Vesicle for inducing immunity against each of plurality of immunogenic peptides

Abstract

The present invention provides a fusion protein of an extracellular vesicle surface marker protein or an extracellular vesicle transport signal peptide and a plurality of immunogenic peptides. An extracellular vesicle obtained from a cell expressing the fusion protein can be advantageously used in inducing antigen-specific immunity against each of the plurality of immunogenic peptides.

Description

Vesicles that induce immunity against each of multiple immunogenic peptides

[0001] This disclosure relates to vesicles (particularly extracellular vesicles) that induce immunity against multiple immunogenic peptides.

[0002] Patent Document 1 discloses a DNA vaccine that expresses an antigen formed by fusing CD63 with a vaccine antigen. Patent Document 1 discloses that this solves the problem of low immunogenicity, which was a problem with conventional DNA vaccines, and that it was possible to induce antigen-specific cytotoxic T cells. However, Patent Document 1 does not envision the clinical use of exosomes as a vaccine.

[0003] WO2017 / 175253A

[0004] In this invention, vesicles (e.g., extracellular vesicles such as exosomes) are used as the active ingredient of a vaccine. We have found that by expressing one or more antigens in these extracellular vesicles, specific immunity to one or more antigens can be induced. Furthermore, we have found that extracellular vesicles, such as exosomes, expressing a fusion protein containing an extracellular vesicle marker and multiple antigens, can induce specific immunity to each of the multiple antigens in mammalian individuals. We have also clarified that antigen competition does not occur in this process.

[0005] The present disclosure may provide the following inventions: (1) an immunogenic fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal, and a plurality of immunogenic peptides; (2) the fusion protein according to (1), wherein, if the immunogenic peptide is derived from a protein having a transmembrane domain, the transmembrane domain is deleted in the immunogenic peptide; (3) an extracellular vesicle expressing the fusion protein according to (1) or (2) on its surface; (4) an immunogenic composition comprising the extracellular vesicle according to (3); (5) the immunogenic composition according to (4), which is adjuvant-free; (6) the immunogenic composition according to (4) or (5), for use in inducing specific antibodies against a plurality of immunogenic peptides and / or specific cellular immunity; (7) the protein, extracellular vesicle, or immunogenic composition according to any of (1) to (6), wherein the plurality of immunogenic peptides are derived from a virus. (1) The protein, extracellular vesicle, or immunogenic composition according to any one of (1) to (7) above, comprising the full length or a portion thereof. (9) The protein, extracellular vesicle, or immunogenic composition according to any one of (1) to (8) above, wherein a plurality of immunogenic peptides comprises the full length or a portion thereof of hemagglutinin of influenza virus. (10) The protein, extracellular vesicle, or immunogenic composition according to any one of (1) to (9) above, wherein antigen-specific antibodies against each of the plurality of immunogenic peptides are induced in vivo after administration to an individual, and the induced antibody titer for each peptide is at least twice the pre-induced titer. (11) The immunogenic composition according to any one of (1) to (10) above, wherein antigen-specific antibodies against each of the plurality of immunogenic peptides are induced in vivo after administration to an individual, and the induced antibody titer for each antibody is within the range of the mean value ± 25% of the antibody titer. (12) A nucleic acid encoding the fusion protein according to (1) or (2) above.

[0006] The present disclosure may provide the following inventions: (1A) an immunogenic fusion protein comprising a membrane protein (e.g., a surface marker protein of an extracellular vesicle) or a portion thereof including its transmembrane domain, or an extracellular vesicle-transition peptide such as an extracellular vesicle-transition signal, and a plurality of immunogenic peptides. (2A) the fusion protein according to (1A) above, wherein, if the immunogenic peptide is derived from a protein having a transmembrane domain, the transmembrane domain is deleted in the immunogenic peptide. (3A) a vesicle expressing the fusion protein according to (1A) or (2A) above on its surface. (4A) an immunogenic composition comprising the vesicle according to (3A) above. (5A) the immunogenic composition according to (4A) above, free of adjuvants. (6A) the immunogenic composition according to (4A) or (5A) above for use in inducing specific antibodies against a plurality of immunogenic peptides, and / or specific cellular immunity. (7A) A protein, vesicle, or immunogenic composition according to any one of (1A) to (6A) above, wherein the plurality of immunogenic peptides are derived from a virus. (8A) A protein, vesicle, or immunogenic composition according to any one of (1A) to (7A) above, wherein the plurality of immunogenic peptides include the full length or a portion thereof of influenza virus neuraminidase. (9A) A protein, vesicle, or immunogenic composition according to any one of (1A) to (8A) above, wherein the plurality of immunogenic peptides include the full length or a portion thereof of influenza virus hemagglutinin. (10A) A protein, vesicle, or immunogenic composition according to any one of (1A) to (9A) above, wherein after administration to an individual, antigen-specific antibodies against each of the plurality of immunogenic peptides are induced in vivo, and the antibody titer induced for each peptide is at least twice the pre-induction titer. (11A) An immunogenic composition according to any one of (1A) to (10A) above, wherein antigen-specific antibodies against each of several immunogenic peptides are induced in vivo after administration to an individual, and the amount of each induced antibody falls within the range of the mean value ± 25% of the total amount of antibodies. (12A) A nucleic acid encoding the fusion protein according to (1A) or (2A) above.

[0007] The present disclosure may provide the following inventions: (1B) an immunogenic fusion protein comprising an extracellular vesicle marker protein or a part thereof, or an extracellular vesicle transition peptide such as an extracellular vesicle transition signal, and a plurality of immunogenic peptides. (2B) the fusion protein according to (1B), wherein the extracellular vesicle marker protein is a protein in the lumen of the extracellular vesicle. (3B) the fusion protein according to (1B), wherein the extracellular vesicle marker protein is a membrane protein of the extracellular vesicle. (4B) the fusion protein according to any one of (1B) to (3B), wherein, if the immunogenic peptide is derived from a protein having a transmembrane domain, the transmembrane domain is deleted in the immunogenic peptide. (5B) a vesicle (particularly an extracellular vesicle) expressing the fusion protein according to (1B) or (2B) on its surface. (6B) an immunogenic composition comprising the vesicle (particularly an extracellular vesicle) according to (5B). (7B) the immunogenic composition according to (6B), which does not contain an adjuvant. (8B) The immunogenic composition according to (6B) or (7B) above for use in inducing specific antibodies and / or specific cellular immunity against multiple immunogenic peptides. (9B) The fusion protein or vesicle (especially extracellular vesicle) according to any of (1B) to (5B) above, wherein the multiple immunogenic peptides are derived from a virus, or the immunogenic composition according to any of (6B) to (8B) above. (10B) The fusion protein or vesicle (especially extracellular vesicle) according to any of (1B) to (5B) above, wherein the multiple immunogenic peptides include the full-length or partial-length neuraminidase of influenza virus, or the immunogenic composition according to any of (6B) to (8B) above. (11B) The fusion protein or vesicle (especially extracellular vesicle) according to any of (1B) to (5B) above, wherein the multiple immunogenic peptides include the full-length or partial-length hemagglutinin of influenza virus, or the immunogenic composition according to any of (6B) to (10B) above. (12B) An immunogenic composition according to any one of (1B) to (11B) above, wherein antigen-specific antibodies against each of several immunogenic peptides are induced in vivo after administration to an individual, and the antibody titer induced for each peptide is at least twice the titer before induction.(13B) An immunogenic composition according to any one of (1B) to (12B) above, wherein antigen-specific antibodies against each of several immunogenic peptides are induced in vivo after administration to an individual, and the amount of each induced antibody falls within the range of the mean value ± 25% of the total amount of antibodies. (14B) A nucleic acid encoding a fusion protein according to any one of (1B) to (5B) above.

