extracellular vesicle" described herein may be derived from any animal species. Examples include those derived from animals such as rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivora such as dogs and cats; and mammals such as primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. The "membrane protein or transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle" or "protein or domain thereof that can bind to the membrane of a cell or extracellular vesicle" described herein are preferably derived from rodents or mammals, more preferably from mice or humans.

[0073] According to Non-Patent Document 2, markers for mammalian extracellular vesicles are classified as follows. 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), other multi-membrane spanning membrane proteins (CD47, heterotrimeric G proteins (GNA: Guanine nucleotide-binding proteins), etc.), MHC class I (HLA-A / B / C, H2-K / D / Q), integrins (ITGA / ITGB), transferrin receptor (TFR2); LAMP1 / 2; heparan sulfate proteoglycans (including syndecan (SDC)); extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as BSG or CD147); ADAM10; CD73 (NT5E), a GPI-anchored 5'-nucleotidase; CD55 and CD59, GPI-anchored complement-binding proteins; Sonic Hedgehog protein (SHH); 2) Cell / tissue-specific: several tetraspanins: TSPAN8 (epithelial cell-specific), CD37 and CD53 (leukocyte-specific); PECAM1 (endothelial cell-specific); ERBB2 (breast cancer-specific); EPCAM (epithelial-specific); CD90 (THY1) (mesenchymal stem cell-specific); CD45 (PTPRC) (immune cell-specific), CD41 (ITGA2B) or CD42a (GP9) (platelet-specific); glycophorin A (GYPA) (erythrocyte-specific); CD14 (monocyte-specific), MHC class II (HLA-DR / DP / DQ, H2-A); Examples of such proteins include CD3 (T cell-specific), acetylcholinesterase / AChE-S (neuron-specific), AChE-E (erythrocyte-specific), and amyloid βA4 / APP (neuron-specific). Therefore, although not limited to these, proteins that are markers of 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.

[0074] As used herein, "tetraspanin" refers to proteins belonging to the tetraspanin family (e.g., but not limited to, CD9, CD53, CD63, CD81, CD82, CD151, etc.). Tetraspanins generally have, from the N-terminus, transmembrane domain 1 (hereinafter also referred to as "TM1"), small extracellular loop (hereinafter also referred to as "SEL"), transmembrane domain 2 (hereinafter also referred to as "TM2"), small intracellular loop (hereinafter also referred to as "SIL"), transmembrane domain 3 (hereinafter also referred to as "TM3"), large extracellular loop (hereinafter also referred to as "LEL"), and transmembrane domain 4 (hereinafter also referred to as "TM4"), and are therefore four-transmembrane proteins, with both the N-terminus and C-terminus located on the cytoplasmic side. For example, when the tetraspanin is mouse CD63 (amino acid sequence 1 to 238: SEQ ID NO: 27), the amino acid sequence from about 1 to about 110 typically contains TM1, SEL, TM2, SIL, and TM3, the amino acid sequence from about 111 to about 200 contains LEL, and the amino acid sequence from about 201 to about 238 contains TM4.

[0075] Each domain (e.g., TM1, SEL, SIL, LTL, etc.) in the "tetraspanin" described herein may be derived from the same tetraspanin, or may be derived entirely or partially from different tetraspanins.

[0076] 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 to their wild-type amino acid sequences (e.g., in the case of full-length CD9, for example, SEQ ID NO: 21; in the case of full-length CD63, for example, SEQ ID NO: 27; in the case of full-length CD81, for example, SEQ ID NO: 15), so long as they can be expressed on the membrane of extracellular vesicles. Alternatively, the tetraspanins described herein may have one or more amino acids deleted, inserted, added, and / or substituted from their wild-type amino acid sequences, so long as they can be expressed on the membrane of extracellular vesicles.

[0077] The partial sequences of the tetraspanins described herein (e.g., partial sequences including each domain; TM1, SEL, TM2, SIL, and TM3 (e.g., SEQ ID NO: 57 for CD63; SEQ ID NO: 61 for CD81); partial sequences including TM4 (e.g., 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 still more preferably 99% or more to the wild-type amino acid sequence. Alternatively, the partial sequences of the tetraspanins described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions, etc., of the wild-type amino acid sequence.

[0078] 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 to its wild-type amino acid sequence (e.g., SEQ ID NO: 49, etc.), so long as it is capable of binding to the membrane of extracellular vesicles. Alternatively, the MFG-E8 described herein may have one or more amino acids deleted, inserted, added, and / or substituted, etc., from its wild-type amino acid sequence, so long as it is capable of binding to the membrane of extracellular vesicles.

[0079] CD58, PTGFRN, etc. 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 the wild-type amino acid sequence, so long as they are capable of being expressed on the membrane of extracellular vesicles or capable of binding to the membrane of extracellular vesicles. Alternatively, CD58, PTGFRN, etc. described herein may have one or more amino acid deletions, insertions, additions, and / or substitutions, etc., relative to the wild-type amino acid sequence, so long as they are capable of being expressed on the membrane of extracellular vesicles or capable of binding to the membrane of extracellular vesicles.

[0080] Spacer Sequence As used herein, the term "spacer sequence" refers to any sequence having at least one amino acid residue that is present between two or more proteins, or subsequences or domains thereof. Spacer sequences can be used, for example, to link two or more proteins, or subsequences or domains thereof. Spacer sequences include peptide linkers. The length of a spacer sequence is usually 1 to about 50 amino acid residues, preferably about 2 to about 28, and more preferably about 4 to about 25. Examples of spacer sequences include, but are not limited to, (GGGXS) ｎ G ｍ wherein each occurrence of X is independently A or G, n is 1-8, and m is 0-3 (e.g., SEQ ID NOs: 5, 11, 29, 39, etc.); (GGGS) ｎ G ｍ (wherein n is 1 to 10 and m is 0 to 3); T ａ S ｂ (GGX) ｎ G ｍ (wherein, each occurrence of X is independently S or T, n is 1 to 8, m is 0 to 3, a is 0 or 1, and b is 0 or 1) (e.g., SEQ ID NO: 77, etc.); and the like.

[0081] In one embodiment of the antigen-presenting extracellular vesicles described herein, extracellular vesicles that present antigen-presenting MHC molecules and T cell-stimulating cytokines on the extracellular membrane are provided (a model thereof is shown in Figure 2K(1)). Such extracellular vesicles may present antigen-presenting MHC molecules and T cell-stimulating cytokines on the extracellular membrane by incorporating proteins defined in (A) and (B) below into their membranes, or may present antigen-presenting MHC molecules and T cell-stimulating cytokines on the extracellular membrane by incorporating proteins defined in (D) into their membranes. Alternatively, antigen-presenting MHC molecules and T cell-stimulating cytokines may be attached to the membrane surface of isolated extracellular vesicles afterward. The attachment method is not particularly limited, and 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 phospholipid moieties into the membrane of the extracellular vesicles. Phosphatidylserine is present on the surface of extracellular vesicles. Therefore, extracellular vesicles that present antigen-presenting MHC molecules and T cell-stimulating cytokines on their membrane surface can be prepared by synthesizing and purifying proteins in which the antigen-presenting MHC molecules or T cell-stimulating cytokines to be presented are fused to phosphatidylserine-binding MFG-E8, and then mixing the fusion proteins with extracellular vesicles. Alternatively, peptide neoepitope (PNE) nanobodies may be pre-expressed in extracellular vesicles, and PNE-tagged antigen-presenting MHC molecules and T cell-stimulating cytokines may be added later to present them on the membrane surface of the extracellular vesicles. Biotinylated antigen-presenting MHC molecules and T cell-stimulating cytokines may be added to streptavidin-expressed extracellular vesicles, and then presented on the membrane surface of the extracellular vesicles.

[0082] In one embodiment of the present invention, the extracellular vesicles may present multiple (2, 3, 4, 5) types of antigen-presenting MHC molecules and multiple (2, 3, 4, 5) types of T cell-stimulating cytokines (hereinafter, to distinguish between the individual T cell-stimulating cytokines, they may be referred to as a first T cell-stimulating cytokine, a second T cell-stimulating cytokine, or more T cell-stimulating cytokines, etc.) on the extracellular membrane. Alternatively, the extracellular vesicles may present one type of antigen-presenting MHC molecule and multiple types of T cell-stimulating cytokines on the extracellular membrane (Figure 2K(3) shows an example of a model of an extracellular vesicle presenting one type of antigen-presenting MHC molecule and two types of T cell-stimulating cytokines on the extracellular membrane).

[0083] In one embodiment of the present invention, a cell that presents an antigen-presenting MHC molecule and a T cell-stimulating cytokine on the extracellular membrane is provided (corresponding to the model of extracellular vesicles in Figure 2K(1)). Such an antigen-presenting cell may present the antigen-presenting MHC molecule and the T cell-stimulating cytokine on the extracellular membrane by containing in its membrane the proteins defined in (A) and (B) below, or may present the antigen-presenting MHC molecule and the T cell-stimulating cytokine on the extracellular membrane by containing in its membrane the protein defined in (D).

[0084] In one embodiment of the present invention, the cells may be those that present multiple (2, 3, 4, 5) types of antigen-presenting MHC molecules and multiple (2, 3, 4, 5) types of T cell-stimulating cytokines (hereinafter, to distinguish between the individual T cell-stimulating cytokines, they may be referred to as a first T cell-stimulating cytokine, a second T cell-stimulating cytokine, or further T cell-stimulating cytokines, etc.) on the outside of their membranes. Alternatively, the cells may be those that present one type of antigen-presenting MHC molecule and multiple types of cell-stimulating cytokines on the outside of their membranes (corresponding to the model of extracellular vesicles in Figure 2K(3)).

[0085] One embodiment of the present invention provides an antigen-presenting cell or an antigen-presenting extracellular vesicle, the antigen-presenting cell or the antigen-presenting extracellular vesicle comprising, in its membrane, the following: (A) a protein capable of extramembrane presentation of an antigen, the protein comprising an antigen-presenting MHC molecule; and (B) a protein capable of extramembrane presentation of a first T cell-stimulating cytokine, the protein comprising a first T cell-stimulating cytokine.

[0086] Constituent Requirement (A) The above-mentioned (A) "protein capable of presenting an antigen extracellularly, including an antigen-presenting MHC molecule" may include, in addition to the antigen-presenting MHC molecule, other proteins or domains thereof, etc., as long as the protein is capable of presenting an antigen extracellularly on the membrane of a cell or extracellular vesicle.

[0087] In one embodiment of the present invention, (A) is a fusion protein or protein complex capable of presenting the antigen extracellularly, comprising an antigen-presenting MHC molecule and a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a domain thereof.

[0088] In one embodiment of the present invention, the (A) is: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, comprising an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC molecule comprising a transmembrane domain, in this order; or (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) an MHC class I α chain, β chain, or ２ a fusion protein comprising an amino acid sequence consisting of a fusion protein of microglobulin and an MHC class I α chain, an MHC class II α chain, or an MHC class II β chain; and (A-6) β ２a protein comprising the amino acid sequence of microglobulin, MHC class I α chain, MHC class II β chain, or MHC class II α chain; and a protein complex comprising (A-3) and (A-6), wherein the combination of (A-3) and (A-6) constitutes an MHC class I molecule or an MHC class II molecule.

[0089] In one embodiment of the present invention, the above-mentioned (A) is a fusion protein capable of presenting an antigenic peptide extramembrane, comprising an amino acid sequence comprising, in this order from the N-terminus: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC class I molecule comprising a transmembrane domain. Also, in one embodiment of the present invention, the above-mentioned (A) is a fusion protein capable of presenting an antigenic peptide extramembrane, comprising an amino acid sequence comprising, in this order from the N-terminus: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC class II molecule comprising a transmembrane domain.

[0090] In one embodiment of the present invention, the above-mentioned (A) is a protein complex capable of extramembrane presentation of an antigenic peptide, which comprises, from the N-terminus thereof: (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; (A-3) a β ２ In one embodiment of the present invention, the above (A) comprises an amino acid sequence comprising, in this order, (A) a protein complex capable of extramembrane presentation of an antigenic peptide, and (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) an amino acid sequence of an MHC class I α chain. In another embodiment of the present invention, the above (A) comprises: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, and (A-4) an amino acid sequence comprising, in this order from the N-terminal side, (A-5) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-6) an amino acid sequence of an MHC class I α chain; and (A-7) an amino acid sequence of an MHC class I α chain. ２In one embodiment of the present invention, the (A) is a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of an MHC class II β chain; and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain. In one embodiment of the present invention, the above-mentioned (A) is a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; and (A-3) the amino acid sequence of an MHC class II α chain; and (A-6) a protein comprising the amino acid sequence of an MHC class II β chain.

[0091] In one embodiment of the present invention, (A) is a fusion protein or protein complex comprising an antigen-presenting MHC molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof, and capable of presenting the antigen outside the membrane.

[0092] In one embodiment of the present invention, the above-mentioned (A) is a fusion protein comprising an amino acid sequence comprising, from its N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a membrane-binding domain thereof, in this order; or ２(A-4) an amino acid sequence of an extramembrane region of a microglobulin, an MHC class II α chain, or an extramembrane region of an MHC class II β chain; (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; and (A-6) a β ２ a protein comprising the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain; and

[0093] In one embodiment of the present invention, the above-mentioned (A) is a fusion protein capable of extramembrane presentation of an antigenic peptide, comprising an amino acid sequence comprising, in this order from the N-terminus: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class I molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. Also, in one embodiment of the present invention, the above-mentioned (A) is a fusion protein capable of extramembrane presentation of an antigenic peptide, comprising an amino acid sequence comprising, in this order from the N-terminus: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class II molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof.

[0094] In one embodiment of the present invention, the above-mentioned (A) comprises, from the N-terminal side thereof, (A-1) an amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) an optionally present spacer sequence, and (A-3) β ２(A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order, and (A-6) a protein comprising the amino acid sequence of an MHC class I α chain. In one embodiment of the present invention, the (A) is a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of the extramembrane region of the MHC class I α chain, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a protein comprising the amino acid sequence of an MHC class I α chain. ２In one embodiment of the present invention, the (A) is a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of the extramembrane region of an MHC class II β chain, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain. In one embodiment of the present invention, the above-mentioned (A) is a protein complex capable of presenting an antigenic peptide extramembrane, comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence including, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of the extramembrane region of the MHC class II α chain, (A-4) an optional spacer sequence, and (A-5) tetraspanin; and (A-6) a protein comprising the amino acid sequence of an MHC class II β chain.

[0095] In one embodiment of the present invention, when the "single-chain MHC molecule" is a "single-chain MHC class I molecule", the "single-chain MHC class I molecule" is ２The antibody comprises a microglobulin (e.g., SEQ ID NO: 7, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still 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 (e.g., SEQ ID NO: 9, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more). In one embodiment of the present invention, when the above (A-3) is a "single-chain MHC class I molecule," the "single-chain MHC class I molecule" is SEQ ID NO: 65, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0096] In one embodiment of the present invention, when the "single-chain MHC molecule" is a "single-chain MHC class II molecule," the "single-chain MHC class II molecule" consists of, from the N-terminus, an MHC class II β chain, an optional spacer sequence, and an MHC class II α chain.

[0097] In one embodiment of the present invention, the above (A-3) and / or (A-6) are "β ２ In the case of "β microglobulin," ２ "Microglobulin" is SEQ ID NO: 7, or one having an amino acid sequence identity thereto 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.

[0098] In one embodiment of the present invention, when the above (A-3) and / or (A-6) is an "MHC class I α chain", the "MHC class I α chain" is SEQ ID NO: 9, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0099] In one embodiment of the present invention, when the above (A-3) and / or (A-6) is an "MHC class II β chain," the "MHC class II β chain" is SEQ ID NO: 37, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0100] In one embodiment of the present invention, when the above (A-3) and / or (A-6) is an "MHC class II α chain," the "MHC class II α chain" is SEQ ID NO: 71, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0101] In each of the above embodiments, the "optional spacer sequences" of (A-2) and (A-4), when present, can be independently selected. When present, (A-2) may be, for example, a spacer sequence such as SEQ ID NO: 5, 11, 29, 39, or 77. When present, (A-4) may be, for example, a spacer sequence such as SEQ ID NO: 5, 11, 29, 39, or 77.

[0102] In one embodiment of the present invention, the tetraspanin (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 (A-5) in each of the above embodiments is CD81 (preferably SEQ ID NO: 15, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0103] In one embodiment of the present invention, the above-mentioned (A) is a fusion protein comprising an amino acid sequence comprising, from its N-terminus, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto).

[0104] In one embodiment of the present invention, the above-mentioned (A) is a fusion protein comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) A protein complex comprising the amino acid sequence of the MHC class II α chain of SEQ ID NO: 71 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0105] Constituent Requirement (B) The "protein comprising a first T cell-stimulating cytokine and capable of extramembrane presentation of the first T cell-stimulating cytokine" in (B) above may comprise, in addition to the first T cell-stimulating cytokine, another protein or a domain thereof, etc., as long as it is a protein capable of extramembrane presentation of the first T cell-stimulating cytokine.

[0106] In one embodiment of the present invention, (B) is a fusion protein capable of presenting a first T cell-stimulating cytokine on the extracellular surface, the fusion protein comprising a first T cell-stimulating cytokine and a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof.

[0107] In one embodiment of the present invention, (B) is a fusion protein comprising a first T cell-stimulating cytokine and CD8 or a transmembrane domain thereof, and capable of presenting the first T cell-stimulating cytokine on the extramembrane.

[0108] In one embodiment of the present invention, the above-mentioned (B) is a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, comprising, from its N-terminus, an amino acid sequence consisting of: (B-3) a first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) CD8 or a transmembrane domain thereof.

[0109] In one embodiment of the present invention, (B) is: (B) a fusion protein comprising a first T cell-stimulating cytokine and a partial sequence of a tetraspanin, capable of extramembrane display of the first T cell-stimulating cytokine, wherein the partial sequence of the tetraspanin has at least two transmembrane domains and the first T cell-stimulating cytokine is disposed between the two transmembrane domains; or (B) a fusion protein comprising a first T cell-stimulating cytokine and MFG-E8 or a domain thereof, capable of extramembrane display of the first T cell-stimulating cytokine.

[0110] As used herein, "a partial tetraspanin sequence having at least two transmembrane domains, and the first T cell-stimulating cytokine being disposed between the two transmembrane domains" means, for example, a case where the partial tetraspanin sequence includes at least TM1 and TM2 of the tetraspanin, and the first T cell-stimulating cytokine being disposed between TM1 and TM2, or a case where the partial tetraspanin sequence includes at least TM3 and TM4 of the tetraspanin, and the first T cell-stimulating cytokine being disposed between TM3 and TM4.

[0111] In one embodiment of the present invention, (B) is a fusion protein capable of extramembrane display of a first T cell-stimulating cytokine, comprising an amino acid sequence consisting of, from its N-terminus: (B-1) a partial sequence of a tetraspanin comprising, from the N-terminus: transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3; (B-2) an optional spacer sequence; (B-3) a first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) a partial sequence of a tetraspanin comprising transmembrane domain 4; or (B) a fusion protein capable of extramembrane display of a first T cell-stimulating cytokine, comprising, from its N-terminus: (B-3) the first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) MFG-E8.

[0112] As disclosed in WO 2016 / 139354, it has been reported that tetraspanins can be expressed on membranes even when their large extracellular loops (LEL) have been replaced in whole or in part with different amino acid sequences. Therefore, the first T cell-stimulating cytokine (B-3) may be inserted in place of the LEL of the tetraspanin via an optionally present spacer sequence, or may be inserted at any position in the LEL of the tetraspanin or in a partial sequence thereof.

[0113] The "partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop and transmembrane domain 3" in (B-1) generally does not comprise transmembrane domain 4 of the tetraspanin. The "partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop and transmembrane domain 3" in (B-1) may comprise a large extracellular loop or a partial sequence thereof. Each of the domains, transmembrane domain 1, the small extracellular loop, transmembrane domain 2, the small intracellular loop and transmembrane domain 3 in (B-1), may each be derived from a different tetraspanin, or may all be derived from the same tetraspanin. Preferably, each of the domains, transmembrane domain 1, the small extracellular loop, transmembrane domain 2, the small intracellular loop and transmembrane domain 3 in (B-1) are all derived from the same tetraspanin.

[0114] In one embodiment of the present invention, all of the tetraspanin partial sequences in (B-1) comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3 are partial sequences derived from CD9, CD63, or CD81. In one embodiment of the present invention, all of the tetraspanin partial sequences in (B-1) comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3 are partial sequences derived from CD63 or CD81 (preferably SEQ ID NO: 57 or SEQ ID NO: 61, etc., or those having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0115] The "partial sequence of a tetraspanin comprising transmembrane domain 4" in (B-5) generally does not comprise transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, or transmembrane domain 3 of the tetraspanin. The "partial sequence of a tetraspanin comprising transmembrane domain 4" in (B-5) may comprise a large extracellular loop or a partial sequence thereof. The transmembrane domain 4 in (B-5) may be a sequence derived from a tetraspanin other than (B-1), or may be a sequence derived from the same tetraspanin as (B-1). Preferably, the transmembrane domain 4 in (B-5) is a sequence derived from the same tetraspanin as (B-1). In one embodiment of the present invention, the partial sequence of a tetraspanin comprising transmembrane domain 4 in (B-5) is a partial sequence derived from CD9, CD63, or CD81. In one embodiment of the present invention, the partial sequence of the tetraspanin comprising 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, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0116] In one embodiment of the present invention, the "tetraspanin partial sequence comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3" in (B-1) is a partial sequence derived from CD63 (preferably, SEQ ID NO: 57, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), and the "tetraspanin partial sequence comprising transmembrane domain 4" in (B-5) is a partial sequence derived from CD63 (preferably, SEQ ID NO: 59, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more). In one embodiment of the present invention, the "partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3" in (B-1) is a partial sequence derived from CD81 (preferably, SEQ ID NO: 61, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), and the "partial sequence of a tetraspanin comprising transmembrane domain 4" in (B-5) is a partial sequence derived from CD81 (preferably, SEQ ID NO: 63, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0117] When the fusion protein (B) above is a fusion protein containing a partial sequence of a tetraspanin, and one or more of the fusion proteins (A) above and the optional (C) described below contain fusion proteins containing the amino acid sequence of a tetraspanin, the fusion protein (B) above may be a separate fusion protein from the fusion proteins (A) above and / or the optional (C) described below, or may constitute a part of the fusion protein (A) above and / or the optional (C) described below. The fusion protein (B) "constitutes a part of the fusion protein (A) above and / or the optional (C) described below" means, for example, when the tetraspanin (A-5) constitutes the fusion protein (B) and / or when the tetraspanin (C-3) described below constitutes the fusion protein (B).

[0118] The "MFG-E8" in (B-5) above is preferably SEQ ID NO: 49, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0119] In one embodiment of the present invention, the first T cell-stimulating cytokine in (B-3) of each of the above embodiments is IL-2, IL-4, IL-6, IL-12, IL-15, or TGF-β. In one embodiment of the present invention, the first T cell-stimulating cytokine in (B-3) of each of the above embodiments is IL-2 (preferably SEQ ID NO: 25, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), IL-4 (preferably SEQ ID NO: 53, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), or TGF-β (preferably SEQ ID NO: 73, or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0120] The "optional spacer sequences" in (B-2) and (B-4) of each of the above embodiments, when present, can be independently selected. (B-2), when present, may be, for example, a spacer sequence such as SEQ ID NO: 5, 11, 29, 39, or 77. (B-4), when present, may be, for example, a spacer sequence such as SEQ ID NO: 5, 11, 29, 39, or 77.

[0121] In one embodiment of the present invention, (B) comprises, from its N-terminal side: (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 57 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) a first T cell-stimulating cytokine which is IL-2 of SEQ ID NO: 25 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 59 (or one having 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 still more preferably 99% or more thereto). The fusion protein is capable of extramembrane presentation of the first T cell-stimulating cytokine, which has an amino acid sequence consisting of:

[0122] In one embodiment of the present invention, (B) is a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine of SEQ ID NO: 31 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0123] In one embodiment of the present invention, (B) comprises, from its N-terminal side: (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 61 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-2) a spacer sequence of SEQ ID NO: 29; (B-3) a first T cell-stimulating cytokine which is IL-4 of SEQ ID NO: 53 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 29; and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 63 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more). The fusion protein is capable of extramembrane presentation of the first T cell-stimulating cytokine, which has an amino acid sequence consisting of:

[0124] In one embodiment of the present invention, (B) is a fusion protein capable of extramembrane display of the first T cell-stimulating cytokine of SEQ ID NO: 55 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0125] In one embodiment of the present invention, (B) above is a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, the fusion protein having an amino acid sequence consisting of, from its N-terminus: (B-3) a first T cell-stimulating cytokine which is TGF-β of SEQ ID NO: 73 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 29; and (B-5) MFG-E8 of SEQ ID NO: 49 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0126] In one embodiment of the present invention, (B) is a fusion protein capable of extramembrane display of the first T cell-stimulating cytokine of SEQ ID NO: 75 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0127] 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. Thus, in one embodiment of the present invention, the antigen-presenting cells or antigen-presenting extracellular vesicles described herein further comprise a second T cell-stimulating cytokine. In particular, when the antigen-presentable MHC molecule is an antigen-presentable MHC class II molecule, it is preferable that the antigen-presenting cells or antigen-presenting extracellular vesicles described herein comprise a second T cell-stimulating cytokine.

[0128] The second (or more) T cell-stimulating cytokine may be inserted into (B) above, for example (for example, the second (or more) T cell-stimulating cytokine may be linked to the N-terminus and / or C-terminus of the "first T cell-stimulating cytokine" of (B-3), optionally via a spacer sequence or the like). Alternatively, the second (or more) T cell-stimulating cytokine may have a structure similar to that of constituent feature (B) described herein, and thus may be contained in the membrane of the antigen-presenting cell or antigen-presenting extracellular vesicle described herein, similar to the first T cell-stimulating cytokine, as a protein (or fusion protein) separate from the protein (or fusion protein) of constituent feature (B) described herein.

[0129] In one embodiment of the invention, the second T cell stimulating cytokine is IL-2, IL-4, IL-6, IL-12 or TGF-β. In one embodiment of the invention, the second T cell stimulating cytokine is TGF-β (preferably SEQ ID NO: 73, or one having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% amino acid sequence identity thereto).

[0130] In one embodiment of the present invention, the first T cell stimulatory cytokine is IL-2 or IL-4 (preferably SEQ ID NO: 25 or SEQ ID NO: 53, or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more amino acid sequence identity thereto), and the second T cell stimulatory cytokine is TGF-β (preferably SEQ ID NO: 73, or one having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more amino acid sequence identity thereto).

[0131] In one embodiment of the present invention, the antigen-presenting cell or antigen-presenting extracellular vesicle is an antigen-presenting cell or antigen-presenting extracellular vesicle described herein, comprising, in its membrane, the following: (A) a fusion protein or protein complex capable of presenting the antigen on the extracellular side, the fusion protein comprising an antigen-presenting MHC molecule and a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein that can bind to the membrane of an extracellular vesicle or a domain thereof; and (B) a fusion protein capable of presenting a first T-cell stimulating cytokine on the extracellular side, the fusion protein comprising a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein that can bind to the membrane of an extracellular vesicle or a domain thereof.

[0132] In one embodiment of the present invention, there is provided an antigen-presenting cell as described herein, comprising, on its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) an amino acid sequence of an MHC class I α chain; and (A-6) a β ２ and (B) a protein complex comprising: a protein comprising the amino acid sequence of a T-cell stimulating cytokine; and (B) an antigen-presenting cell comprising a fusion protein capable of extramembrane presentation of the first T-cell stimulating cytokine, the fusion protein comprising, from the N-terminus, an amino acid sequence comprising, in this order: (B-3) the amino acid sequence of a first T-cell stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) the amino acid sequence of CD8 or a transmembrane domain thereof.

[0133] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, the membrane of which comprises the following: (A) a fusion protein or protein complex comprising an antigen-presenting MHC molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof, capable of presenting the antigen outside the membrane; and (B) a fusion protein comprising a first T cell-stimulating cytokine and a partial sequence of a tetraspanin, capable of presenting the first T cell-stimulating cytokine outside the membrane, wherein the partial sequence of the tetraspanin has at least two transmembrane domains and the first T cell-stimulating cytokine is disposed between the two transmembrane domains, or (B) a fusion protein comprising a first T cell-stimulating cytokine and MFG-E8 or a domain thereof, capable of presenting the first T cell-stimulating cytokine outside the membrane.

[0134] In one embodiment of the present invention, the present invention provides an antigen-presenting extracellular vesicle described herein, the membrane of which comprises the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; or (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) an MHC class I α chain, β ２ (A-4) an amino acid sequence of a microglobulin, an MHC class II α chain, or an MHC class II β chain; (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; and (A-6) a β ２and (B) a protein complex comprising: a protein comprising the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain; and (B) a fusion protein capable of extramembrane presentation of a first T cell stimulating cytokine, comprising an amino acid sequence comprising, in this order from the N-terminus: (B-1) a partial sequence of a tetraspanin comprising, from the N-terminus, transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3; (B-2) an optionally present spacer sequence; (B-3) the amino acid sequence of a first T cell stimulating cytokine; (B-4) an optionally present spacer sequence; and (B-5) a partial sequence of a tetraspanin comprising transmembrane domain 4; or (B) from the N-terminus: (B-3) the amino acid sequence of a first T cell stimulating cytokine; (B-4) an optionally present spacer sequence; and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof. and a fusion protein capable of extracellularly presenting the first T cell-stimulating cytokine, the fusion protein comprising an amino acid sequence comprising the above in this order.

[0135] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, comprising, in its membrane, the following: (A) a fusion protein capable of extramembrane-presenting an antigenic peptide, the fusion protein comprising an amino acid sequence comprising, from the N-terminus, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class I molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order.

[0136] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, comprising, in its membrane, the following: (A) a protein complex capable of extramembrane-presenting an antigenic peptide, comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of an MHC class II β chain, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain.

[0137] One embodiment of the 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, IL-15, or TGF-β.

[0138] In one embodiment of the present invention, the extracellular vesicles described herein further present a T cell costimulatory molecule on the extracellular membrane (a model thereof is illustrated in Figure 2K(2)). Such extracellular vesicles may present a T cell costimulatory molecule on the extracellular membrane by including in their membrane a protein defined in (C) below. 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, and antigen-presenting MHC molecules and T cell stimulatory cytokines may be attached to the membrane surface by binding phospholipids to the T cell costimulatory molecules and incorporating the phospholipid moiety into the membrane of the extracellular vesicles. In one embodiment of the present invention, the antigen-presenting extracellular vesicles described herein further comprise the following in their membrane: (C) a protein comprising a T cell costimulatory molecule, capable of interacting with T cells.

[0139] Constituent Requirement (C) The "protein comprising a T cell costimulatory molecule, capable of interacting with a T cell" in (C) above may comprise, in addition to the T cell costimulatory molecule, other proteins or domains thereof, etc., as long as the protein is capable of interacting with a T cell and the T cell.

[0140] In one embodiment of the present invention, (C) is a fusion protein capable of interacting with a T cell, the fusion protein comprising a T cell costimulatory molecule and a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof.

[0141] In one embodiment of the present invention, (C) is a protein comprising a T cell costimulatory molecule containing a transmembrane domain, and capable of interacting with the T cell costimulatory molecule and a T cell.

[0142] In one embodiment of the present invention, (C) is a fusion protein comprising a T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof, which is capable of interacting with the T cell costimulatory molecule and a T cell.

[0143] In one embodiment of the present invention, the above-mentioned (C) is a fusion protein capable of interacting with a T cell, comprising an amino acid sequence comprising, from its N-terminus, (C-1) the amino acid sequence of a T cell costimulatory molecule, (C-2) an optional spacer sequence, and (C-3) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order.

[0144] 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, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0145] The "spacer sequence that may be present" in (C-2) above, when present, may be, for example, a spacer sequence such as SEQ ID NO: 5, 11, 29, 39, 77, etc.