[0008] The results of Western blotting analysis of isolated EV, recombinant hemagglutinin (rHA) in EV, and recombinant neuraminidase (rNA) are shown. The results of antigen-specific antibody production in mice immunized with EV vaccine or PR8 vaccine are shown. The results of antigen-specific cellular immune responses in mice immunized with EV vaccine or PR8 vaccine are shown. The amount of anti-HA IgG antibody produced by isolated EV containing various antigens is shown. Isolated EV containing a fusion protein containing HA and NP demonstrates antigen-specific cellular immune induction ability. Isolated EV containing a fusion protein containing HA and N demonstrates antigen-specific cellular immune induction ability. Isolated EV containing a fusion protein containing N and RBD demonstrates antigen-specific cellular immune induction ability, and isolated EV expressing a fusion protein containing S and N also demonstrates antigen-specific cellular immune induction ability. Isolated EV containing a fusion protein containing HA and RBD demonstrates antigen-specific cellular immune induction ability.

[0009] <Definition> In this specification, "subject" refers to mammals, rodents such as mice and rats, domestic animals such as pigs and cats, pet animals such as dogs and cats, birds such as chickens, and primates such as humans and monkeys. The subject is preferably a human. The subject is a healthy person, a subject at risk of developing a disease or condition, or a subject with a disease or condition. The disease may be, for example, a tumor, particularly a malignant tumor. The disease may also be, for example, an infectious disease. The disease may also be, for example, an autoimmune disease, such as rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, scleroderma, neuromyelitis optica, celiac disease, vitiligo, multiple sclerosis, Crohn's disease, etc.

[0010] In this specification, “treatment” includes both therapeutic treatment and preventive treatment. In this specification, “treatment” means the treatment, cure, prevention, or improvement of remission of a disease or disorder, or the reduction of the rate of progression of a disease or disorder. In this specification, “prevention” means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition. Treatment may be performed on a person with a disease or condition. Prevention may be performed on a healthy person or a person at risk of developing a disease or condition (a person before the onset of the disease).

[0011] In this specification, “therapeutically effective dose” means the amount of drug that is effective in treating (preventing or curing) a disease or condition. A therapeutically effective dose of drug can slow the rate of progression of symptoms of a disease or condition, halt the progression of such symptoms, improve such symptoms, cure such symptoms, or suppress the onset or development of such symptoms.

[0012] In this specification, "effective amount" refers to the amount of active ingredient that produces the desired effect.

[0013] In this specification, “pharmaceutical composition” means a composition that exerts a medicinal effect and has a composition suitable for administration to a target. A pharmaceutical composition may contain excipients in addition to a pharmacoactive ingredient (API). A pharmaceutical composition may be formulated for, for example, intravenous, nasal, respiratory, subcutaneous, intramuscular, or topical administration, and can be administered intravenously, nasally, respiratoryly, subcutaneously, intramuscularly, or topically, respectively.

[0014] In this specification, “mesenchymal stem cells” refer to a type of stem cell present in adults that is a mesodermal tissue and possesses the ability to differentiate into bone, cartilage, and fat (i.e., multipotency). Mesenchymal stem cells can typically be obtained from tissues such as bone marrow, fat, dental pulp, menstrual blood, placenta, Wharton jelly, umbilical cord, and umbilical cord blood. Human mesenchymal stem cells (human MSCs) can be identified by their adherence to plastic under standard culture conditions, their positivity for the cell surface markers CD73, CD90, and CD105, and their negativeness for CD11b or CD14, CD19 or CD79α, CD34, and CD45, and their HLA-DR.

[0015] In this specification, “passaging” refers to the process of removing some or all of the cultured cells from a culture vessel after they have been cultured in the vessel, and transferring them to a new culture vessel containing fresh culture medium. Passaging is typically performed before the cells reach confluence, as this increases the risk of altering the properties of the cells due to close contact between cells, or the risk of depletion of nutrients in the culture medium or an increase in the concentration of cellular waste products into the medium. In this specification, “passaging number” is expressed as the cumulative number of passages, with the number of passages being 0 at the time of removal from the living organism.

[0016] In this specification, “extracellular vesicles” are vesicles secreted by cells. The membrane of an extracellular vesicle is derived from the membrane of the cell and therefore contains at least one of the components of the cell membrane (e.g., lipids and membrane proteins that make up the cell membrane). Examples of extracellular vesicles include apoptotic vesicles (diameter 1 μm to 5 μm), microvesicles (diameter 100 nm to 1000 nm), and exosomes (diameter 50 nm to 150 nm). In vivo, cells secrete extracellular vesicles into the extracellular space (particularly into body fluids), and under culture conditions, cells secrete extracellular vesicles into the extracellular space (particularly into the culture medium). Extracellular vesicles are secreted by many cells, including mesenchymal stem cells (MSCs). Extracellular vesicles are typically obtained from cell culture supernatant or tissue supernatant by affinity chromatography, size exclusion chromatography, polymer precipitation, ultracentrifugation, and ultrafiltration, and combinations thereof. The characteristics of the extracellular vesicles obtained vary greatly depending on the acquisition method. Furthermore, since cells secrete various extracellular vesicles into the extracellular space, it is not always easy to identify useful extracellular vesicles with specific properties from among them, or to concentrate, enrich, isolate, or purify them. Mesenchymal stem cell-derived exosomes generally have anti-aging, skin-whitening, skin-regenerating, immunomodulatory, anti-fibrotic, anti-inflammatory, antitumor, and therapeutic effects against neurodegenerative diseases (e.g., Alzheimer's disease). Exosomes, depending on the cell from which they originate, typically express one or more, or all, of the following: Alix, Tsg101, tetraspanins (including major markers such as CD81, CD63, and CD9, as well as CD37, CD82, CD151, TSPAN1, TSPAN2, TSPAN3, TSPAN4, TSPAN5, TSPAN6, TSPAN7, TSPAN8, TSPAN14, and TSPAN15), heat shock proteins (such as HSP60, HSP70, HSC70, and HSP90), and flochilins. The amount of exosomes or their quantity can be determined, for example, by the expression of one or more of the tetraspanins. The amount of exosomes or their quantity can also be determined by their particle size. Microvesicles (MVs) are formed when the cell membrane buds outward and have a diameter of approximately 100–1000 nm.MVs are known to express selectins, CD40, and other molecules. Apoptotic vesicles are vesicles produced by programmed cell death (apoptosis) and have a diameter of 50 to 5000 nm. Apoptotic vesicles are known to express annexin V, phosphatidylserine, and other molecules. These molecules are sometimes used as markers for extracellular vesicles or as extracellular vesicle transition signal peptides.