[0146] 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, etc., or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0147] In one embodiment of the present invention, the above-mentioned (C) is a fusion protein capable of interacting with a T cell, comprising, from its N-terminus, an amino acid sequence comprising: (C-1) the amino acid sequence of a T cell costimulatory molecule, that is CD80, as represented by SEQ ID NO: 67 (or one having 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 still more preferably 99% or more thereto), and (C-3) the amino acid sequence of a tetraspanin or transmembrane domain thereof, as represented by SEQ ID NO: 21 (or one having 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 still more preferably 99% or more thereto).

[0148] In one embodiment of the present invention, (C) is a fusion protein capable of interacting with a T cell, the T cell costimulatory molecule of SEQ ID NO: 69 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0149] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, comprising in its membrane the following: (A) a fusion protein capable of extramembrane presentation of an antigen peptide, comprising, from the N-terminus, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto); and (B) from the N-terminus, (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 57 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) a first T cell-stimulating cytokine which is IL-2 of SEQ ID NO: 25 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 59 (or one having 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 still more preferably 99% or more thereto), in this order, capable of extramembrane presentation of the first T cell-stimulating cytokine.

[0150] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, comprising in its membrane the following: (A) a fusion protein capable of extramembrane presentation of an antigen peptide, comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto); and (B) a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine of SEQ ID NO: 31 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0151] In one embodiment of the present invention, the present invention provides an antigen-presenting extracellular vesicle described herein, comprising the following amino acid sequences in its membrane: (A) a fusion protein capable of extramembrane presentation of an antigen peptide, the amino acid sequences consisting of, from the N-terminus, (A-1) an MHC class I molecule-restricted antigen peptide, (A-2) a spacer sequence of SEQ ID NO: 5, (A-3) a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) a tetraspanin of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto); (B) a fusion protein comprising, from the N-terminus, (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 57 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) a first T cell-stimulating cytokine which is IL-2 of SEQ ID NO: 25 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 59 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more);and (C) an antigen-presenting extracellular vesicle comprising, from the N-terminus, (C-1) a T cell costimulatory molecule that is CD80 of SEQ ID NO: 67 (or one having 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 still more preferably 99% or more thereto), and (C-3) a fusion protein capable of interacting with a T cell, the fusion protein having an amino acid sequence consisting of:

[0152] In one embodiment of the present invention, the antigen-presenting extracellular vesicle is an antigen-presenting extracellular vesicle described herein, comprising in its membrane the following: (A) a fusion protein capable of extramembrane presentation of an antigen peptide, comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigen peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto); (B) a fusion protein of SEQ ID NO: 31 (or one having 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 still more preferably 99% or more thereto) capable of extramembrane presentation of the first T cell stimulatory cytokine; and (C) a fusion protein of SEQ ID NO: 69 (or one having 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 still more preferably 99% or more thereto) capable of interacting with the T cell costimulatory molecule and a T cell.

[0153] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) A protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having 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 still more preferably 99% or more thereto); (B) A protein complex consisting of, from the N-terminus, (B-1) a partial sequence of tetraspanin of SEQ ID NO: 57 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) an amino acid sequence of a first T cell stimulating cytokine, which is IL-2 of SEQ ID NO: 25 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, comprising an amino acid sequence comprising, in this order, a partial sequence of tetraspanin of SEQ ID NO: 59 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more);and (C) a fusion protein capable of interacting with a T cell, comprising, from the N-terminus, an amino acid sequence comprising: (C-1) the amino acid sequence of a T cell costimulatory molecule that is CD80 of SEQ ID NO: 67 (or one having 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 still more preferably 99% or more thereto), and (C-3) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 21 (or one having 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 still more preferably 99% or more thereto).

[0154] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) a protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having 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 still more preferably 99% or more thereto); (B) the amino acid sequence of a fusion protein capable of extramembrane presentation of the first T cell stimulatory cytokine of SEQ ID NO: 31 (or one having 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 still more preferably 99% or more thereto); and (C) the amino acid sequence of a fusion protein capable of interacting with the T cell costimulatory molecule of SEQ ID NO: 69 (or one having 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 still more preferably 99% or more thereto).

[0155] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) A protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having 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 still more preferably 99% or more thereto); (B) A protein complex consisting of, from the N-terminus, (B-1) a partial sequence of tetraspanin of SEQ ID NO: 57 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) an amino acid sequence of a first T cell stimulating cytokine, which is IL-2 of SEQ ID NO: 25 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, comprising an amino acid sequence comprising, in this order, a partial sequence of tetraspanin of SEQ ID NO: 59 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more);(B') a fusion protein capable of extramembrane presentation of a second T cell-stimulating cytokine, comprising an amino acid sequence comprising, in this order from the N-terminus: (B-3) the amino acid sequence of a second T cell-stimulating cytokine, which is TGF-β of SEQ ID NO: 73 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 29; and (B-5) the amino acid sequence of MFG-E8 of SEQ ID NO: 49 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); and (C) from the N-terminus: An antigen-presenting extracellular vesicle comprising: (C-1) an amino acid sequence of a T cell costimulatory molecule, which is CD80, as represented by SEQ ID NO: 67 (or one having 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 still more preferably 99% or more thereto); and (C-3) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, as represented by SEQ ID NO: 21 (or one having 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 still more preferably 99% or more thereto), in this order, which fusion protein is capable of interacting with the T cell costimulatory molecule and a T cell.

[0156] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or membrane-binding domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) a protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B) a fusion protein capable of extramembrane display of the first T cell stimulating cytokine of SEQ ID NO: 31 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B') a fusion protein capable of extramembrane display of the second T cell stimulating cytokine of SEQ ID NO: 75 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); and (C) a fusion protein of SEQ ID NO: 69 (or one having 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 still more preferably 99% or more thereto), capable of interacting with a T cell.

[0157] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) A protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having 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 still more preferably 99% or more thereto); (B) A protein complex consisting of, from the N-terminus, (B-1) a partial sequence of tetraspanin of SEQ ID NO: 61 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) an amino acid sequence of a first T cell stimulating cytokine, which is IL-4 of SEQ ID NO: 53 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, comprising an amino acid sequence comprising, in this order, a partial sequence of tetraspanin of SEQ ID NO: 63 (or one having an amino acid sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more);and (C) a fusion protein capable of interacting with a T cell, comprising, from the N-terminus, an amino acid sequence comprising: (C-1) the amino acid sequence of a T cell costimulatory molecule that is CD80 of SEQ ID NO: 67 (or one having 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 still more preferably 99% or more thereto), and (C-3) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 21 (or one having 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 still more preferably 99% or more thereto).

[0158] In one embodiment of the present invention, the antigen-presenting extracellular vesicle described herein comprises a fusion protein comprising, in its membrane, the following: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto). (A-6) a protein complex consisting of an MHC class II α chain of SEQ ID NO: 71 (or one having 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 still more preferably 99% or more thereto); (B) a fusion protein of SEQ ID NO: 55 (or one having 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 still more preferably 99% or more thereto) capable of extramembrane presentation of the first T cell stimulatory cytokine; and (C) a fusion protein of SEQ ID NO: 69 (or one having 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 still more preferably 99% or more thereto) capable of interacting with the T cell costimulatory molecule of SEQ ID NO: 69 (or one having 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 still more preferably 99% or more thereto) capable of interacting with a T cell.

[0159] In one embodiment of the present invention, with respect to (A), (B), and (C), (A) and (B) may be fused to form a single molecule, (B) and (C) may be fused to form a single molecule, or (A), (B), and (C) may be fused to form a single molecule. Such a fusion molecule may be translated as a single protein molecule with or without a spacer sequence between (A), (B), and (C), or may be fused to form a single molecule by chemically crosslinking the proteins (A), (B), and (C) (e.g., a disulfide bond between cysteine ​​residues). Alternatively, (A), (B), and (C) may be functionally fused by sharing an element for localizing the protein to a cell or extracellular vesicle, i.e., a "membrane protein or a transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle" or a "protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle." For example, in one embodiment of the present invention, the fusion protein (D) may include: (1) an antigen-presenting MHC molecule; (2) at least one T cell stimulatory cytokine; and (3) a "membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof" or a "protein that can bind to the membrane of a cell or extracellular vesicle or a domain thereof" fused between (A) and (B) in a manner that they share the portion "a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof" or "a protein that can bind to the membrane of a cell or extracellular vesicle or a domain thereof"; or (A) and (C) in a manner that they share the portion "a membrane protein that can be expressed on the membrane of an extracellular vesicle or a transmembrane domain thereof" or "a protein that can bind to the membrane of an extracellular vesicle or a domain thereof". (3) A fusion protein (F) containing a "membrane protein or a transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle" or a "protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle";(B) and (C) may contain: (1) at least one T cell stimulatory cytokine; (2) a T cell costimulatory molecule; and (3) a fusion protein (G) containing a "membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof" or a "protein capable of binding to the membrane of a cell or extracellular vesicle or a domain thereof" portion, which are fused together; or (A) to (C) may contain: (1) an antigen-presenting MHC molecule; (2) at least one T cell stimulatory cytokine; (3) a T cell costimulatory molecule; and (4) It may contain a fusion protein (E) containing a "membrane protein or its transmembrane domain that can be expressed in the membrane of a cell or extracellular vesicle" or a "protein or its domain that can bind to the membrane of a cell or extracellular vesicle."

[0160] In one embodiment of the present invention, the present invention may be a cell or antigen-presenting extracellular vesicle cell comprising fusion protein (D) having the functions of constituent requirement (A) and constituent requirement (B), using constituent requirement (B) "a protein capable of presenting the first T cell-stimulating cytokine on the extramembrane, including a first T cell-stimulating cytokine," instead of constituent requirement (A) "a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle, or a transmembrane domain thereof, or a protein capable of binding to the membrane of an extracellular vesicle."

[0161] The fusion protein (D) having the functions of the constituent features (A) and (B) may be a fusion protein that contains an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine and is capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine. The fusion protein may contain the antigen-presenting MHC molecule, the at least one T cell-stimulating cytokine, and a membrane protein capable of localizing in the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof.

[0162] In fusion protein (D) having the functions of constituent features (A) and (B), the membrane protein capable of localizing in the cell membrane or the protein capable of binding to the cell membrane may be CD8, or an MHC molecule containing a transmembrane domain may perform that function. The fusion protein may contain, from its N-terminus, an amino acid sequence encoding, in this order: (D-1) an amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) an amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) a fusion peptide containing CD8 or a transmembrane domain thereof and at least one T cell-stimulating cytokine. The fusion protein may comprise, from its N-terminus, an amino acid sequence encoding, in this order: (D-1) an amino acid sequence of an MHC molecule-restricted antigen peptide; (D-2) an optional spacer sequence; (D-3) an amino acid sequence of a single-chain MHC molecule comprising a transmembrane domain; (D-4) an optional spacer sequence; and (D-5) at least one T cell-stimulating cytokine.

[0163] In fusion protein (D) having the functions of constituent features (A) and (B), the membrane protein capable of localizing in the membrane of extracellular vesicles or the protein capable of binding to the membrane of extracellular vesicles may be a tetraspanin or MFG-E8. The fusion protein may comprise, from its N-terminus, an amino acid sequence encoding, in this order: (D-1) an amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) an amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and a fusion peptide comprising at least one T cell-stimulating cytokine. The fusion protein may comprise, from its N-terminus, an amino acid sequence encoding, in this order: (D-1) a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof and at least one T cell-stimulating cytokine, (D-2) an optional spacer sequence, (D-3) the amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) an MHC molecule-restricted antigenic peptide. Here, the fusion peptide may comprise, from its N-terminus, an amino acid sequence encoding, in this order: (1) a partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (2) an optional spacer sequence, (3) the amino acid sequence of at least one T cell-stimulating cytokine, (4) an optional spacer sequence, and (5) a partial sequence of a tetraspanin comprising transmembrane domain 4. The fusion peptide may comprise, from its N-terminus, an amino acid sequence encoding, in this order, (1) the amino acid sequence of at least one T cell-stimulating cytokine, (2) an optional spacer sequence, and (3) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof.

[0164] In one embodiment of the present invention, the MHC molecule-restricted antigenic peptide may be an MHC class I molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular region of the MHC class I α chain; or the MHC molecule-restricted antigenic peptide may be an MHC class II molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular domain of the MHC class II α chain and / or the extracellular domain of the MHC class II β chain.

[0165] In an embodiment comprising a fusion protein (D) having the functions of constituent features (A) and (B); (C) the fusion protein may further comprise, in its membrane, a protein comprising at least one T cell costimulatory molecule and capable of interacting with a T cell; the protein capable of interacting with a T cell may comprise the at least one T cell costimulatory molecule and a membrane protein capable of being expressed on the membrane of a cell or an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or an extracellular vesicle or a domain thereof; the protein capable of interacting with a T cell may comprise the at least one T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof. The protein capable of interacting with a T cell may comprise one T cell costimulatory molecule comprising a transmembrane domain.

[0166] In one embodiment of the present invention, the extracellular vesicles are exosomes.

[0167] The antigen-presenting cells or antigen-presenting extracellular vesicles described herein may contain or be bound to a potentially therapeutically beneficial substance (e.g., a low-molecular-weight compound, a nucleic acid, etc.) within or within their membranes. Methods for encapsulating the substance within the membrane of cells or extracellular vesicles include, but are not limited to, a method in which the substance is mixed with the cells or extracellular vesicles described herein in a suitable solvent. In one embodiment of the present invention, the antigen-presenting cells or antigen-presenting extracellular vesicles may contain any protein preparation. Protein preparations may include, but are not limited to, naturally occurring proteins such as erythropoietin, non-naturally occurring synthetic proteins such as immunoglobulin-CTLA4 fusion proteins, or monoclonal antibodies or active fragments thereof. These protein preparations may be localized on the surface of the antigen-presenting extracellular vesicles as fusion proteins with membrane proteins or transmembrane domains thereof that can be localized on the membrane of cells or extracellular vesicles, or proteins or membrane-binding domains thereof that can bind to the membrane of cells or extracellular vesicles. Such antigen-presenting cells or antigen-presenting extracellular vesicles can be produced by transfecting a vector for expressing the fusion protein into: 1) any cell; or 2) a cell that produces extracellular vesicles, which can be secreted by transfecting the vector into a cell that produces extracellular vesicles.

[0168] Each fusion protein, protein complex, or protein preparation contained in the membrane of the antigen-presenting cell or 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, radioactive material, or enzyme (e.g., peroxidase, phosphatase), etc. These may be linked, for example, to the N-terminus or C-terminus of the fusion protein, protein complex, or protein preparation as a component of the fusion protein, protein complex, or protein preparation.

[0169] Polynucleotides

[0170] In one embodiment of the present invention, there are provided polynucleotides encoding each of the fusion proteins or protein complexes (A) and (B), and optionally (C), contained in the membrane of the antigen-presenting cell or antigen-presenting extracellular vesicle described herein. In one embodiment of the present invention, there are provided polynucleotides encoding each of the fusion proteins or protein complexes (A) to (G) defined herein.

[0171] In one embodiment of the present invention, there are provided: (a) a sequence encoding a fusion protein (A) comprising an antigen-presenting MHC molecule and capable of presenting the antigen-presenting MHC molecule on the extracellular membrane of a cell or extracellular vesicle; (b) a sequence encoding a fusion protein (B) comprising at least one T cell stimulatory cytokine or a subunit thereof, capable of presenting the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle; (c) a sequence encoding a fusion protein (C) comprising a T cell costimulatory molecule, capable of presenting the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle; (d) a sequence encoding a fusion protein (D) comprising an antigen-presenting MHC molecule and at least one T cell stimulatory cytokine or a subunit thereof, capable of presenting the antigen and the T cell stimulatory cytokine on the extracellular membrane of a cell or extracellular vesicle; and (e) a sequence encoding a fusion protein (E) comprising an antigen-presenting MHC molecule and at least one T cell stimulatory cytokine or a subunit thereof, and a T cell costimulatory molecule, and capable of presenting the antigen, the T cell stimulatory cytokine, and the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle. The sequences (a) to (e) include, but are not limited to, sequences specifically described herein and sequences highly homologous thereto (preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology). As long as they have equivalent functions, they may be paralogs (gene sequences resulting from gene duplication) or orthologs (gene groups with homologous functions present in different organisms), and also include sequences with altered sequence information (such as deletions, substitutions, or deletions).

[0172] As used herein, "polynucleotide" refers to a single-stranded or double-stranded DNA molecule, an RNA molecule, or a DNA-RNA chimeric molecule. Polynucleotides include genomic DNA, cDNA, hnRNA, mRNA, and the like, as well as all naturally occurring or artificially modified derivatives thereof. Polynucleotides may be linear or circular.

[0173] A polynucleotide encoding each of the fusion proteins or protein complexes (A) to (G) described above can be appropriately determined by a person skilled in the art with reference to the amino acid sequence of the fusion protein or protein complex. The amino acid sequence of each of the fusion proteins or protein complexes (A) to (G) can be appropriately determined with reference to the amino acid sequences of each component of each fusion protein or protein complex (e.g., 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 taking into consideration the codon frequency of cells to be transformed using a vector containing the polynucleotide.

[0174] If necessary, a polynucleotide encoding a signal peptide (signal sequence) may be added to the N-terminus of the polynucleotide encoding each of the fusion proteins or protein complexes (A) to (G) described above.

[0175] Any amino acid sequence can be used for the signal peptide, and may be determined taking into consideration, for example, the amino acid sequence of the fusion protein to be expressed. Examples of polynucleotides encoding signal peptides include, for example, β ２ Examples of such polynucleotides include a polynucleotide (e.g., SEQ ID NO: 2) encoding a signal peptide of a microglobulin (e.g., SEQ ID NO: 1), a polynucleotide (e.g., SEQ ID NO: 3) encoding a signal peptide of an MHC class I α chain, a polynucleotide (e.g., SEQ ID NO: 3) encoding a signal peptide of an MHC class II α chain, and a polynucleotide (e.g., SEQ ID NO: 34) encoding a signal peptide of an MHC class II β chain (e.g., SEQ ID NO: 33).

[0176] Information on the amino acid sequences of the components of each of the fusion proteins or protein complexes (A) to (G) above (for example, in the case of (A), (A-1) to (A-5), and (A-6) if present), signal peptides, etc., as well as the polynucleotides encoding them, may be appropriately obtained, for example, by searching publicly known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Furthermore, WO 2016 / 139354 may be used as a reference for the amino acid sequences of tetraspanin partial sequences (for example, partial sequences in (C-1) and (C-5)) and the polynucleotides encoding them.

[0177] In one embodiment of the present invention, (a) may comprise a sequence encoding the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, comprising, from its N-terminus, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC molecule comprising a transmembrane domain. In one embodiment of the present invention, (a) may comprise: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from its N-terminus, an amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) an MHC class I α chain, β chain, or both. ２ a fusion protein comprising an amino acid sequence including, in this order, an amino acid sequence consisting of a fusion protein of microglobulin and an MHC class I α chain, an MHC class II α chain, or an MHC class II β chain; and (A-6) β ２and a protein comprising the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain; wherein, when translated, it is preferred that (A-3) and (A-6) pair to form an MHC class I molecule or an MHC class II molecule. In one embodiment of the present invention, (a) may comprise a sequence in which the amino acid sequence consisting of the above (A-1) to (A-3) and the sequence of (A-6) become a sequence encoding a single fusion protein via at least one of the following 2A peptide sequences: T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 211) P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 212) E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 213) F2A (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 214). The 2A peptide sequence causes ribosomal skipping, and when the sequence encoding the fusion protein is actually translated, a fusion protein containing the independent amino acid sequences (A-1) to (A-3) and a protein containing the independent sequence (A-6) are translated, and it is preferred that the two translated proteins form an MHC class I molecule or an MHC class II molecule.

[0178] In one embodiment of the present invention, (a) may comprise: (A) a sequence encoding a fusion protein capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising an amino acid sequence comprising, in this order from the N-terminus, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC class I molecule comprising a transmembrane domain; or (A) a sequence encoding a fusion protein capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising an amino acid sequence comprising, in this order from the N-terminus, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of a single-chain MHC class II molecule comprising a transmembrane domain.

[0179] In one embodiment of the present invention, the above-mentioned (a) is: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus thereof: (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; and (A-3) a β ２ In one embodiment of the present invention, the above (a) may comprise an amino acid sequence comprising, in this order, (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising, from the N-terminus thereof, (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of an MHC class I α chain; and (A-6) a β ２and a protein comprising the amino acid sequence of an MHC class II α chain, or may comprise a sequence in which the amino acid sequence consisting of (A-1) to (A-3) and the amino acid sequence of (A-6) form a sequence encoding a single fusion protein via at least one 2A peptide sequence. In one embodiment of the present invention, (a) above may comprise a sequence encoding a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising an amino acid sequence comprising, from the N-terminus thereof, (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of an MHC class II β chain, in this order; and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain, or may comprise a sequence in which the amino acid sequence consisting of (A-1) to (A-3) and the amino acid sequence of (A-6) form a sequence encoding a single fusion protein via at least one 2A peptide sequence. In one embodiment of the present invention, the above (a) may comprise a sequence encoding a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, and (A-3) the amino acid sequence of an MHC class II α chain; and (A-6) a protein comprising the amino acid sequence of an MHC class II β chain.

[0180] In one embodiment of the present invention, (A) is a fusion protein or protein complex comprising an antigen-presenting MHC molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof, and capable of presenting the antigen outside the membrane.

[0181] In one embodiment of the present invention, the (a) may comprise a sequence encoding a fusion protein comprising, from the N-terminus thereof, the amino acid sequence of: (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; (A-3) the amino acid sequence of a single-chain MHC molecule; (A-4) an optional spacer sequence; and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; or (A) a protein complex capable of presenting an antigenic peptide outside a membrane, comprising, from the N-terminus thereof, the amino acid sequence of: (A-1) the amino acid sequence of an MHC molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; and (A-3) an MHC class I α chain, β chain, or ２ (A-4) an amino acid sequence of a microglobulin, an MHC class II α chain, or an MHC class II β chain; (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; and (A-6) a β ２ and a protein comprising the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain.

[0182] In one embodiment of the present invention, the above (a) may comprise a sequence encoding (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, the fusion protein comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class I molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof. In one embodiment of the present invention, the above (a) may comprise a sequence encoding: (A) a fusion protein capable of presenting an antigenic peptide outside a membrane, the fusion protein comprising, from its N-terminus, an amino acid sequence consisting of: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of a single-chain MHC class II molecule, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof.

[0183] In one embodiment of the present invention, the above (a) comprises the following: (A) a fusion protein capable of extramembrane presentation of an antigen peptide, which comprises, from the N-terminus thereof: (A-1) an amino acid sequence of an MHC molecule-restricted antigen peptide; (A-2) an optional spacer sequence; (A-3) an MHC class I α chain, β chain, ２ (A-4) an amino acid sequence of a microglobulin, an MHC class II α chain, or an MHC class II β chain; (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; and (A-6) a β ２It may also include a sequence encoding the amino acid sequence of microglobulin, MHC class I α chain, MHC class II β chain, or MHC class II α chain. When translated, it is preferred that (A-3) and (A-6) pair to form an MHC class I molecule or an MHC class II molecule. In one embodiment of the present invention, the above (a) may comprise a sequence in which the amino acid sequence consisting of the above (A-1) to (A-5) and the sequence of (A-6) become a sequence encoding a single fusion protein via at least one of the following 2A peptide sequences: T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 211) P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 212) E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 213) F2A (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 214). The 2A peptide sequence causes ribosomal skipping, and when the sequence encoding the fusion protein is actually translated, a fusion protein containing the independent amino acid sequences (A-1) to (A-5) and a protein containing the independent sequence (A-6) are translated, and the two translated proteins preferably form an MHC class I molecule or an MHC class II molecule. In one embodiment of the present invention, the above (a) is a polynucleotide comprising the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, which comprises, from the N-terminus thereof, (A-1) an amino acid sequence of an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) an MHC class I α chain, a β chain, an MHC class I β chain, an MHC class I α chain, an MHC class I ... ２ (A-4) an amino acid sequence of a microglobulin, an MHC class II α chain, or an MHC class II β chain; (A-5) an amino acid sequence of a tetraspanin or a membrane-binding domain thereof; (A-5.5) a 2A peptide sequence; (A-6) β ２In one embodiment of the present invention, (a) may be a polynucleotide that encodes a fusion protein comprising an amino acid sequence comprising, in this order, the amino acid sequence of a microglobulin, an MHC class I α chain, an MHC class II β chain, or an MHC class II α chain. In one embodiment of the present invention, (a) may be a polynucleotide that encodes the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus, an amino acid sequence comprising, in this order: (A-1) an MHC molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of an MHC class II β chain, (A-4) an optional spacer sequence, (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, (A-5.5) a 2A peptide sequence, and (A-6) the amino acid sequence of an MHC class II α chain. An example of such an embodiment is HLADR-1sc-TPI1-hCD81 (amino acid sequence: SEQ ID NO: 165; polynucleotide sequence: SEQ ID NO: 166).

[0184] In one embodiment of the present invention, the above (a) is: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus thereof: (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; (A-3) β ２(A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) an amino acid sequence of an MHC class I α chain, (A-4) an optional spacer sequence, and (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order, and (A-6) a protein comprising the amino acid sequence of an MHC class I α chain. In one embodiment of the present invention, the (a) above may comprise a sequence encoding a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus thereof, an amino acid sequence comprising, in this order: (A-1) an amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) an amino acid sequence of an MHC class I α chain, (A-4) an optional spacer sequence, and (A-5) an amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a protein comprising the amino acid sequence of an MHC class I α chain. ２In one embodiment of the present invention, the (a) may comprise a sequence encoding a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, a fusion protein comprising an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide, (A-2) an optional spacer sequence, (A-3) the amino acid sequence of an MHC class II β chain, (A-4) an optional spacer sequence, and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof; and (A-6) a protein comprising the amino acid sequence of an MHC class II α chain. In one embodiment of the present invention, the above (a) may comprise a sequence encoding a protein complex comprising: (A) a protein complex capable of extramembrane presentation of an antigenic peptide, the protein complex comprising, from the N-terminus thereof, an amino acid sequence comprising: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigenic peptide; (A-2) an optional spacer sequence; (A-3) the amino acid sequence of an MHC class II α chain; (A-4) an optional spacer sequence; and (A-5) the amino acid sequence of a tetraspanin or a transmembrane domain thereof, in this order; and (A-6) a protein comprising the amino acid sequence of an MHC class II β chain.

[0185] In one embodiment of the present invention, (a) is a sequence encoding a fusion protein comprising, in this order from the N-terminus, the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto), or (A) a fusion protein constituting a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus, The fusion protein may comprise a sequence encoding an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigen peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto).

[0186] In one embodiment of the present invention, (a) is a sequence encoding a fusion protein comprising, in this order from the N-terminus, the following: (A) a fusion protein capable of extramembrane presentation of an antigenic peptide, (A-1) the amino acid sequence of an MHC class I molecule-restricted antigenic peptide, (A-2) the spacer sequence of SEQ ID NO: 5, (A-3) the amino acid sequence of a single-chain MHC class I molecule of SEQ ID NO: 65 (or one having 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 still more preferably 99% or more thereto), and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto), or (A) a fusion protein constituting a protein complex capable of extramembrane presentation of an antigenic peptide, comprising, from the N-terminus, The fusion protein may comprise a sequence encoding an amino acid sequence comprising, in this order: (A-1) the amino acid sequence of an MHC class II molecule-restricted antigen peptide; (A-2) the spacer sequence of SEQ ID NO: 39; (A-3) the amino acid sequence of an MHC class II β chain of SEQ ID NO: 37 (or one having 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 still more preferably 99% or more thereto); and (A-5) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 15 (or one having 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 still more preferably 99% or more thereto).

[0187] In one embodiment of the present invention, (a) is a sequence comprising, in this order from the 5' end: (A) a polynucleotide encoding a fusion protein capable of extramembrane presentation of an antigenic peptide, (A-1) a sequence encoding an MHC class I molecule-restricted antigenic peptide, (A-2) a spacer sequence of SEQ ID NO: 6, (A-3) a sequence encoding a single-chain MHC class I molecule of SEQ ID NO: 66 (or one having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more thereto), and (A-5) a sequence encoding a tetraspanin or transmembrane domain thereof of SEQ ID NO: 16 (or one having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more thereto), or (A) a polynucleotide encoding a fusion protein constituting a protein complex capable of extramembrane presentation of an antigenic peptide, (A-1) a polynucleotide encoding an MHC class II molecule-restricted antigenic peptide, (A-2) a spacer sequence of SEQ ID NO: 40; (A-3) a sequence encoding an MHC class II β chain of SEQ ID NO: 38 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); and (A-5) a sequence encoding a tetraspanin or transmembrane domain thereof of SEQ ID NO: 16 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).In one embodiment of the present invention, the above-mentioned (a) is a sequence comprising, in this order from the 5' end: (A) a polynucleotide encoding a fusion protein capable of extramembrane presentation of an antigenic peptide, (A-1) a polynucleotide encoding an MHC class I molecule-restricted antigenic peptide, (A-2) a spacer sequence of SEQ ID NO: 6, (A-3) a sequence encoding a single-chain MHC class I molecule of SEQ ID NO: 66 (or one having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more thereto), and (A-5) a sequence encoding a tetraspanin or a transmembrane domain thereof of SEQ ID NO: 16 (or one having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more thereto), or (A-2) a spacer sequence of SEQ ID NO: 40, (A-3) a sequence encoding an MHC class II β chain of SEQ ID NO: 38 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more), and (A-5) a sequence encoding a tetraspanin or transmembrane domain thereof of SEQ ID NO: 16 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more).

[0188] In one embodiment of the present invention, the above (b) may comprise (B) a sequence encoding a fusion protein comprising a first T cell-stimulating cytokine and CD8 or a transmembrane domain thereof, capable of extramembrane presentation of the first T cell-stimulating cytokine.

[0189] In one embodiment of the present invention, the above (b) may comprise a sequence encoding a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, the fusion protein comprising, from the N-terminus, an amino acid sequence comprising, in this order: (B) (B-3) the amino acid sequence of a first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) the amino acid sequence of CD8 or a transmembrane domain thereof.

[0190] In one embodiment of the present invention, (b) above may comprise: (B) a sequence encoding a fusion protein comprising a first T cell-stimulating cytokine and a partial sequence of a tetraspanin, the fusion protein being capable of extramembrane display of the first T cell-stimulating cytokine, wherein the partial sequence of the tetraspanin has at least two transmembrane domains and the first T cell-stimulating cytokine is disposed between the two transmembrane domains; or (B) a sequence encoding a fusion protein comprising a first T cell-stimulating cytokine and MFG-E8 or a domain thereof, the fusion protein being capable of extramembrane display of the first T cell-stimulating cytokine.

[0191] In one embodiment of the present invention, the above (b) may comprise: (B) a sequence encoding a fusion protein capable of extramembrane display of a first T cell-stimulating cytokine, the sequence comprising an amino acid sequence comprising, in this order from the N-terminus: (B-1) a partial sequence of a tetraspanin comprising, from the N-terminus, transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3; (B-2) an optional spacer sequence; (B-3) the amino acid sequence of a first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) a partial sequence of a tetraspanin comprising transmembrane domain 4; or (B) a sequence encoding a fusion protein capable of extramembrane display of a first T cell-stimulating cytokine, the sequence comprising, in this order from the N-terminus: (B-3) the amino acid sequence of a first T cell-stimulating cytokine; (B-4) an optional spacer sequence; and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof.