[0017] In this specification, “aqueous composition” means a composition containing water as a solvent, or an aqueous solution. Aqueous compositions have a composition suitable for the retention of extracellular vesicles (for example, the composition of physiological saline). In one preferred embodiment, an aqueous composition is a pharmaceutical composition. Aqueous compositions also include dispersion formulations containing a dispersed phase dispersed in water or an aqueous solution.

[0018] In this specification, "CD9" is a member of a protein family called tetraspanins, possessing a transmembrane domain and located on the cell membrane or exosome membrane. The standard sequence of human CD9 is registered, for example, as GenBank registry number AAC60586.1. CD9 includes proteins that have a sequence corresponding to the amino acid sequence registered as GenBank registry number AAC60586.1, or that have 90% or more sequence identity with said amino acid sequence, or are isoforms thereof, and that have the ability to translocate to exosomes. CD9 may be the full-length or fragments thereof that have the ability to translocate to exosomes.

[0019] In this specification, "CD63" is a member of a protein family called tetraspanins, possessing a transmembrane domain and located on the cell membrane and exosome membrane. The standard sequence of human CD63 is registered with the National Center for Biotechnology Information (NCBI) as GenBank Registry No. AAH13017.1. CD63 includes proteins having a sequence corresponding to the amino acid sequence registered as GenBank Registry No. AAH13017.1, or having 90% or more sequence identity with said amino acid sequence, or being an isoform thereof, and possessing the ability to translocate to exosomes. CD63 may also be a fragment thereof that possesses the ability to translocate to exosomes.

[0020] In this specification, "CD81" is a member of a protein family called tetraspanins, possessing a transmembrane domain and located on the cell membrane and exosome membrane. The standard sequence of human CD81 is registered with the National Center for Biotechnology Information (NCBI) as GenBank Registry No. AAH93047.1. CD81 includes proteins having a sequence corresponding to the amino acid sequence registered as GenBank Registry No. AAH93047.1, or having 90% or more sequence identity with said amino acid sequence, or being an isoform thereof, and possessing the ability to translocate to exosomes. CD81 may also be a fragment thereof that possesses the ability to translocate to exosomes.

[0021] In this specification, "extracellular vesicle translocation signal" refers to a peptide having an amino acid sequence that facilitates the uptake of the peptide into extracellular vesicles (EVs) when fused to the peptide. While not particularly limited, examples of extracellular vesicle translocation signals include tetraspanin family proteins, Alix, TSG101, HSP70, LAMP2B, WW domain-binding peptides, CXCR4 peptide motifs, RVG peptides, C1C2 domains (e.g., derived from Annexin V), CD63 transmembrane region peptides, and glycosaminoglycan (GAG)-binding peptides. Furthermore, binding proteins designed to bind to the above-mentioned signal peptides can also be extracellular vesicle translocation signals.

[0022] In this specification, "peptide" is a polymer of amino acids linked by peptide bonds, and is a concept that includes proteins. Peptides are not particularly limited, but may have lengths of, for example, 10 to 3000 amino acids, 30 to 1000 amino acids, or 100 to 500 amino acids. Extracellular vesicle-transport peptides are peptides that have the ability to transfer to extracellular vesicles. Extracellular vesicle-transport peptides are not particularly limited, but examples include those that exhibit binding affinity to the Endosomal Sorting Complex Required for Transport (ESCRT). Peptides that exhibit ESCRT binding affinity include motifs that bind to TSG101 (e.g., L-domain motifs such as PPXY, PTAP, PSAP, and YPXnL), motifs that readily bind to ALIX (e.g., YPXnL motifs such as YPDL, YPQRL, and YPLSVL), ubiquitinated peptides, WW domain binding motifs such as PPXY, and peptides possessing lipid draft enrichment signals (e.g., GPI-anchored proteins, palmitoylation signals). Peptides that bind to any of the extracellular vesicle markers may also be extracellular vesicle-transport peptides.

[0023] The fragments may have a length of, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total length.

[0024] In this specification, “isolation” refers to the removal of at least one other component from the environment in which it was produced. Therefore, isolation does not require purification until only a single component remains, and allows for the presence of other components (e.g., pharmaceutically acceptable impurities) in the same composition. “Isolated microRNA” means microRNA in its free form. Free form is used to distinguish it from microRNA in vesicles or cells, and means microRNA outside of vesicles or cells (including microRNA isolated from vesicles or cells and artificially synthesized microRNA).

[0025] In this specification, "immunogenicity" refers to the property of inducing antigen-specific immunity in an individual. An immunogenic peptide is a peptide that possesses immunogenic properties. An immunogenic composition is a composition that possesses immunogenic properties.

[0026] In this specification, "vaccine" or "vaccine composition" are interchangeable and refer to compositions used in vaccine applications. Vaccine applications are those that induce antigen-specific immunity in an individual. By inducing antigen-specific immunity, for example, cancer and infectious diseases can be treated.

[0027] In this specification, "adjuvant" refers to a substance that promotes immune induction against an antigen (e.g., an immunogenic peptide). Examples of adjuvants include aluminum hydroxide and aluminum salts such as aluminum phosphate; monophosphoryl lipid A, QS-1, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG oligonucleotides, and MF59. In many cases, adjuvants cause inflammation.

[0028] <Fusion Proteins of the Disclosure> The Disclosure provides immunogenic fusion proteins comprising an extracellular vesicle-transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle-transition signal peptide, and an immunogenic peptide. In one preferred embodiment, the immunogenic fusion protein comprises two or more different immunogenic peptides.

[0029] In one preferred embodiment, in a fusion protein, different peptides are linked directly or via a linker. The linker may be a peptide linker. The linker may also be a flexible linker. GS linkers are commonly used as flexible linkers and can be preferably used in this disclosure. A GS linker may have two to five consecutive G and S units of two to four units. For example, a GS linker may have two to four units, e.g., three units, each containing four Gs and one S.

[0030] In one preferred embodiment, the fusion protein does not have an internal sequence that cleaves under physiological conditions.

[0031] In one preferred embodiment, the extracellular vesicle marker protein is a non-surface marker protein of the extracellular vesicle (e.g., a marker expressed inside the extracellular vesicle). By including an extracellular vesicle marker protein or an extracellular vesicle transition peptide such as an extracellular vesicle transition signal peptide, the immunogenic fusion protein can efficiently translocate to extracellular vesicles. The extracellular vesicle marker protein or the extracellular vesicle transition peptide such as an extracellular vesicle transition signal peptide may be a fragment thereof, as long as it retains its ability to translocate to extracellular vesicles.