[0192] In one embodiment of the present invention, the above (b) is: (B) from the N-terminus: (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 57 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) an amino acid sequence of a first T cell-stimulating cytokine that is IL-2 of SEQ ID NO: 25 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 59 (or one having 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 still more preferably 99% or more thereto). a sequence encoding a fusion protein capable of extramembrane presentation of the first T cell-stimulating cytokine, the fusion protein comprising an amino acid sequence comprising, in this order:(B) From the N-terminus, (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 61 (or one having 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 still more preferably 99% or more thereto), (B-2) a spacer sequence of SEQ ID NO: 29, (B-3) an amino acid sequence of a first T cell-stimulating cytokine, which is IL-4 of SEQ ID NO: 53 (or one having 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 still more preferably 99% or more thereto), (B-4) a spacer sequence of SEQ ID NO: 29, and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 63 (or one having 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 still more preferably 99% or more thereto). (B) a sequence encoding a fusion protein capable of extramembrane display of the first T cell-stimulating cytokine, comprising an amino acid sequence comprising, in this order from the N-terminus: (B-3) the amino acid sequence of a second T cell-stimulating cytokine, which is TGF-β of SEQ ID NO: 73 (or one having 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 still more preferably 99% or more thereto); (B-4) a spacer sequence of SEQ ID NO: 29; and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof of SEQ ID NO: 49 (or one having 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 still more preferably 99% or more thereto);

[0193] In one embodiment of the present invention, the above (b) is: (B) a polynucleotide encoding a fusion protein capable of extramembrane presentation of a first T cell-stimulating cytokine, comprising from the 5' end: (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 58 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-2) a spacer sequence of SEQ ID NO: 30; (B-3) a sequence encoding the first T cell-stimulating cytokine, which is IL-2 of SEQ ID NO: 26 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 30; and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 60 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more). (B) a polynucleotide encoding a fusion protein capable of extramembrane presentation of a first T cell-stimulating cytokine, comprising, from the 5' end: (B-1) a partial sequence of the tetraspanin of SEQ ID NO: 62 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-2) a spacer sequence of SEQ ID NO: 30; (B-3) a first T cell-stimulating cytokine which is IL-4 of SEQ ID NO: 54 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 30; and (B-5) a partial sequence of the tetraspanin of SEQ ID NO: 64 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); an array containing, in this order;or (B) a polynucleotide encoding a fusion protein capable of extramembrane presentation of a second (or first) T cell-stimulating cytokine, comprising, from the 5' end, in this order: (B-3) the second (or first) T cell-stimulating cytokine, which is TGF-β of SEQ ID NO: 74 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B-4) a spacer sequence of SEQ ID NO: 30; and (B-5) a sequence encoding MFG-E8 of SEQ ID NO: 50 (or one having sequence identity thereto of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more); (B) a sequence encoding a fusion protein capable of extramembrane display of the first (or second) T cell-stimulating cytokine, which is represented by SEQ ID NO: 31, 75, or 55 (or one having 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 still more preferably 99% or more thereto); or (B) a sequence encoding a fusion protein capable of extramembrane display of the first (or second) T cell-stimulating cytokine, which is represented by SEQ ID NO: 32, 76, or 56 (or one having 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 still more preferably 99% or more thereto);

[0194] When a T cell-stimulating cytokine functions through a heterogeneous combination of subunits, it is preferable that in (b) above, the sequence of one subunit is used as the sequence of the T cell-stimulating cytokine in (B) above, and the sequence of the remaining subunit is separately included in (b), and when this sequence is translated, it is preferable that an active T cell-stimulating cytokine is formed outside the membrane by the fusion protein of (B) and the remaining subunit. In one embodiment of the present invention, when a T cell-stimulating cytokine functions through a heterogeneous combination of subunits, (b) may include a sequence that is translated as a protein formed by fusing the fusion protein of (B) and the remaining subunit. Such a fusion protein of (B) and the remaining subunit may be fused via a spacer sequence or a 2A peptide sequence. In one embodiment of the present invention, (b) may comprise a sequence encoding a fusion protein capable of presenting IL-12 outside the cell membrane, comprising an amino acid sequence comprising, from the N-terminus, in this order: (B) (B-6) the amino acid sequence of an IL-12β subunit; (B-7) an optional spacer sequence; (B-3) the amino acid sequence of an IL-12α subunit; (B-4) an optional spacer sequence; and (B-5) the amino acid sequence of CD8 or a transmembrane domain thereof. In one embodiment of the present invention, (b) may comprise a sequence encoding a fusion protein capable of presenting IL-12 on the extracellular membrane of extracellular vesicles, the fusion protein comprising, from the N-terminus, the amino acid sequence comprising, in this order: (B) from the N-terminus: (B-6) the amino acid sequence of an IL-12β subunit, (B-7) an optional spacer sequence, (B-3) the amino acid sequence of an IL-12α subunit, (B-4) an optional spacer sequence, and (B-5) the amino acid sequence of MFG-E8 or a membrane-binding domain thereof. Such an embodiment is exemplified by hIL-12sc-MFGe8 (amino acid sequence: SEQ ID NO: 177; polynucleotide sequence 178).

[0195] In one embodiment of the present invention, (c) may comprise a sequence encoding: (c) a T cell costimulatory molecule comprising a transmembrane domain; or a fusion protein comprising a T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof. In one embodiment of the present invention, (c) may comprise: (C) a sequence encoding a fusion protein capable of interacting with a T cell, comprising an amino acid sequence comprising, from the N-terminus: (C-1) the amino acid sequence of a T cell costimulatory molecule, (C-2) an optional spacer sequence, and (C-3) the amino acid sequence of a tetraspanin or a membrane-binding domain thereof, in this order.

[0196] In one embodiment of the present invention, (c) may comprise an amino acid sequence encoding a fusion protein capable of interacting with a T cell, comprising, from the N-terminus, the amino acid sequence comprising: (C-1) the amino acid sequence of a T cell costimulatory molecule that is CD80 of SEQ ID NO: 67 (or one having 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 still more preferably 99% or more to that effect); and (C-3) the amino acid sequence of a tetraspanin or transmembrane domain thereof of SEQ ID NO: 21 (or one having 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 still more preferably 99% or more to that effect). In one embodiment of the present invention, (c) may comprise: (C) a sequence encoding a fusion protein capable of interacting with the T cell costimulatory molecule of SEQ ID NO: 23 (or one having 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 still more preferably 99% or more thereto). In one embodiment of the present invention, (c) may comprise: (C) a sequence comprising, from the 5' end: (C-1) a sequence encoding a T cell costimulatory molecule that is CD80 of SEQ ID NO: 68 (or one having 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 still more preferably 99% or more thereto), and (C-3) a sequence encoding a tetraspanin or membrane-binding domain thereof of SEQ ID NO: 22 (or one having 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 still more preferably 99% or more thereto). In one embodiment of the present invention, (c) may comprise: (C) a sequence encoding a fusion protein capable of interacting with the T cell costimulatory molecule of SEQ ID NO: 24 (or a sequence having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more sequence identity thereto).

[0197] In one embodiment of the present invention, (d) comprises: (D) a sequence encoding a fusion protein comprising the antigen-presenting MHC molecule, the at least one T cell-stimulating cytokine or a subunit thereof, and a membrane protein capable of localizing in the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof. The membrane protein capable of localizing in the membrane of an extracellular vesicle or the protein capable of binding to the membrane of an extracellular vesicle may be tetraspanin or MFG-E8. The membrane protein capable of localizing in the membrane of a cell or the protein capable of binding to the membrane of a cell may be CD8, or an MHC molecule comprising a transmembrane domain may perform that function. In one embodiment of the present invention, (d) may comprise: (D) a sequence encoding a fusion protein, comprising an amino acid sequence comprising, from the N-terminus, (D-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) the amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) the amino acid sequence of a fusion peptide comprising CD8 or a transmembrane domain thereof and at least one T cell-stimulating cytokine, in this order. In one embodiment of the present invention, (d) may comprise: (D) a sequence encoding a fusion protein, comprising an amino acid sequence comprising, from the N-terminus, (D-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) the amino acid sequence of a single-chain MHC molecule comprising a transmembrane domain, (D-4) an optional spacer sequence, and (D-5) the amino acid sequence of a fusion peptide comprising at least one T cell-stimulating cytokine, in this order.

[0198] In one embodiment of the present invention, (d) may comprise a sequence encoding a fusion protein, comprising an amino acid sequence comprising, from the N-terminus, (D-1) the amino acid sequence of an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) the amino acid sequence of a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) the amino acid sequence of a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and at least one T cell-stimulating cytokine or a subunit thereof (here, each of the components (D-1) to (D-5) includes aspects described herein). Alternatively, in one embodiment of the present invention, (d) may comprise: (D) a sequence encoding a fusion protein comprising an amino acid sequence comprising, from the N-terminus, (D-1) the amino acid sequence of a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and at least one T cell-stimulating cytokine or a subunit thereof; (D-2) an optionally present spacer sequence; (D-3) the amino acid sequence of a single-chain MHC molecule; (D-4) an optionally present spacer sequence; and (D-5) the amino acid sequence of an MHC molecule-restricted antigen peptide (wherein each of the components (D-1) to (D-5) includes aspects described herein).In this embodiment, the fusion peptide comprising the tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof and the at least one T cell stimulating cytokine or a subunit thereof may comprise an amino acid sequence encoding, in this order from the N-terminus, (1) a partial sequence of the tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (2) an optional spacer sequence, (3) the at least one T cell stimulating cytokine or a subunit thereof, (4) an optional spacer sequence, and (5) a partial sequence of the tetraspanin comprising transmembrane domain 4; or the fusion peptide comprising the tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof and the at least one T cell stimulating cytokine or a subunit thereof may comprise, in this order from the N-terminus, (1) the at least one T cell stimulating cytokine or a subunit thereof, (2) an optional spacer sequence, and (3) MFG-E8 or a membrane-binding domain thereof, in this order. Here, the MHC molecule-restricted antigenic peptide may be an MHC class I molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular domain of the MHC class I α chain; or the MHC molecule-restricted antigenic peptide may be an MHC class II molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular domain of the MHC class II α chain and / or the extracellular domain of the MHC class II β chain.

[0199] In one embodiment of the present invention, there is provided a polynucleotide comprising the sequence defined in (a) above and the sequence defined in (b) above, and optionally further comprising the sequence defined in (c) above.

[0200] In one embodiment of the present invention, there is provided a polynucleotide comprising the sequence defined in (d) above. An example of such a sequence is the nucleic acid sequence of SEQ ID NO: 136, which encodes the amino acid sequence of SEQ ID NO: 135. In this embodiment, the polynucleotide may comprise the sequence defined in (c) above.

[0201] In one embodiment of the present invention, there is provided a polynucleotide comprising the sequence defined in (e) above.

[0202] In one embodiment of the present invention, the above (A), (B), and (C) may be a polynucleotide encoding a fusion protein formed by fusing (A) and (B) to form a single molecule, or a polynucleotide encoding a fusion protein formed by fusing (B) and (C) to form a single molecule, or a polynucleotide encoding a fusion protein formed by fusing (A), (B), and (C) to form a single molecule. Such a polynucleotide may encode a single fusion protein with or without a spacer sequence between (A), (B), and (C). The sequences encoding the fusion proteins (A) to (C) may be fused via at least one sequence independently selected from the following 2A peptide sequences: T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 211) P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 212) E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 213) F2A (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 214) The 2A peptide sequence causes ribosome skipping, and when the sequences encoding the fusion proteins are actually translated, they may exist in cells or extracellular vesicles as independent molecules (A), (B), and (C). Alternatively, in one embodiment of the present invention, the polynucleotide may encode a functionally fused protein in which the above (A), (B), and (C) share an element for localizing the protein in a cell or extracellular vesicle, i.e., a portion of "a membrane protein that can be expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof" or "a protein that can bind to the membrane of a cell or extracellular vesicle or a domain thereof." For example, in one embodiment of the present invention, the polynucleotide may encode: (1) an antigen-presenting MHC molecule; (2) at least one T cell-stimulating cytokine; (3) at least one T cell-stimulating cytokine; (4) at least one T cell-stimulating cytokine; (5) at least one T cell-stimulating cytokine; (6) at least one T cell-stimulating cytokine; (7) at least one T cell-stimulating cytokine; (8) at least one T cell-stimulating cytokine; (9) at least one T cell-stimulating cytokine; (10) at least one T cell-stimulating cytokine; (11) at least one T cell-stimulating cytokine; (12) at least one T cell-stimulating cytokine; (13) at least one T cell-stimulating cytokine; (14) at least one T cell-stimulating cytokine; (15) at least one T cell-stimulating cytokine; (16) at least one T cell-stimulating cytokine; (17) at least one T cell-stimulating cytokine; (18) at least one T cell-stimulating cytokine; (19) at least one T cell-stimulating cytokine; (20) at least one T cell-stimulating cytokine; (21) at least one T cell-stimulating cytokine; (22) at least one T cell-stimulating cytokine; (23) at least one T cell-stimulating cytokine; (24) at least one T cell-stimulating cytokine; (25) at least one T cell-stimulating cytokine; (26) at least one T cell-stimand (3) a polynucleotide encoding a fusion protein (D) comprising "a membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or "a protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle"; or (A) and (C) are fused together in a manner that they share the portion "a membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or "a protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle"; or (B) and (C) are fused together in a manner that they share the portion "a membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or "a protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle"; The polynucleotide may be a polynucleotide encoding a fusion protein (G) comprising: (1) at least one T cell stimulatory cytokine; (2) a T cell costimulatory molecule; and (3) a "membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or a "protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle"; or the polynucleotide may be a polynucleotide encoding a fusion protein (E) comprising: (1) an antigen-presenting MHC molecule; (2) at least one T cell stimulatory cytokine; (2) a T cell costimulatory molecule; and (4) a "membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or a "protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle" that are fused together by sharing the "membrane protein or a transmembrane domain thereof that can be expressed on the membrane of a cell or extracellular vesicle" or "protein or a domain thereof that can bind to the membrane of a cell or extracellular vesicle" portion.

[0203] One embodiment of the present invention may be a polynucleotide encoding a fusion protein (D) comprising the functions of constituent requirement (A) and constituent requirement (B), in which a protein capable of extramembrane presentation of the first T cell-stimulating cytokine comprising constituent requirement (B) is used instead of constituent requirement (A) a membrane protein capable of being expressed on the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle, wherein the first T cell-stimulating cytokine is present on the extramembrane.

[0204] The fusion protein (D) having the functions of the constituent features (A) and (B) may be a fusion protein that contains an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine and is capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine. The fusion protein may contain the antigen-presenting MHC molecule, the at least one T cell-stimulating cytokine, and a membrane protein capable of localizing in the membrane of a cell or extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of a cell or extracellular vesicle or a membrane-binding domain thereof.

[0205] In the fusion protein (D) having the functions of the constituent feature (A) and the constituent feature (B), the membrane protein capable of being localized in 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.

[0206] In fusion protein (D) having the functions of constituent features (A) and (B), the membrane protein capable of localizing in the membrane of extracellular vesicles or the protein capable of binding to the membrane of extracellular vesicles may be a tetraspanin or MFG-E8. The fusion protein may comprise, from its N-terminus, an amino acid sequence encoding, in this order: (D-1) an MHC molecule-restricted antigen peptide, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule, (D-4) an optional spacer sequence, (D-5) a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof, and the fusion peptide comprising at least one T cell-stimulating cytokine. The fusion protein may comprise an amino acid sequence encoding, in this order from its N-terminus: (D-1) a fusion peptide comprising a tetraspanin or a transmembrane domain thereof or MFG-E8 or a transmembrane domain thereof and the at least one T cell-stimulating cytokine, (D-2) an optional spacer sequence, (D-3) a single-chain MHC molecule, (D-4) an optional spacer sequence, and (D-5) an MHC molecule-restricted antigenic peptide. Here, the fusion peptide may comprise an amino acid sequence encoding, in this order from its N-terminus: (1) a partial sequence of a tetraspanin comprising transmembrane domain 1, a small extracellular loop, transmembrane domain 2, a small intracellular loop, and transmembrane domain 3, (2) an optional spacer sequence, (3) the at least one T cell-stimulating cytokine, (4) an optional spacer sequence, and (5) a partial sequence of a tetraspanin comprising transmembrane domain 4. The fusion peptide may contain an amino acid sequence encoding, in this order from its N-terminus, (1) at least one T cell-stimulating cytokine, (2) an optional spacer sequence, and (3) MFG-E8.

[0207] In one embodiment of the present invention, the MHC molecule-restricted antigenic peptide may be an MHC class I molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular region of the MHC class I α chain; or the MHC molecule-restricted antigenic peptide may be an MHC class II molecule-restricted antigenic peptide, and the single-chain MHC molecule may comprise the extracellular domain of the MHC class II α chain and / or the extracellular domain of the MHC class II β chain.

[0208] In an embodiment comprising a fusion protein (D) having the functions of constituent feature (A) and constituent feature (B); (C) the membrane may further comprise a protein comprising at least one T cell costimulatory molecule and capable of interacting with a T cell; the protein capable of interacting with a T cell may comprise the at least one T cell costimulatory molecule and a membrane protein capable of being expressed in the membrane of an extracellular vesicle or a transmembrane domain thereof, or a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof; the protein capable of interacting with a T cell may comprise the at least one T cell costimulatory molecule and a tetraspanin or a transmembrane domain thereof, or MFG-E8 or a domain thereof.

[0209] Vectors, Kits

[0210] In one embodiment of the present invention, there is provided a vector comprising at least one polynucleotide selected from the polynucleotides described herein.

[0211] As used herein, the term "vector" refers to any vector (including, but 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 expression of the operably linked coding sequence in a host organism (e.g., a plant, insect, animal, etc.) or in an in vitro expression system. Cloning vectors may be used to manipulate and / or amplify a desired polynucleotide fragment. Cloning vectors may lack functional sequences required for expression of the desired polynucleotide fragment.

[0212] 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 that combines two or more vectors each containing at least one polynucleotide selected from the polynucleotides described herein.

[0213] transformed cells

[0214] In one embodiment of the present invention, there is provided a cell transformed with a vector comprising: (i) a polynucleotide encoding a fusion protein or protein complex of (A) described herein; (ii) a polynucleotide encoding a fusion protein of (B) described herein; or (iii) a polynucleotide encoding a fusion protein of (C) described herein.

[0215] In one embodiment of the present invention, there is provided a cell transformed with a single vector or a combination of two or more vectors comprising: (i) a polynucleotide encoding a fusion protein or protein complex of (A) described herein, and (ii) a polynucleotide encoding a fusion protein of (B) described herein, and optionally (iii) a polynucleotide encoding a fusion protein of (C) described herein.

[0216] In one embodiment of the present invention, the cell may be transformed with a vector comprising a polynucleotide encoding a fusion protein in which (A) and (B) are fused to form a single molecule, a vector comprising a polynucleotide encoding a fusion protein in which (B) and (C) are fused to form a single molecule, or a vector comprising a polynucleotide encoding a fusion protein in which (A), (B) and (C) are fused to form a single molecule. Such a polynucleotide may encode a single fusion protein with or without a spacer sequence between (A), (B), and (C). Alternatively, (A), (B), and (C) may encode a functionally fused fusion protein by sharing an element for localizing the protein to extracellular vesicles, i.e., a "membrane protein or a transmembrane domain thereof that can be expressed on the membrane of extracellular vesicles" or a "protein or a domain thereof that can bind to the membrane of extracellular vesicles." For example, in one embodiment of the present invention, a vector comprising a polynucleotide encoding a fusion protein (D) comprising: (1) an antigen-presenting MHC molecule; (2) at least one T cell stimulatory cytokine; and (3) a fusion protein (D) comprising: (A) and (B) fused together in a manner that they share the portion "a membrane protein capable of being expressed on the membrane of an extracellular vesicle or a transmembrane domain thereof" or "a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof"; (A) and (C) fused together in a manner that they share the portion "a membrane protein capable of being expressed on the membrane of an extracellular vesicle or a transmembrane domain thereof" or "a protein capable of binding to the membrane of an extracellular vesicle or a domain thereof"; (1) an antigen-presenting MHC molecule; (2) a T cell costimulatory molecule; and (3) A vector containing a polynucleotide encoding a fusion protein (F) containing a "membrane protein or a transmembrane domain thereof capable of being expressed in the membrane of an extracellular vesicle" or a "protein or a domain thereof capable of binding to the membrane of an extracellular vesicle";A vector comprising a polynucleotide encoding a fusion protein (G) comprising: (1) at least one T cell stimulatory cytokine; (2) a T cell costimulatory molecule; and (3) a "membrane protein capable of being expressed on the membrane of extracellular vesicles or a transmembrane domain thereof" or a "protein capable of binding to the membrane of extracellular vesicles or a domain thereof"; (B) and (C) are fused together in a manner that they share the portion "a membrane protein capable of being expressed on the membrane of extracellular vesicles or a transmembrane domain thereof" or "a protein capable of binding to the membrane of extracellular vesicles or a domain thereof"; or (1) an antigen-presenting MHC molecule; (2) at least one T cell stimulatory cytokine; (3) a T cell costimulatory molecule; and (A) to (C) are fused together in a manner that they share the portion "a membrane protein capable of being expressed on the membrane of extracellular vesicles or a transmembrane domain thereof" or "a protein capable of binding to the membrane of extracellular vesicles or a domain thereof". (4) The vector may be transformed with a polynucleotide encoding a fusion protein (E) containing a "membrane protein or a transmembrane domain thereof that can be expressed in the membrane of an extracellular vesicle" or a "protein or a domain thereof that can bind to the membrane of an extracellular vesicle."

[0217] Alternatively, in one embodiment of the present invention, there is provided a cell transformed with a vector comprising a polynucleotide encoding a fusion protein comprising an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine as described in (D) herein and capable of presenting the antigen and the T cell-stimulating cytokine on the extracellular membrane, which is a fusion protein having the functions of both constituent features (A) and (B), using a protein capable of presenting the first T cell-stimulating cytokine on the extracellular membrane, comprising the first T cell-stimulating cytokine of constituent feature (B), instead of the membrane protein capable of being expressed on the membrane of the extracellular vesicle or its transmembrane domain, or the protein capable of binding to the membrane of the extracellular vesicle as described in constituent feature (A).

[0218] The phrase "transformed with a single vector or a combination of two or more vectors" means, for example, that the cell may be transformed with all of the polynucleotides (i) to (iv) above inserted into the same vector, or may be transformed with a combination of two or more vectors in which two or more of these polynucleotides are inserted into separate vectors.

[0219] 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 comprising a polynucleotide encoding the fusion protein of (A) and a vector comprising 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 (B); - A combination of a vector comprising a polynucleotide encoding the fusion protein of (A), a vector comprising a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding the fusion protein of (C).

[0220] Alternatively, examples of "a single vector or a combination of two or more vectors" when (A) is a protein complex include the following: - a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein comprising (A-6), and a polynucleotide encoding the fusion protein of (B); - a combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5) and a polynucleotide encoding a protein comprising (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 comprising the amino acid sequence consisting of (A-1) to (A-5) and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding a protein comprising (A-6); - a combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5), and a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding a protein comprising (A-6); a combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein comprising (A-6), and a vector comprising a polynucleotide encoding the fusion protein of (B); a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence of (A-1) to (A-5), a polynucleotide encoding a protein comprising (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 comprising the amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein comprising (A-6), and a polynucleotide encoding the fusion protein of (B), with a vector comprising a polynucleotide encoding the fusion protein of (C); - A combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding the fusion protein of (B), and a polynucleotide encoding the fusion protein of (C), with a vector comprising a polynucleotide encoding a protein comprising (A-6); - A combination of a vector comprising a polynucleotide encoding a protein comprising (A-6), a polynucleotide encoding the fusion protein of (B), and a polynucleotide encoding the fusion protein of (C), with a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5); - A combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5), a polynucleotide encoding a protein comprising (A-6), and a polynucleotide encoding the fusion protein of (C), with a vector comprising a polynucleotide encoding the fusion protein of (B); - A combination of a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5) and a polynucleotide encoding a protein comprising (A-6), with 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 comprising the amino acid sequence consisting of (A-1) to (A-5), and a polynucleotide encoding the fusion protein of (B), with a vector comprising a polynucleotide encoding a protein comprising (A-6), and a polynucleotide encoding the fusion protein of (C);a combination of a vector comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence consisting of (A-1) to (A-5), and a polynucleotide encoding the fusion protein of (C), with a vector comprising a polynucleotide encoding a protein comprising (A-6), and a polynucleotide encoding the fusion protein of (B); a combination of a vector comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein comprising (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 comprising an amino acid sequence consisting of (A-1) to (A-5), a vector comprising 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 comprising the amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding the fusion protein of (C), a vector comprising a polynucleotide encoding a protein comprising (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 comprising (A-6), a vector comprising a polynucleotide encoding the fusion protein of (B), and a vector comprising a polynucleotide encoding a fusion protein comprising the amino acid sequence consisting of (A-1) to (A-5), and a polynucleotide encoding the fusion protein of (C);a combination of a vector comprising a polynucleotide encoding a protein comprising (A-6), a vector comprising a polynucleotide encoding the fusion protein of (C), a vector comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence consisting of (A-1) to (A-5), 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), a vector comprising a polynucleotide encoding the fusion protein of (C), a vector comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence consisting of (A-1) to (A-5), and a vector comprising a polynucleotide encoding a protein comprising (A-6); or a combination of a vector comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence consisting of (A-1) to (A-5), a vector comprising a polynucleotide encoding a protein comprising (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);

[0221] The cells to be transformed are not particularly limited as long as they can produce the antigen-presenting extracellular vesicles described herein after transformation, and may be primary culture cells, passaged cells, or established cell lines. These may be normal cells or diseased cells, including cancerous or tumorigenic cells. The origin of the cells to be transformed is also not particularly limited, and examples include cells derived from animals such as rodents (e.g., mice, rats, hamsters, and guinea pigs), lagomorphs (e.g., rabbits), ungulates (e.g., pigs, cattle, goats, horses, and sheep), carnivores (e.g., dogs and cats), and mammals (e.g., primates (e.g., humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees); plant-derived cells; and insect-derived cells. Cells to be transformed are preferably animal-derived cells. Examples of animal-derived cells include, but are not limited to, human embryonic kidney cells (including HEK293T cells), human FL cells, Chinese hamster ovary cells (CHO cells), COS-7, Vero, mouse L cells, and rat GH3.

[0222] The method for transforming cells is not particularly limited as long as it is a method that can introduce a target polynucleotide into cells, and may be, for example, electroporation, microinjection, calcium phosphate, cationic lipid, liposome-based methods, non-liposomal methods such as polyethyleneimine, or viral infection.

[0223] The transformed cells may be transformed cells that transiently express the fusion proteins or protein complexes of (A), (B), (C), (D), (E), (F) and / or (G), or may be transformed cells that stably express the fusion proteins or protein complexes of (A), (B), (C), (D), (E), (F) and / or (G) (stable cell lines).

[0224] The culture conditions for transformed cells are not particularly limited. For example, when the transformed cells are derived from animals, a medium commonly used for cell culture (e.g., RPMI 1640 medium, Eagle's MEM medium, Dulbecco's modified Eagle's medium (DMEM medium), Ham's F12 medium, or any combination thereof) or a medium supplemented with other components such as fetal bovine serum, antibiotics, and amino acids may be used, for example, under conditions of about 1 to about 10% (preferably about 2 to about 5%) CO ２ The culture may be performed (e.g., stationary or shaking) in the presence of at about 30 to about 40°C (preferably about 37°C) for a desired period of time (e.g., about 0.5 hours to about 240 hours (preferably, about 5 to about 120 hours, more preferably, about 12 to about 72 hours)).

[0225] 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 medium from which extracellular vesicles such as exosomes have been removed (e.g., Dulbecco's modified Eagle's medium containing about 1 to about 5% fetal bovine serum from which exosomes have been removed) may be used, if necessary.

[0226] Culture supernatant

[0227] In one embodiment of the present invention, a culture supernatant is provided, which is obtained by culturing the transformed cells described herein.

[0228] The antigen-presenting extracellular vesicles contained in the culture supernatant described herein can be recovered, for example, by purifying (e.g., by centrifugation, chromatography, etc.), concentrating, isolating, etc. the culture supernatant.

[0229] In one embodiment of the present invention, there is provided an antigen-presenting extracellular vesicle obtained from the culture supernatant described herein.

[0230] Methods for producing antigen-presenting cells or antigen-presenting extracellular vesicles as described herein

[0231] The antigen-presenting cells or antigen-presenting extracellular vesicles described herein may be obtained by, but are not limited to, means such as genetic recombination techniques known to those skilled in the art (for example, by the methods described below, or by the methods described in the Examples, or by methods similar thereto). Polynucleotides encoding the above-mentioned proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), can be obtained by standard genetic recombination techniques (alternatively, (A) to (a) to (d) can be operably inserted into the same or separate vectors). When two or more of the polynucleotides encoding 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 promoters. The obtained single or two or more vectors for antigen-presenting cells can be simultaneously or sequentially transformed into cells to obtain antigen-presenting cells (which may be transformed cells that transiently express these fusion proteins or transformed cells (stable strains) that stably express these fusion proteins). The resulting single or two or more vectors for antigen-presenting extracellular vesicles can be simultaneously or sequentially transformed into cells to obtain transformed cells (which may be transformed cells that transiently express these fusion proteins or transformed cells (stable strains) that stably express these fusion proteins). The resulting transformed cells are cultured under desired conditions to obtain a culture supernatant, which can then be purified as needed (for example, by centrifugation, purification using an antibody (for example, an antibody that recognizes a protein contained in the membrane of the extracellular vesicles), chromatography, flow cytometry, etc.), concentrated (for example, ultrafiltration, etc.), dried, etc., to obtain the antigen-presenting extracellular vesicles described herein.

[0232] Alternatively, when soluble proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are used, the antigen-presenting cells or 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 commercially available products. Next, cells or extracellular vesicles are obtained from the desired cells, for example, by known methods, methods described herein, or methods similar thereto. Next, the obtained cells or extracellular vesicles are reacted with one or more of the above-mentioned soluble proteins in a desired solvent under desired conditions (for example, the method described in JP 2018-104341 A may be used as a reference). This procedure can be 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 cells or antigen-presenting extracellular vesicles described herein.

[0233] Alternatively, when soluble proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally (C), are used, 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 attaching a desired tag (e.g., a His tag, a FLAG tag, a PNE tag of SEQ ID NO: 79, etc., which may all be the same tag or all be different types of tags) to the N-terminus or C-terminus thereof using conventional genetic engineering techniques. Next, cells or extracellular vesicles are obtained from the desired cells, for example, by a known method or a method described herein, or a method similar thereto, and an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody, for example, the anti-PNE tag nanobody of SEQ ID NO: 83) or the like is bound to the cells or extracellular vesicles via a peptide linker or the like as needed; alternatively, an antibody against the tag or an antigen-binding fragment thereof (e.g., s A polynucleotide (e.g., SEQ ID NO: 88, 90, etc.) encoding a fusion protein (e.g., 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 this is operably inserted into a vector, which is then used to transform a cell to obtain a transformed cell (which may be a transformed cell that transiently expresses the fusion protein, or a transformed cell (stable strain) that stably expresses the fusion protein). The resulting transformed cell is then cultured, and extracellular vesicles are recovered by the method described above, etc. The antigen-presenting extracellular vesicles described herein may be obtained by mixing, under desired conditions, soluble tagged proteins (A) and (B), and optionally (C), with extracellular vesicles containing, in their membranes, proteins comprising, for example, an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody).

[0234] Alternatively, transformation may be performed using a combination of polynucleotides encoding the fusion proteins (A) to (G) described above, and the antigen-presenting cells or antigen-presenting extracellular vesicles described in the specification may be obtained from the resulting transformed cells.

[0235] Alternatively, the antigen-presenting extracellular vesicles described herein may be obtained by a combination of two or more of the above-mentioned methods.

[0236] The antigen-presenting extracellular vesicles described herein may be confirmed to contain proteins (A) and (B) (or (D) instead of (A) and (B)), and optionally proteins (C) in their membranes, for example, by techniques such as flow cytometry, ELISA, and Western blotting.

[0237] In one embodiment of the present invention, there is provided a method for producing the antigen-presenting extracellular vesicles described herein, the method comprising culturing the transformed cells described herein and recovering the culture supernatant obtained.