[0032] In one preferred embodiment, the extracellular vesicle marker protein is a surface marker protein of the extracellular vesicle. The surface marker protein is expressed on the surface of the extracellular vesicle. Therefore, using a surface marker protein as a marker protein has the advantage of facilitating the examination of protein expression levels. In this disclosure, for example, tetraspanin family proteins may be preferably used as surface marker proteins. When an antigen peptide is fused to a tetraspanin family protein, the antigen peptide may be ligated, for example, at its N-terminus, C-terminus, or between its EC1 domain and EC2 domain.

[0033] Therefore, in a preferred embodiment, an immunogenic fusion protein is provided comprising (i) an extracellular vesicle-transfer peptide such as an extracellular vesicle surface marker protein or an extracellular vesicle-transfer signal peptide, and (ii) two or more different immunogenic peptides. Typically, when immunizing with two or more antigenic peptides simultaneously, strong immune induction occurs against one antigenic peptide, but poor immune induction occurs against the other antigenic peptide. This phenomenon is called antigen competition.

[0034] According to this disclosure, extracellular vesicles expressing an immunogenic fusion protein comprising (i) an extracellular vesicle transition peptide such as an extracellular vesicle surface marker protein or an extracellular vesicle transition signal peptide, and (ii) two or more different immunogenic peptides, showed good immune induction against each of the two or more different immunogenic peptides without antigen competition. Therefore, the above extracellular vesicles can be preferably used when inducing immune response to each of the two or more immunogenic peptides.

[0035] In one embodiment, an extracellular vesicle expressing an immunogenic fusion protein comprising (i) an extracellular vesicle surface marker protein or an extracellular vesicle transition signal peptide, and (ii) two or more different immunogenic peptides, induces antigen-specific immunity to each of the two or more different immunogenic peptides, and the antibody titer of antigen-specific IgG1 antibody against the antigen increases by 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, and 40 times the antibody titer before induction. It is possible that the multiplier is 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more, or 10000 times or more. The antibody titers of antigen-specific IgG1 antibodies against the antigen may be 1 million times or less, 500,000 times or less, 100,000 times or less, 50,000 times or less, 10,000 times or less, 9,000 times or less, 8,000 times or less, 7,000 times or less, 6,000 times or less, 5,000 times or less, 4,000 times or less, 3,000 times or less, 2,000 times or less, 1,000 times or less, 900 times or less, 800 times or less, 700 times or less, 600 times or less, 500 times or less, 400 times or less, 300 times or less, 200 times or less, 10 times or less, 90 times or less, 80 times or less, 70 times or less, 60 times or less, 50 times or less, 40 times or less, 30 times or less, 20 times or less, or 10 times or less compared to the antibody titer before induction. The antibody titers of antigen-specific IgG1 antibodies against the antigen can be 2 to 1 million times, 5 to 1 million times, 10 to 500,000 times, 100 to 1 million times, 1,000 to 1 million times, and 10,000 to 1 million times, respectively, compared to the antibody titer before induction.

[0036] In one embodiment, an extracellular vesicle expressing an immunogenic fusion protein comprising an extracellular vesicle surface marker protein or an extracellular vesicle transition signal peptide, and two or more different immunogenic peptides, induces antigen-specific immunity to each of the two or more different immunogenic peptides, and the antibody titer of the antigen-specific IgG1 antibody against the antigen may be within ±100%, ±90%, ±80%, ±70%, ±60%, preferably within ±50%, more preferably within ±40%, even more preferably within ±30%, even more preferably within ±20%, or particularly preferably within ±10% of the average antibody titer of the antigen-specific IgG1 antibody induced by the fusion protein.

[0037] Antibody titer can be measured by methods well known to those skilled in the art. For example, antibody titer can be determined by immobilizing an antigen on the surface of a plate, blocking it, then contacting serum or a dilution of it with the immobilized antigen, and measuring the amount of antibody bound to the immobilized antigen by a labeled secondary antibody. For example, horseradish peroxidase (HRP) can be used as the label. When using HRP, it can be detected by developing a color in the presence of a substrate (e.g., TMB) and measuring the optical density (OD450). Antibody titer can also be defined, for example, as the highest serum dilution ratio such that {labeled amount of diluted serum (e.g., OD450 value) > labeled amount of negative control serum (e.g., OD450 value)}. Those skilled in the art can select a secondary antibody and label as appropriate and measure the antibody titer.

[0038] Extracellular vesicles include, but are not particularly limited, one or more extracellular vesicles selected from the group consisting of, for example, exosomes, apoptotic vesicles, and microvesicles. In a certain preferred subject, the extracellular vesicles include, for example, exosomes.

[0039] Since the fusion protein of the present disclosure contains an extracellular vesicle marker protein or an extracellular vesicle translocation peptide such as an extracellular vesicle translocation signal peptide, when expressed in cells, extracellular vesicles are released from the cells and translocate to the extracellular vesicles. Therefore, the extracellular vesicles released from the cells contain extracellular vesicles expressing the marker protein or an extracellular vesicle translocation peptide such as an extracellular vesicle translocation signal peptide. These extracellular vesicles can induce, for example, a Th1-type and / or Th2-type immune response against the immunogenic peptide contained in the fusion protein expressed in the extracellular vesicles.

[0040] The immunogenic peptide can be an endogenous protein or a part thereof, or an exogenous protein or a part thereof. The immunogenic peptide can be, for example, a cancer antigen. The immunogenic peptide can also be, for example, a protein or a part thereof possessed by a pathogen (e.g., virus, bacterium, fungus, and parasite, etc.).

[0041] As the cancer antigen that can be incorporated into the fusion protein of the present disclosure, any protein that is expressed in cancer cells can be used. For example, Wilmus Tumor 1 (WT-1), Human Carbohydrate Antigen 125 (CA-125), Carcinoembryonic Antigen (CEA), Human Telomerase Reverse Transcriptase (hTERT), Mucin-1 (Muc-1), Mucin-2 (Muc-2), Cancer / Testis antigen 1B (CTAG1B / NY-ESO-1), Prostatic Acid Phosphatase (PAP), Prostate Specific Antigen (PSA), Prostate Specific Membrane Antigen (PSMA), Survivin b, mutant ras, mutant p53, etc. can be mentioned. When administered to humans, it is desirable that these proteins are human proteins.

[0042] The pathogen antigens that can be incorporated into the fusion proteins of this disclosure may be any protein possessed by the pathogen, but examples include bacteria such as Escherichia coli, Mycobacterium tuberculosis, Streptococcus pneumoniae, Salmonella, and Clostridium botulinum; viruses such as influenza virus, coronavirus (e.g., β-coronavirus such as SARS-CoV-2), human immunodeficiency virus, human papillomavirus (e.g., types 16 and 18), and herpes simplex virus; fungi such as Candida, Aspergillus, and Cryptococcus; protozoa such as Plasmodium malariae, Trichomonas, Toxoplasma gondii, and Entamoeba histolytica; and parasites such as Ascaris, filariasis, Schistosoma japonicum, and house dust mites. The pathogen antigens that can be incorporated into the fusion proteins of this disclosure may, for example, be E6 and / or E7 of human papillomavirus (HPV). The pathogen antigens that can be incorporated into the fusion proteins of this disclosure may, for example, be S1 and / or S2 proteins of coronavirus. Examples of pathogenic antigens that may be incorporated into the fusion proteins of this disclosure include hemagglutinin (HA) and / or neuraminidase (NA) of various types of influenza viruses.