[0238] In one embodiment of the present invention, there is provided a method for producing antigen-presenting extracellular vesicles described herein, the method comprising: simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising: (i) a polynucleotide encoding the fusion protein (A) or protein complex for antigen-presenting extracellular vesicles described herein; and (ii) a polynucleotide encoding the fusion protein (B) for antigen-presenting extracellular vesicles described herein; and optionally (iii) a polynucleotide encoding the fusion protein (C) for antigen-presenting extracellular vesicles described herein; and culturing the resulting transformed cells to collect the resulting culture supernatant. Alternatively, one embodiment of the present invention provides a method for producing the antigen-presenting extracellular vesicles described herein, comprising: (iv) a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule (D) for the antigen-presenting extracellular vesicles described herein and at least one T cell-stimulating cytokine, and capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine; and optionally (iii) a polynucleotide encoding the fusion protein (C) for the antigen-presenting extracellular vesicles described herein; and (iv) a polynucleotide encoding a fusion protein (D) for the antigen-presenting extracellular vesicles described herein; and (iii) a polynucleotide encoding the fusion protein (C) for the antigen-presenting extracellular vesicles described herein. The method comprises: simultaneously or sequentially (preferably simultaneously) transfecting cells with a single vector or a combination of two or more vectors; and culturing the resulting transformed cells and collecting the resulting culture supernatant. Alternatively, one embodiment of the present invention provides a method for producing the antigen-presenting extracellular vesicles described herein, comprising: (v) transforming cells with a vector comprising a polynucleotide encoding a fusion protein that contains the antigen-presenting MHC molecule (E) for the antigen-presenting extracellular vesicles described herein, at least one T cell stimulatory cytokine, and a T cell costimulatory molecule, and that is capable of extramembrane presentation of the antigen and the T cell stimulatory cytokine; and culturing the resulting transformed cells to collect the resulting culture supernatant.

[0239] In one embodiment of the invention, a method is provided for producing the antigen-presenting cells described herein.

[0240] In one embodiment of the present invention, there is provided a method for producing an antigen-presenting cell described herein, comprising simultaneously or sequentially (preferably simultaneously) transforming a cell with a single vector or a combination of two or more vectors comprising: (i) a polynucleotide encoding the fusion protein or protein complex (A) for the antigen-presenting cell described herein; and (ii) a polynucleotide encoding the fusion protein (B) for the antigen-presenting cell described herein; and optionally (iii) a polynucleotide encoding the fusion protein (C) for the antigen-presenting cell described herein. Alternatively, one embodiment of the present invention provides a method for producing the antigen-presenting cells described herein, comprising simultaneously or sequentially (preferably simultaneously) transfecting a cell with a single vector or a combination of two or more vectors comprising: (iv) a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule of (D) for the antigen-presenting cells described herein and at least one T cell stimulatory cytokine, wherein the fusion protein is capable of extramembrane presentation of the antigen and the T cell stimulatory cytokine, and optionally (iii) a polynucleotide encoding the fusion protein of (C) for the antigen-presenting cells described herein. Alternatively, one embodiment of the present invention provides a method for producing the antigen-presenting extracellular vesicles described herein, comprising: (v) transforming a cell with a vector comprising a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule of (E) for the antigen-presenting cells described herein, at least one T cell stimulatory cytokine, and a T cell costimulatory molecule, wherein the fusion protein is capable of extramembrane presentation of the antigen and the T cell stimulatory cytokine.

[0241] In one embodiment of the present invention, there is provided an antigen-presenting extracellular vesicle obtained from the culture supernatant described herein.

[0242] In one embodiment of the present invention, there are provided antigen-presenting extracellular vesicles obtained by a method comprising: simultaneously or sequentially (preferably simultaneously) transforming cells with a single vector or a combination of two or more vectors comprising: (i) a polynucleotide encoding the fusion protein (A) or protein complex for use with antigen-presenting extracellular vesicles described herein, and (ii) a polynucleotide encoding the fusion protein (B) for use with antigen-presenting extracellular vesicles described herein, and optionally (iii) a polynucleotide encoding the fusion protein (C) for use with antigen-presenting extracellular vesicles described herein; and culturing the resulting transformed cells and collecting the resulting culture supernatant. Alternatively, one embodiment of the present invention provides antigen-presenting extracellular vesicles obtained by a method comprising: simultaneously or sequentially (preferably simultaneously) transfecting cells with a single vector or a combination of two or more vectors comprising: (iv) a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule (D) for antigen-presenting extracellular vesicles described herein and at least one T cell-stimulating cytokine, and capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine; and optionally (iii) a polynucleotide encoding the fusion protein (C) for antigen-presenting extracellular vesicles described herein; and culturing the obtained transformed cells and collecting the resulting culture supernatant. Alternatively, one embodiment of the present invention provides antigen-presenting extracellular vesicles obtained by a method comprising the following steps: (v) transforming cells with a polynucleotide encoding a fusion protein that contains the antigen-presenting MHC molecule (E) for antigen-presenting extracellular vesicles described herein, at least one T cell stimulatory cytokine, and a T cell costimulatory molecule, and that is capable of extramembrane presentation of the antigen and the T cell stimulatory cytokine; and culturing the resulting transformed cells to collect the resulting culture supernatant.

[0243] In one embodiment of the present invention, there is provided an antigen-presenting cell obtained by a method comprising: simultaneously or sequentially (preferably simultaneously) transfecting a cell with a single vector or a combination of two or more vectors comprising: (i) a polynucleotide encoding the fusion protein or protein complex (A) for use in an antigen-presenting cell described herein, and (ii) a polynucleotide encoding the fusion protein (B) for use in an antigen-presenting cell described herein, and optionally (iii) a polynucleotide encoding the fusion protein (C) for use in an antigen-presenting cell described herein. Alternatively, one embodiment of the present invention provides an antigen-presenting cell obtained by a method comprising: simultaneously or sequentially (preferably simultaneously) transforming a cell with a single vector or a combination of two or more vectors comprising the following: (iv) a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule of (D) for an antigen-presenting cell described herein and at least one T cell-stimulating cytokine, wherein the fusion protein is capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine, and optionally (iii) a polynucleotide encoding the fusion protein of (C) described herein. Alternatively, one embodiment of the present invention provides an antigen-presenting cell obtained by a method comprising: (v) transforming a cell with a polynucleotide encoding a fusion protein comprising the antigen-presenting MHC molecule of (E) for an antigen-presenting cell described herein, at least one T cell-stimulating cytokine, and a T cell costimulatory molecule, wherein the fusion protein is capable of extramembrane presentation of the antigen and the T cell-stimulating cytokine.

[0244] Composition, use

[0245] In one embodiment of the present invention, there is provided a composition (e.g., a pharmaceutical composition) comprising the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants described herein. In one embodiment of the present invention, there is provided a pharmaceutical composition comprising the antigen-presenting cells or antigen-presenting extracellular vesicles described herein, or the culture supernatant described herein.

[0246] The compositions (e.g., pharmaceutical compositions) described herein may contain additives such as, but not limited to, excipients, lubricants, binders, disintegrants, pH adjusters, solvents, solubilizers, suspending agents, isotonicity adjusters, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners, and surfactants. The type and amount of these additives can be appropriately selected by those skilled in the art depending on the purpose. When used as a pharmaceutical composition, these additives are preferably pharmacologically acceptable carriers. Furthermore, when the compositions described herein contain polynucleotides, it is preferable, although not essential, to include a carrier suitable for drug delivery (DD) of nucleic acids. Examples of such carriers include lipid nanoparticles (LNPs) and polymers (e.g., PEI).

[0247] The compositions (e.g., pharmaceutical compositions) described herein can be formulated together with the above-mentioned additives by methods known per se 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. The formulated products may further contain other beneficial ingredients (e.g., other therapeutically beneficial ingredients) depending on the purpose.

[0248] As shown in the test examples, the composition of one embodiment of the present invention is capable of enhancing adaptive immunity (cellular immunity and / or humoral immunity) against a specific antigen, and when a peptide derived from an infectious pathogen (such as a pathogenic bacterium or virus) is used as the antigen, it can be used as a pharmaceutical composition for treating or preventing an infectious disease caused by the infectious pathogen. Furthermore, as shown in the test examples, the composition of one embodiment of the present invention is capable of inducing inflammatory cytokines and activating innate immunity (including recruiting and activating neutrophils, monocytes, macrophages, etc. to phagocytose pathogens, etc.), thereby eliminating the infectious pathogen, and can be used as a pharmaceutical composition for treating or preventing an infectious disease caused by the infectious pathogen.

[0249] The antigen-presenting cells, antigen-presenting extracellular vesicles (preferably antigen-presenting cells or antigen-presenting extracellular vesicles comprising an MHC class I-restricted antigenic peptide and an MHC class I molecule in their membranes), polynucleotides thereof and / or vectors comprising the same, and / or transformed cells and / or culture supernatants thereof, or compositions comprising any of these (e.g., pharmaceutical compositions) may be useful for treating or preventing cancer.

[0250] Therefore, one embodiment of the present invention provides antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing these (e.g., pharmaceutical compositions) described herein for treating or preventing cancer. As shown in the test examples, the antigen-presenting extracellular vesicles, etc., which are one embodiment of the present invention, are capable of proliferating and activating cytotoxic T cells specific to the antigen used, and when a tumor-associated antigen peptide is used as the antigen, the proliferated and activated cytotoxic T cells recognize and attack cancer cells, thereby killing them.

[0251] In another embodiment of the present invention, there is provided use of antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing these (e.g., pharmaceutical compositions) described herein, for the manufacture of a medicament for treating or preventing cancer.

[0252] In another embodiment of the present invention, there is provided a method for treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising same, and / or transformed cells and / or their culture supernatants, or compositions comprising any of the above, as described herein.

[0253] Cancers include any solid cancer and blood cancer, but are not limited to, for example, small cell lung cancer, non-small cell lung cancer, breast cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, pancreatic cancer, prostate cancer, bone marrow cancer, kidney cancer (including renal cell cancer, etc.), parathyroid cancer, adrenal cancer, ureter cancer, liver cancer, bile duct cancer, cervical cancer, ovarian cancer (for example, the histological types of which are 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, Merkel cell cancer, Examples of cancers that can be treated include melanoma (such as malignant melanoma), epithelial cancer, squamous cell carcinoma, basal cell carcinoma, childhood cancer, cancer of unknown primary origin, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, synovioma, mesothelioma, Ewing's tumor, seminoma, Wilms' tumor, brain tumor, glioma, glioblastoma, astrocytoma, medulloblastoma, meningioma, neuroblastoma, medulloblastoma, retinoblastoma, spinal tumor, malignant lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, etc.), chronic or acute lymphocytic leukemia, adult T-cell leukemia, and the like.

[0254] In one embodiment of the present invention, immune checkpoint inhibitors can be used in combination to treat or prevent cancer. Immune checkpoint inhibitors may be administered to a patient simultaneously or sequentially, or may be contained in the medicament of 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 nibulumab and pembrolizimab), 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). When the immune checkpoint inhibitor is an antibody or an active fragment thereof, the antibody or an active fragment thereof may be bound to a membrane protein capable of localizing in the membrane of the extracellular vesicle or its transmembrane domain, or to a protein capable of binding to the membrane of the extracellular vesicle or its membrane-binding domain, and then present on the membrane of the extracellular vesicle of the present invention. Such combined use of immune checkpoint inhibitors enhances cytotoxicity against cancer cells.

[0255] The antigen-presenting cells, antigen-presenting extracellular vesicles (preferably antigen-presenting cells or antigen-presenting extracellular vesicles comprising an MHC class II-restricted antigenic peptide and an MHC class II molecule on their membranes), polynucleotides thereof and / or vectors comprising the same, and / or transformed cells and / or culture supernatants thereof, or compositions comprising any of these, described herein may be useful for treating or preventing autoimmune diseases. As illustrated in the test examples, the antigen-presenting extracellular vesicles of one embodiment of the present invention are capable of proliferating and activating regulatory T cells (Tregs) specific to the antigen used, and when an autoantigen peptide is used as the antigen, the proliferated and activated Tregs induce immune tolerance to the autoantigen, allowing for the treatment or prevention of autoimmune diseases.

[0256] Therefore, one embodiment of the present invention provides antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or culture supernatants thereof, or compositions containing any of these (e.g., pharmaceutical compositions) described herein for treating or preventing autoimmune diseases.

[0257] In another embodiment of the present invention, there is provided use of the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising same, and / or transformed cells and / or culture supernatants thereof, or compositions comprising any of these (e.g., pharmaceutical compositions) described herein, for the manufacture of a medicament for treating or preventing an autoimmune disease.

[0258] In another embodiment of the present invention, there is provided a method for treating or preventing an autoimmune disease, comprising administering to a subject in need thereof an effective amount of the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising same, and / or transformed cells and / or their culture supernatants, or compositions comprising any of the above, described herein.

[0259] Examples of autoimmune diseases include, but are not limited to, asthma, psoriasis, systemic lupus erythematosus, Guillain-Barré syndrome, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, pernicious anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, Crohn's disease, inflammatory bowel disease, and rheumatoid arthritis.

[0260] The antigen-presenting cells, antigen-presenting extracellular vesicles (preferably antigen-presenting cells or antigen-presenting extracellular vesicles comprising an MHC class II-restricted antigenic peptide and an MHC class II molecule on their membranes), polynucleotides thereof and / or vectors comprising the same, and / or transformed cells and / or culture supernatants thereof, or compositions comprising any of these (e.g., pharmaceutical compositions) described herein may be useful for treating or preventing allergic diseases. As shown in the test examples, the antigen-presenting extracellular vesicles, etc., which are one embodiment of the present invention, are capable of proliferating and activating regulatory T cells (Tregs) specific to the antigen used, and when an allergen is used as the antigen, the proliferated and activated Tregs induce immune tolerance to the allergen, allowing the treatment or prevention of allergic diseases.

[0261] Therefore, one embodiment of the present invention provides antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing these (e.g., pharmaceutical compositions) described herein for treating or preventing allergic diseases.

[0262] In another embodiment of the present invention, there is provided use of the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing the same, and / or transformed cells and / or their culture supernatants, or compositions containing any of these (e.g., pharmaceutical compositions) described herein, for the manufacture of a medicament for treating or preventing an allergic disease.

[0263] In another embodiment of the present invention, there is provided a method for treating or preventing an allergic disease, comprising administering to a subject in need thereof an effective amount of the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors comprising same, and / or transformed cells and / or their culture supernatants, or compositions comprising any of the above, described herein.

[0264] Allergic diseases include, but are not limited to, allergic rhinitis, atopic dermatitis, allergic asthma, allergic conjunctivitis, allergic gastroenteritis, food allergies, drug allergies, and urticaria.

[0265] Subjects to be treated or prevented for the various diseases described above include, but are not limited to, animals such as mammals, including 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 primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees; or plants, but are preferably animals, more preferably rodents or primates, and even more preferably mice or humans.

[0266] The dosage of the antigen-presenting cells, antigen-presenting extracellular vesicles, polynucleotides and / or vectors containing them, and / or transformed cells and / or culture supernatants thereof described herein, or compositions containing them or formulations thereof, can be determined appropriately taking into consideration the sex, age, weight, health condition, severity of disease, or diet of the subject to be administered; the time of administration; the method of administration; combination with other drugs; and other factors.

[0267] Method for activating, proliferating, and / or differentiating T cells against a specific antigen The antigen-presenting cells or antigen-presenting extracellular vesicles described herein can be contacted with T cells (including, but not limited to, T cells or T cell populations obtained from peripheral blood, the spleen, etc.) in vitro, ex vivo, and / or in vivo to activate, proliferate, differentiate, etc., T cells against a specific antigen.

[0268] In one embodiment of the present invention, there is provided a method for activating, proliferating and / or differentiating T cells against a specific antigen, comprising contacting the T cells in vitro or ex vivo with an antigen-presenting cell or antigen-presenting extracellular vesicle described herein.

[0269] In one embodiment of the invention, there is provided a T cell obtained by the method described above.

[0270] The T cells obtained by the above-described method may be administered to a subject to treat and / or prevent a disease (e.g., cancer, autoimmune disease, allergic disease, etc.).

[0271] The present invention will be described in more detail below using examples, but these examples are not intended to limit the scope of the present invention in any way.

[0272] Plasmid Preparation 1: Using the pCAG-puro vector, a vector was created to express MHC class I molecules capable of extracellularly presenting antigens on the membrane of extracellular vesicles. ２ A polynucleotide (SEQ ID NO: 2) encoding the signal peptide of microglobulin (amino acids 1 to 20; SEQ ID NO: 1), a polynucleotide (SEQ ID NO: 4) encoding the OVA peptide (SEQ ID NO: 3) which is a model antigen peptide, a peptide linker (amino acid sequence: SEQ ID NO: 5, polynucleotide: SEQ ID NO: 6), and a β ２A single-chain trimer (sc-Trimmer) was constructed from a polynucleotide (SEQ ID NO: 8) encoding the full-length sequence of microglobulin (amino acids 21-119; SEQ ID NO: 7), a polynucleotide (SEQ ID NO: 12) encoding a peptide linker (SEQ ID NO: 11), and a polynucleotide (SEQ ID NO: 10) encoding the full-length sequence of the MHC class I α chain (amino acids 22-369; SEQ ID NO: 9) excluding the signal peptide (amino acid sequence: SEQ ID NO: 13; polynucleotide: SEQ ID NO: 14). Next, the sc-Trimmer was linked to a polynucleotide (SEQ ID NO: 16) encoding the full-length sequence of the tetraspanin CD81 (amino acids 1-236; SEQ ID NO: 15), resulting in a polynucleotide (SEQ ID NO: 18; corresponding amino acid sequence: SEQ ID NO: 17) inserted into the pCAG-puro vector (Figures 1A and 1B; hereafter referred to as sc-Trimmer-CD81). In a similar manner, to express CD80, a T cell costimulatory molecule, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 24; corresponding amino acid sequence: SEQ ID NO: 23) was prepared by linking a polynucleotide (SEQ ID NO: 20) encoding the full-length sequence of CD80 (amino acids 1 to 306; SEQ ID NO: 19) with a polynucleotide (SEQ ID NO: 22) encoding the full-length sequence of the tetraspanin CD9 (amino acids 1 to 306; SEQ ID NO: 21) and inserted the resulting polynucleotide into a pCAG-puro or pMX vector (Figures 1C and 1D; hereinafter, referred to as CD80-CD9). Using a similar method, in order to express IL-2, a T cell-stimulating cytokine, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 26) encoding the full-length sequence of IL-2 excluding the signal peptide (amino acids 21 to 169; SEQ ID NO: 25) was inserted between amino acids 170C and 171I in the large extracellular loop of mouse tetraspanin CD63 (amino acids 1 to 238: SEQ ID NO: 27; polynucleotide: SEQ ID NO: 28) (i.e., the IL-2 sequence was inserted between a polynucleotide (SEQ ID NO: 58) encoding the partial sequence of CD63 of SEQ ID NO: 57 and a polynucleotide (SEQ ID NO: 60) encoding the partial sequence of CD63 of SEQ ID NO: 59).A polynucleotide (SEQ ID NO: 30) encoding a peptide linker (amino acid sequence GGGGS: SEQ ID NO: 29) was added to the N-terminus and C-terminus of IL-2. This polynucleotide (SEQ ID NO: 32; corresponding amino acid sequence: SEQ ID NO: 31) was inserted into the pCAG-puro vector (Fig. 1E and 1F; hereafter referred to as CD63-IL-2).

[0273] Plasmid Preparation 2: A vector for expressing MHC class II molecules capable of extracellular antigen presentation on the membrane of extracellular vesicles was prepared using the pCAG-puro vector. Using established cloning techniques, a polynucleotide (SEQ ID NO: 34) encoding the signal peptide of the MHC class II β chain (amino acids 1-27; SEQ ID NO: 33), a polynucleotide (SEQ ID NO: 36) encoding the model antigen peptide OVA peptide (SEQ ID NO: 35), and a polynucleotide (SEQ ID NO: 38) encoding the full-length sequence of the MHC class II β chain excluding the signal peptide (amino acids 28-265; SEQ ID NO: 37) were linked together with a polynucleotide (SEQ ID NO: 40) encoding a peptide linker (SEQ ID NO: 39) to prepare a single-chain dimer (sc-Dimer) (amino acid sequence: SEQ ID NO: 41; polynucleotide: SEQ ID NO: 42). Next, a polynucleotide (SEQ ID NO: 44; corresponding amino acid sequence: SEQ ID NO: 43) encoding the full-length sequence of the tetraspanin CD81 (amino acids 1-236; SEQ ID NO: 15) linked to sc-Dimer was inserted into a pCAG-puro vector (Figs. 1G and 1H; hereafter referred to as sc-Dimer-CD81).A polynucleotide (SEQ ID NO: 46) encoding the full-length sequence of the MHC class II α chain (amino acids 1-256; SEQ ID NO: 45), a component of the MHC class II molecule, was inserted into another pCAG-puro vector (Fig. 1I; hereafter referred to as MHC class II α chain). In a similar manner, to express TGF-β1, a T cell-stimulating cytokine, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 48) encoding the full-length sequence of TGF-β1 (amino acids 1 to 390; SEQ ID NO: 47) in which three Cs at positions 33, 223, and 225 of the LAP domain were changed to S was linked to a polynucleotide (SEQ ID NO: 50) encoding the full-length sequence of MFG-E8 (amino acids 23 to 463; SEQ ID NO: 49) excluding the signal peptide in which D at position 89 was changed to E, 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 the pCAG-puro vector (Figures 1J and 1K; hereinafter, referred to as TGF-β-MFG-E8). Using a similar method, to express IL-4, a T cell-stimulating cytokine, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO:54) encoding the full-length sequence of IL-4 excluding the signal peptide (amino acids 21-140; SEQ ID NO:53) was inserted between amino acids 177S and 178G in the large extracellular loop of mouse tetraspanin CD81 (amino acids 1-236: SEQ ID NO:15; polynucleotide: SEQ ID NO:16). (That is, the IL-4 sequence was inserted between a polynucleotide (SEQ ID NO:62) encoding the partial sequence of CD81 represented by SEQ ID NO:61 and a polynucleotide (SEQ ID NO:64) encoding the partial sequence of CD81 represented by SEQ ID NO:63.) A polynucleotide (SEQ ID NO: 30) encoding a peptide linker (amino acid sequence GGGGS; SEQ ID NO: 29) was added to the N-terminus and C-terminus of IL-4. This polynucleotide (SEQ ID NO: 56; corresponding amino acid sequence: SEQ ID NO: 55) was inserted into the pCAG-puro vector (Figures 1L and 1M; hereafter referred to as CD81-IL-4).

[0274] Plasmid Preparation 3: sc-Dimer-CD81-IL-12p40 A polynucleotide (SEQ ID NO: 92) encoding a protein (SEQ ID NO: 91) in which CD81 and IL-12p40, a subunit of the T cell-stimulating cytokine IL-12, were fused to the sc-Dimer was inserted into the pCAG-puro vector to prepare a vector expressing 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 the pCAG-puro or pMX vector to prepare a vector expressing IL-12p35.

[0275] CD81-IL-6 To express IL-6, a T cell-stimulating cytokine, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 100) encoding the full-length sequence (SEQ ID NO: 99) of IL-6 excluding its signal peptide was introduced into a polynucleotide encoding the extracellular loop of CD81, a tetraspanin, and a polynucleotide (SEQ ID NO: 102) encoding a CD81-IL-6 fusion protein (SEQ ID NO: 101) was inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein.

[0276] hCD80-hCD9 To express human CD80, a T cell costimulatory molecule, 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 expressing the fusion protein. sc-Trimer-CD81-IL-2 To express IL-2, a T cell stimulatory cytokine, on the membrane of extracellular vesicles, a polynucleotide encoding a CD81-IL2 fusion peptide was prepared in the same manner as for CD81-IL-4 described above. The polynucleotide sequence was linked to a nucleotide sequence encoding sc-Trimer- to prepare a polynucleotide (SEQ ID NO: 136) encoding sc-Trimer-CD81-IL-2 (SEQ ID NO: 135). This polynucleotide was then inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein.

[0277] hsc-Trimer-hCD81 A polynucleotide (SEQ ID NO: 132) encoding hsc-Trimer-hCD81 (SEQ ID NO: 131) was prepared using the human gene sequence of the sc-Trimer-CD81 (HLA-A2402 was used as the MHC-I sequence), and inserted into a pCAG-puro or pMX vector to prepare a vector for expressing the fusion protein. SARS-CoV2sc-Trimer-hCD81: A polynucleotide (SEQ ID NO: 148) encoding an antigen-presenting MHC molecule (SARS-CoV2sc-Trimer; amino acid sequence: SEQ ID NO: 147) was prepared using a 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. This polynucleotide was further linked to a polynucleotide encoding hCD81 to prepare a polynucleotide (SEQ ID NO: 150) encoding SARS-CoV2sc-Trimer-hCD81 (SEQ ID NO: 149). The prepared polynucleotide was inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein. hCD63-hIL-2: The CD63-IL-2 described above was prepared using a human gene sequence. A polynucleotide (SEQ ID NO: 116) encoding hCD63-hIL-2 (SEQ ID NO: 115) was prepared and inserted into a pCAG-puro or pMX vector to prepare a vector that expresses the fusion protein.

[0278] As a negative control, CD63 was fused with Akaluc luciferase to prepare a polynucleotide (SEQ ID NO: 140) for localizing the AlkaLuc fusion protein (SEQ ID NO: 139) in extracellular vesicles. The polynucleotide was inserted into the pCAG-puro or pMX vector to prepare a vector for expressing the fusion protein.

[0279] Plasmid preparation 4: HLADR-1sc-TPI1-hCD81. The signal sequence of the HLA DR1 β chain (amino acid sequence: SEQ ID NO: 151; polynucleotide sequence: SEQ ID NO: 152), the sequence of the TPI-1 peptide (amino acid sequence: SEQ ID NO: 153; polynucleotide sequence: SEQ ID NO: 154), and the sequence of the HLA DR1 β chain (amino acid sequence: SEQ ID NO: 155; polynucleotide sequence: SEQ ID NO: 156) were ligated to prepare a sequence encoding the HLA DR1 β chain that presents the TPI-1 peptide. The sequence of hCD81 (amino acid sequence: SEQ ID NO: 159; polynucleotide sequence: SEQ ID NO: 160) was then ligated to this sequence. Furthermore, a polynucleotide for presenting TPI-1 peptide on the extracellular vesicle membrane was prepared by binding the HLA DR1α chain sequence (amino acid sequence: SEQ ID NO: 163; polynucleotide sequence: SEQ ID NO: 164) via the P2A sequence (amino acid sequence: SEQ ID NO: 161; polynucleotide sequence: SEQ ID NO: 162). The prepared polynucleotide was inserted into a pCAG-puro or pMX vector to prepare a vector expressing the fusion protein HLADR-1sc-TPI1-hCD81 (amino acid sequence: SEQ ID NO: 165; polynucleotide sequence: SEQ ID NO: 166). From mRNA transcribed from this sequence, a fusion protein of the HLA DR1β chain and hCD81 that presents the TPI-1 peptide an...

Claims

1. (a) a sequence encoding a fusion protein (A) that contains an antigen-presenting MHC molecule and is capable of presenting the antigen-presenting MHC molecule on the extracellular membrane of a cell or extracellular vesicle; (b) a sequence encoding a fusion protein (B) that contains at least one T cell-stimulating cytokine or a subunit thereof and is capable of presenting the T cell-stimulating cytokine on the extracellular membrane of a cell or extracellular vesicle; (c) a sequence encoding a fusion protein (C) containing a T cell costimulatory molecule and capable of presenting the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle; (d) a sequence encoding a fusion protein (D) that contains an antigen-presenting MHC molecule and at least one T cell-stimulating cytokine or a subunit thereof and is capable of presenting the antigen and the T cell-stimulating cytokine on the extracellular membrane of a cell or extracellular vesicle; and (e) a sequence encoding a fusion protein (E) that contains an antigen-presenting MHC molecule, at least one T cell-stimulating cytokine or a subunit thereof, and a T cell costimulatory molecule, and that is capable of presenting the antigen, the T cell-stimulating cytokine, and the T cell costimulatory molecule on the extracellular membrane of a cell or extracellular vesicle; A polynucleotide comprising at least one sequence selected from the group consisting of:

2. The fusion protein defined in (A) above comprises an antigen-presenting MHC molecule, a membrane protein or a transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle; Proteins capable of binding to cell or extracellular vesicle membranes or membrane-binding domains thereof The polynucleotide of claim 1, comprising:

3. The fusion protein defined in (B) above comprises at least one T cell stimulating cytokine or a subunit thereof, a membrane protein or a transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle; Proteins or domains thereof capable of binding to the membrane of cells or extracellular vesicles The polynucleotide of claim 1, comprising:

4. The polynucleotide of claim 1, wherein the T cell stimulating cytokine is IL-2, IL-4, IL-6, IL-12, a subunit of IL-12, IL-15, or TGF-β.

5. The fusion protein defined in (C) above comprises a T cell costimulatory molecule, a membrane protein or a transmembrane domain thereof that can be expressed in the membrane of a cell or extracellular vesicle; Proteins capable of binding to cell or extracellular vesicle membranes or membrane-binding domains thereof The polynucleotide of claim 1, comprising:

6. The fusion protein defined in (D) above is the antigen-presenting MHC molecule; the at least one T cell stimulating cytokine or subunit thereof, and A membrane protein or a transmembrane domain thereof capable of localizing in the membrane of a cell or extracellular vesicle, or Proteins capable of binding to cell or extracellular vesicle membranes or membrane-binding domains thereof The polynucleotide of claim 1, comprising:

7. 2. The polynucleotide of claim 1, comprising the sequence defined in (a) and the sequence defined in (b).

8. The polynucleotide according to claim 7, which encodes an amino acid sequence in which the fusion protein (A) and the fusion protein (B) are fused together.

9. 9. The polynucleotide according to claim 8, which encodes an amino acid sequence in which the fusion protein (A) and the fusion protein (B) are fused via at least one 2A peptide.

10. The polynucleotide of claim 7, further comprising the sequence defined in (c).

11. The polynucleotide of claim 10, wherein the fusion protein (A), the fusion protein (B), and the fusion protein (C) encode a fused amino acid sequence.

12. A polynucleotide according to claim 11, encoding an amino acid sequence in which the fusion protein (A), the fusion protein (B), and the fusion protein (C) are fused together via at least one 2A peptide, each of which is independent.

13. The polynucleotide of claim 1, comprising the sequence defined in (d).

14. The polynucleotide of claim 13, further comprising the sequence defined in (c).

15. The polynucleotide of claim 14, which encodes an amino acid sequence in which the fusion protein (D) and the fusion protein (C) are fused together.

16. 16. The polynucleotide according to claim 15, which encodes an amino acid sequence in which the fusion protein (D) and the fusion protein (C) are fused via at least one 2A peptide.

17. A vector comprising the polynucleotide according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising the polynucleotide according to any one of claims 1 to 16 or a vector comprising the polynucleotide according to any one of claims 1 to 16, and a pharmacologically acceptable carrier.

19. for treating or preventing infectious diseases, for treating or preventing cancer, for treating or preventing an autoimmune disease, or A pharmaceutical composition comprising the polynucleotide according to any one of claims 1 to 16 or a vector comprising the polynucleotide according to any one of claims 1 to 16, for treating or preventing an allergic disease.

20. A method for activating and / or proliferating T cells against a specific antigen, the method comprising introducing the polynucleotide according to any one of claims 1 to 16 or a vector comprising the polynucleotide according to any one of claims 1 to 16 into cells in vitro or ex vivo, thereby producing antigen-presenting cells and / or antigen-presenting extracellular vesicles, and contacting the produced antigen-presenting cells and / or antigen-presenting extracellular vesicles with T cells in vitro or ex vivo.