[0043] The subtypes of influenza virus that infect humans can typically be H1N1, H1N2, H2N2, H3N2, H5N1, H5N6, H5N8, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N3, H10N7, and H10N8. Thus, in certain aspects, the extracellular vesicles can express one, two, three, four, five, six, seven, or all selected from the group consisting of H1, H2, H3, H5, H6, H7, H9, and H10. Also, in certain aspects, the extracellular vesicles can express one, two, three, four, five, six, or all selected from the group consisting of N1, N2, N3, N6, N7, N8, and N9. In certain aspects, the extracellular vesicles can express one, two, three, four, five, six, seven, or all selected from the group consisting of H1, H2, H3, H5, H6, H7, H9, and H10, and one, two, three, four, five, six, or all selected from the group consisting of N1, N2, N3, N6, N7, N8, and N9. In certain aspects, from the perspective of corresponding to any subtype that infects humans, H1, H2, H3, H5, H6, H7, H9, and H10, as well as N1, N2, N3, N6, N7, N8, and N9 can be expressed. From the perspective of corresponding to both H1N1 and H3N2 that prevail as seasonal influenza viruses, the extracellular vesicles can express H1, H3, N1, and N2.

[0044] Multiple antigenic peptides may be contained in one fusion protein or may be contained in multiple fusion proteins. The total molecular weight of the antigenic peptides contained in one fusion protein can be, for example, ~100 kDa (corresponding to approximately 900 amino acid lengths) or can be ~200 kDa (corresponding to approximately 1800 amino acid lengths). In certain aspects, the total molecular weight of the antigenic peptides contained in one fusion protein can be 150 kDa to 300 kDa. Those skilled in the art will be able to determine the appropriate size of the fusion protein to achieve a sufficient expression level.

[0045] Protein antigens associated with autoimmune diseases (in particular, protein antigens for the treatment of autoimmune diseases) that can be incorporated into the fusion proteins of this disclosure include, for example, insulin, glutamate decarboxylase (GAD), and ZnT8 (zinc transporter 8) for type 1 diabetes; for example, topoisomerase I, RNA polymerase III, and centromere protein (CENP) for scleroderma; for example, aquaporin 4 (AQP4) for neuromyelitis optica; for example, acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) for myasthenia gravis; and for example, rheumatoid factor (RF), cyclic citrullinated peptide (CCP), and cartilage-derived peptide for rheumatoid arthritis. For celiac disease, for example, gluten (especially gliadin), tissue transglutaminase-modified peptides; for vitiligo, for example, melanocyte-related factors (tyrosinase, TRP1, TRP2); for systemic lupus erythematosus, for example, nuclear antigens (histones, Sm antigen); for multiple sclerosis, for example, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG); for autoimmune hepatitis, for example, liver-specific proteins (F-actin, SLA / LP antigen); for Graves' disease, A Examples of fusion proteins include: thyroid peroxidase (TPO) and thyroglobulin (Tg) for Hashimoto's disease; keratin 17 and IL-17-related peptide for psoriasis; HSP60 (heat shock protein 60) for Behçet's disease; desmoglein 1 and 3 (Dsg1 / Dsg3) for pemphigus; food antigen peptides (milk, egg, and grain-related peptides) for eosinophilic esophagitis; galectin-related protein (galactose-deficient IgA1) for IgA nephropathy; and acetylated myosin-related protein for sarcoidosis. Each antigen is selected based on the etiology and clinical characteristics of the disease and is useful in designing therapies using fusion proteins. Many other autoimmune diseases can be listed, and those skilled in the art can appropriately select antigens and design fusion proteins.

[0046] The fusion proteins of this disclosure may contain multiple (e.g., two) different antigens derived from the same species. This may provide a robust vaccine against a single species or its variants.

[0047] The fusion proteins of this disclosure may contain multiple (e.g., two) different antigens derived from different species. This may enable the provision of vaccines effective against various species.

[0048] Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the flagellar protein (H antigen) of Escherichia coli. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the Ag85 (Antigen 85) complex containing ESAT-6 and CFP-10, Ag85A, Ag85B, and Ag85C of Mycobacterium tuberculosis. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the pneumococcal surface protein A (PspA) and pneumococcal adhesion protein A (PsaA) of Streptococcus pneumoniae. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the flagellar protein and vironen antigen (Vi antigen) of Salmonella. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the botulinum toxin (e.g., types A to G) and flagellar protein (H antigen) of Clostridium botulinum.

[0049] Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the E6 and / or E7 and / or L1 proteins of human papillomavirus (HPV). Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the S1 and / or S2 proteins of coronavirus, or fragments thereof (e.g., fragments containing RBD). Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the N protein of coronavirus. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, hemagglutinin (HA) and / or neuraminidase (NA) of influenza virus. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the NP protein and nucleocapsid protein of influenza virus. Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include, for example, the F protein of respiratory syncytial virus (RSV). Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include HIV envelope proteins (e.g., gp120, pg41, etc.) and capsid proteins (e.g., p24, etc.). Examples of pathogen antigens that can be incorporated into the fusion proteins of this disclosure include glycoprotein D, glycoprotein B, and glycoprotein C of herpes simplex virus.

[0050] Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, the enzole-type protein of Candida. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, Aspergillus Aspf1 and Aspf2. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, Cryptococcus-specific protein (Ccp1) of Cryptococcus. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, the circumsporozoid protein (P. falcipirum CSP), merozoid surface protein (MSP-1), and VAR protein of the malaria parasite. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, Toxoplasma surface antigen 1 (SAG1) and GRA protein. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, the galactose / nacornamine receptor of Entamoeba histolytica, E. Histolytica antigens (EhCP1, EhCP5) are examples.

[0051] Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, Ascaris larval protein 1 (ABA-1) and TES protein of the roundworm. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include Wb123, Bm14, and excretory secretory antigen (ES antigen) of filarial parasites. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include Sm-TSP-2 and Sm28-GST of Schistosoma japonicum. Examples of pathogen antigens that can be incorporated into the fusion protein of this disclosure include, for example, sarcoptin antigen and surface protein of the house dust mite.

[0052] Antigens that can be incorporated into the fusion proteins of this disclosure may, for example, be MHC class I-restricted. Antigens that can be incorporated into the fusion proteins of this disclosure may, for example, be MHC class II-restricted. Whether a peptide is MHC class I-restricted or MHC class II-restricted can be determined by conventional methods by those skilled in the art. The exosomes of this disclosure strongly induce a Th1 response. Therefore, antigens that can be incorporated into the fusion proteins of this disclosure may preferably be MHC class I-restricted. MHC class I restriction is not particularly limited, but can be predicted using, for example, NetMHCpan, IEDB, etc. It may also be predicted using deep learning support vector machines, etc.

[0053] The antigens that can be incorporated into the fusion proteins of this disclosure may be non-membrane proteins, but if the antigens that can be incorporated into the fusion proteins of this disclosure are derived from membrane proteins, the antigens may have a transmembrane domain, and preferably may lack one.

[0054] Extracellular vesicles expressing the fusion protein of this disclosure may or may not express the antigenic portion of the fusion protein (for example, the antigenic portion may be located in the lumen). Extracellular vesicles expressing the fusion protein of this disclosure may express a portion of the antigenic portion of the fusion protein in the lumen and a portion (preferably the remainder) in the open. By interposing a transmembrane domain, a portion of one fusion protein may be expressed in the lumen and a portion (preferably the remainder) may be expressed, or one or more fusion proteins may express the antigen and other fusion proteins may express the antigen in the lumen.

[0055] The fusion proteins of this disclosure may be obtained by introducing the gene encoding them into cells and driving transcription and translation within the cells. The fusion proteins of this disclosure may also be prepared from the gene encoding them using in vitro transcription and translation systems.

[0056] This disclosure provides nucleic acids encoding the fusion proteins of this disclosure. The nucleic acids may be operably ligated to a control sequence. This disclosure provides a vector (e.g., a virus or plasmid) comprising the nucleic acids. The vector is preferably an expression vector and comprises the nucleic acids operably ligated to a control sequence. The control sequence may be, for example, a promoter, which may be, for example, a promoter for RNA polymerase II. Promoters are well known to those skilled in the art, and those skilled in the art can appropriately select and use preferred ones.

[0057] When the cell species and the species from which the immunogenic peptide originates are different, it is preferable that the codons in the nucleic acid encoding the fusion protein be optimized to be suitable for expression in the cell species (i.e., codon-optimized).

[0058] <Vessels of the Disclosure> The Disclosure provides vesicles expressing (including) the fusion protein of the Disclosure. Examples of vesicles include lipid vesicles, lipid nanoparticles, liposomes, micelles, etc. The vesicles are preferably isolated or purified.

[0059] According to this disclosure, the vesicles expressing (including) the fusion protein of this disclosure are not particularly limited, but preferably have the immunogenic peptide in the fusion protein exposed on their surface. This is expected to enhance the immune-inducing effect.

[0060] To expose the immunogenic peptide in the fusion protein to the surface, it is preferable that the fusion protein includes a membrane protein or a portion thereof having a transmembrane domain and the immunogenic peptide. By linking the immunogenic peptide to the N-terminus or C-terminus of the membrane protein or a portion thereof having a transmembrane domain, which is the end that is exposed outside the cell, the immunogenic peptide will be exposed to the surface.

[0061] Such vesicles can be obtained by inducing vesicle formation by mixing the vesicle components with the aforementioned fusion protein during vesicle formation.

[0062] <Extracellular Vesicles of the Disclosure> The Disclosure provides extracellular vesicles expressing (including) the fusion protein of the Disclosure. The extracellular vesicles are preferably isolated or purified, where isolation requires that at least the extracellular vesicles have been separated from the cell that produced them. Purification is an operation in which the extracellular vesicles are concentrated, enriched, or have their proportion of impurities reduced. In a preferred embodiment, the extracellular vesicles contain or are exosomes. Exosomes can efficiently deliver the fusion protein of the Disclosure to immune cells.

[0063] According to this disclosure, the extracellular vesicles expressing (including) the fusion protein of this disclosure are not particularly limited, but preferably have immunogenic peptides in the fusion protein exposed on their surface. This is expected to enhance the immune-inducing effect. In some embodiments, the fusion protein includes a membrane protein, which preferably has an extracellular vesicle translocation signal, such as tetraspanins (e.g., CD9, CD63, CD81).

[0064] In some embodiments, the extracellular vesicles of this disclosure may express the major histocompatibility complex (MHC), or they may not express the MHC, with respect to reducing the risk of immune side effects. Cells that do not express the MHC are not particularly limited, but can be obtained, for example, by β2-microglobulin knockout and / or CIITA knockout. Cells that express the MHC can also be obtained by knocking out the MHC itself.

[0065] In some embodiments, the extracellular vesicles of the present disclosure may or may not express one or more endogenous or exogenous factors selected from the group consisting of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, and TCR. Extracellular vesicles expressing endogenous or exogenous factors can be obtained from cells expressing said factors (e.g., mesenchymal stem cells).

[0066] The extracellular vesicles of this disclosure may or may not express the same marker as the fusion protein separately from the fusion protein. Extracellular vesicles expressing the same marker as the fusion protein separately from the fusion protein can be obtained, for example, from cells in which the gene encoding the marker is not knocked out, while extracellular vesicles in which the marker is not expressed separately from the fusion protein can be obtained, for example, from cells in which the gene encoding the marker is knocked out. Gene knockout can be performed by conventional methods, for example, by genome editing technology.

[0067] Extracellular vesicles can be released into the cell culture supernatant when cells are cultured. Therefore, extracellular vesicles can be recovered from the culture supernatant of cells expressing the fusion protein of this disclosure (preferably mesenchymal cells). The recovered extracellular vesicles can be purified by methods well known to those skilled in the art. In addition to mesenchymal stem cells, other cells, such as human tissue-derived cell lines, can be used. Human tissue-derived cell lines can be prepared using methods known to those skilled in the art, or they can be commercially available. Examples of such cell lines include human embryonic kidney cell 293 (HEK293) cells, HeLa cells, HepG2 cells, MRC-5 cells, K562 cells, iPS cells, or cells derived from such cells. In one embodiment, the human-derived cells used in the present invention are cells derived from HEK293 cells. In one embodiment, cells derived from HEK293 cells include HEK293T cells, HEK293E cells, HEK293S cells, and FreeStyle TM 293-F cells, Expi293 cells, HEK293SF cells, CAP TM cells, Per. C6 TM cells, GPEx TM cells, 293T / 17SF cells, PEAKrapid TM cells, ELEVECTA TM It could be a cell.

[0068] <Immunogenic Compositions or Vaccines of the Disclosure> The Disclosure provides compositions comprising the extracellular vesicles described above. When considering applications as vaccines, the compositions preferably have a composition suitable for administration to a target. In addition to the extracellular vesicles, the compositions of the Disclosure may also contain pharmaceutically acceptable carriers or additives.

[0069] The present disclosure provides an immunogenic composition or vaccine (e.g., a vaccine composition) comprising an extracellular vesicle expressing the fusion protein of the present disclosure.

[0070] In a preferred embodiment, the immunogenic composition or vaccine of the present disclosure may further comprise a pharmaceutically acceptable carrier or excipient.

[0071] Examples of pharmaceutically acceptable carriers or additives include water, salts, isotonic agents, pH adjusters, and preservatives.

[0072] In some embodiments, the immunogenic compositions or vaccines of this disclosure may contain additional adjuvants, but preferably do not contain additional adjuvants. Although not bound by theory, it is desirable to avoid using adjuvants as much as possible because antigen competition may occur when adjuvants are used.

[0073] The immunogenic compositions or vaccines of this disclosure may be used to induce antigen-specific antibodies and / or antigen-specific cellular immunity (particularly antigen-specific T-cell immunity) against each of the multiple immunogenic peptides contained in the fusion protein.

[0074] In one embodiment, an extracellular vesicle expressing an immunogenic fusion protein containing an extracellular vesicle surface marker protein or an extracellular vesicle transition signal peptide, and two or more different immunogenic peptides, induces antigen-specific immunity against each of the two or more different immunogenic peptides, resulting in antibody titers of antigen-specific IgG1 antibodies against the antigen being 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, and 8 times the antibody titer before induction. It may be 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more, or 10000 times or more. The antibody titers of antigen-specific IgG1 antibodies against the antigen may be 1 million times or less, 500,000 times or less, 100,000 times or less, 50,000 times or less, 10,000 times or less, 9,000 times or less, 8,000 times or less, 7,000 times or less, 6,000 times or less, 5,000 times or less, 4,000 times or less, 3,000 times or less, 2,000 times or less, 1,000 times or less, 900 times or less, 800 times or less, 700 times or less, 600 times or less, 500 times or less, 400 times or less, 300 times or less, 200 times or less, 10 times or less, 90 times or less, 80 times or less, 70 times or less, 60 times or less, 50 times or less, 40 times or less, 30 times or less, 20 times or less, or 10 times or less compared to the antibody titer before induction. The antibody titers of antigen-specific IgG1 antibodies against the antigen can be 2 to 1 million times, 5 to 1 million times, 10 to 500,000 times, 100 to 1 million times, 1,000 to 1 million times, and 10,000 to 1 million times, respectively, compared to the antibody titer before induction.

[0075] Following administration of the immunogenic composition or vaccine of this disclosure to an individual, antigen-specific antibodies against each of the immunogenic peptides contained in the fusion protein are induced in vivo, and the amount of each induced antibody (particularly IgG1) falls within the range of ±50%, ±40%, ±30%, ±25%, or ±20% of the mean amount of each antibody (particularly IgG1). The individual is, for example, a human. The individual may also be, for example, an experimental animal (e.g., a mouse).

[0076] According to this disclosure, the immunogenic compositions or vaccines of this disclosure can be administered via various routes of administration, such as intravenous, subcutaneous, intradermal, and intramuscular administration. Accordingly, the immunogenic compositions or vaccines of this disclosure are formulated to be suitable for any of the routes of administration, such as intravenous, subcutaneous, intradermal, intramuscular, and nasal administration.

[0077] In a preferred embodiment, the immunogenic compositions and vaccines of this disclosure are not used in combination with or mixed with other immunogenic compositions and vaccines.

[0078] In a preferred embodiment, the immunogenic compositions and vaccines of this disclosure may be administered to allogeneic subjects.

[0079] In a preferred embodiment, the immunogenic compositions and vaccines of this disclosure are not DNA vaccines, i.e., vaccines having DNA as an active ingredient.

[0080] <Methods for Inducing Antigen-Specific Immunity and Others in This Disclosure> This disclosure provides methods for inducing antigen-specific immunity in a subject. According to this disclosure, a method for inducing antigen-specific immunity in a subject may include administering an effective amount of the extracellular vesicles of this disclosure to the subject.

[0081] According to this disclosure, the immunogenic compositions or vaccines of this disclosure may be administered by various routes of administration, such as intravenous, subcutaneous, intradermal, and intramuscular administration, but are preferably administered intravenously.

[0082] According to this disclosure, antigen-specific immunity may be induced against each immunogenic peptide contained in the fusion protein expressed in the administered extracellular vesicles. Antigen-specific immunity may include the induction of antigen-specific antibodies and / or the induction of antigen-specific cellular immunity (particularly the induction of antigen-specific T cell immunity). The induction of antigen-specific T cell immunity may include, for example, the induction of antigen-specific interferon-γ (IFN-γ) production and / or interleukin (IL)-13 production against T cells.

[0083] According to this disclosure, by performing the above method, antigen-specific immunity against cancer cells or pathogens expressing immunogenic peptides contained in the fusion protein can be induced. As a result, the method of this disclosure can be used to treat cancer in subjects with cancer, or to treat or prevent infection in subjects with an infection.

[0084] Accordingly, the present disclosure provides a method for treating cancer in a subject having cancer, comprising administering to the subject a therapeutically effective amount of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the cancer. The present disclosure also provides a method for treating an infection in a subject having an infection, comprising administering to the subject a therapeutically effective amount of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the causative agent of the infection. The present disclosure further provides a method for preventing an infection in a subject, comprising administering to the subject a therapeutically effective amount of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the causative agent of the infection.

[0085] This disclosure provides immunogenic compositions or vaccines for use in the methods described above.

[0086] The present disclosure provides the use of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the cancer, in the manufacture of an immunogenic composition or vaccine for use in a method for treating cancer in a subject having cancer. The present disclosure provides the use of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the causative agent of the infection, in the manufacture of an immunogenic composition or vaccine for use in a method for treating an infection in a subject having an infection. The present disclosure provides the use of extracellular vesicles expressing a fusion protein comprising an extracellular vesicle transition peptide, such as an extracellular vesicle marker protein or an extracellular vesicle transition signal peptide, and a plurality of immunogenic peptides expressed by the causative agent of the infection, in the manufacture of an immunogenic composition or vaccine for use in a method for preventing an infection in a subject.

[0087] Example 1 Experimental Method A DNA construct was designed to express a full-length CD63 (SEQ ID NO: 1) fused with influenza H1N1 hemagglutinin (with the transmembrane region from 529-549aa of the amino acid sequence of SEQ ID NO: 2 deleted) and neuraminidase (with the transmembrane region from 8-27aa of the amino acid sequence of SEQ ID NO: 3 deleted). Codon optimization was performed using a vector builder, and the gene was synthesized. A common flexible linker, the GS linker {(GGGGS)3} (SEQ ID NO: 5), was inserted between the genes. The fusion gene was designed to be expressed downstream of the CMV promoter. The resulting fusion gene was called CD63-HA-NA, and the amino acid sequence of the fusion protein encoded by this fusion gene was as shown in SEQ ID NO: 4.

[0088]

[0089] HEK293F cells were cultured in Freestyle 293 expression medium (Life Technologies), 8% CO2, and 37°C. The DNA construct was transfected into HEK293F cells for 72 hours. Transfection was performed using PEI MAX (PolySciences) according to the manufacturer's protocol. The culture medium was then collected and centrifuged at 2000 × g for 15 minutes. The supernatant was collected after centrifugation and concentrated using Amicon Ultra-4 100k. Extracellular viable cells (EVs) were isolated from the concentrated culture supernatant using qEV Gen2 original 35 (IZON) according to the manufacturer's protocol. The isolated EVs were used as the vaccine below. The vaccine containing the isolated EVs is referred to as the EV vaccine below. No adjuvants were added to the EV vaccine. The particle concentration of the collected EVs was measured using Zetaview (Vertical Metrics). Antigen loading into the EVs was confirmed by Western blotting. In this extracellular matrix (EV), antigenic portions such as HA and NA were located on the luminal side, not on the EV surface.

[0090] Western blotting was performed as follows: EV, recombinant HA protein (sinobiological), and recombinant NA protein (sinobiological) were added to 4x Laemmli sample buffer (BioRad) and boiled at 95°C for 5 minutes. The sample load was equivalent to 0.01 μg for recombinant HA and 0.02 μg for recombinant NA. The load amount for the EV sample was 2.5 × 10⁻⁶. 10 Samples were analyzed at a rate of one sample per lane. These samples were analyzed by Western blotting using anti-HA antibody (11684-R016, Synobio) and anti-NA antibody (PA5-34949, Invitrogen). Western blotting was performed using Biorad's MiniProtean TGX gel, MiniProtean Tetra cell, and TransBlot Turbo blotting system according to the manufacturer's protocol.

[0091] Six-week-old female C57BL / 6J mice were purchased from CREA Japan Co., Ltd. The mice were immunized intradermally on Day 1 and Day 15 with either EV vaccine (equivalent to 10 ng each of HA and NA) or inactivated influenza virus particles PR8 (A / human / Puerto Rico / 8 / 34 strain, equivalent to 1 μg of HA protein) (100 μL / mouse). Two weeks after the last vaccination, the mice were sacrificed, and the antigen-specific immune response to EV vaccine and PR8 was evaluated. All animal experiments were conducted in accordance with the tissue guidelines of the animal facility at the Institute of Medical Science, University of Tokyo.

[0092] To measure HA- or NA-specific antibody titers, 96-well plates were coated overnight at 4°C with 1 μg / ml HA solution or 5 μg / ml NA solution (Sinobiological). The plates were washed and incubated for 1 hour in blocking buffer (PBS(-) containing 1% BSA and 0.05% Tween20). The plates were washed again, serum dilutions were added, and incubated. After washing the plates, HRP-labeled anti-mouse IgG1Ab (Southern Biotech, Birmingham, AL) was added. After washing the plates, TMB substrate solution was added to each well to initiate the color reaction. The reaction was stopped by adding 2N H2SO4, and the absorbance at 450 nm (OD450) was measured. Ab titer was defined as the highest serum dilution ratio at which {OD450 > OD450 of negative control serum}.

[0093] To evaluate the cellular immune response to EV vaccine and PR8, splenocytes (2 × 10⁶) were used. 6 A sample (per well) was prepared and incubated in either complete RPMI-1640 medium containing 5 μg / ml of NA antigen (sinobiological) or, as a control group, complete RPMI-1640 medium without the antigen. After 24 hours of incubation, the culture supernatant was collected, and the IFN-γ concentration in the supernatant was measured by ELISA (R&D Systems) according to the manufacturer's protocol.

[0094] The results of Western blotting analysis of the isolated EV, recombinant hemagglutinin (rHA), and recombinant neuraminidase (rNA) are shown in Figure 1. The left panel of Figure 1 shows the results of Western blotting using an anti-HA antibody, and the right panel of Figure 1 shows the results of Western blotting using an anti-NA antibody. Figure 1 shows that the EV lane contains bands detected by both anti-HA and anti-NA antibodies. These bands are located in a higher molecular weight region than rHA and rNA and are thought to originate from the CD63-HA-NA fusion protein. Thus, it was suggested that the isolated EV expresses the CD63-HA-NA fusion protein.

[0095] The results of specific antibody production in mice immunized with EV vaccine or PR8 vaccine are shown in Figure 2. According to Figure 2, antibody titers increased approximately 80 times after administration of the EV vaccine compared to before administration. Figure 2 shows that in the PR8 vaccine, IgG antibody titers against HA and NA were biased towards HA, whereas in the EV vaccine, IgG antibody titers against HA and NA were balanced. This suggests that in the PR8 vaccine, multiple antigens compete (antigen competition), whereas in the EV vaccine, multiple antigens do not compete. It has been known that antibodies against NA are difficult to produce. These results also suggest that antibodies against NA can be favorably induced by using the EV vaccine.

[0096] The results of the cellular immune response in mice immunized with either the EV vaccine or the PR8 vaccine are shown in Figure 3. Figure 3 shows that, compared to the PR8 vaccine, the EV vaccine strongly induced interferon-γ from splenocytes in response to the antigen. This suggests that, compared to the PR8 vaccine, the EV vaccine induced a stronger antigen-specific T cell response.

[0097] Example 2 Experimental Method A DNA construct was designed to express influenza H1N1 hemagglutinin (with the transmembrane region from 529-549aa of the amino acid sequence of SEQ ID NO: 2 deleted) or neuraminidase (with the transmembrane region from 8-27aa of the amino acid sequence of SEQ ID NO: 3 deleted) fused to the C-terminus of the full-length CD63 (SEQ ID NO: 1). Codon optimization was performed using a vector builder, and the gene was synthesized. A common flexible linker, the GS linker {(GGGGS)3} (SEQ ID NO: 5), was inserted between each gene. The fusion gene was designed to be expressed downstream of the CMV promoter. The obtained fusion genes were called CD63-HA and CD63-NA, respectively, and the amino acid sequences of the fusion proteins encoded by these fusion genes were as shown in SEQ ID NOs: 6 and 7, respectively. Regarding CD63-HA-NA, it is presumed that the transmembrane region of NA was preserved, and therefore, the fusion protein was expressed in the EV in a manner in which HA was expressed inside the EV and NA was expressed outside the EV.

[0098] The amino acid sequence of CD63-HA (SEQ ID NO: 6) is MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMSGGGGSGGGGSGGGGSVVISAILALVVLTIISLIILISGGGGSGGGGSGGGGSMKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVS SFERFEIFPKESSWPNHNTNGVTAACCHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLR NIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQSFWMCSNGSLQCRICI Amino acid sequence of CD63-NA (SEQ ID NO: 7)MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMSGGGGSGGGGSGGGGSMNPNQKIITIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPYRALMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITETIKSWRKKILRTQESECACVNGSCFTIMTDGPSDGLASYKIFKIEKGKVTKSIELNAPNSHYEECSCYPDTGKVMCVCRDNWHGSNRPWVSFDQNLDYQIGYICSGVFGDNPRPEDGTGSCGPVYVDGANGVKGFSYRYGNGVWIGRTKSHSSRHGFEMIWDPNGWTETDSKFSVRQDVVAMTDWSGYSGSFVQHPELTGLDCMRPCFWVELIRGRPKEKTIWTSASSISFCGVNSDTVDWSWPDGAELPFSIDK

[0099] The anti-HA antibody production ability of extracellular viable cells (EVs) expressing the CD63-HA-NA fusion protein was compared with the anti-HA antibody production ability of a mixture of EVs expressing the CD63-HA-NA fusion protein and EVs expressing the CD63-NA fusion protein. As shown in Figure 4, the anti-HA antibody production ability of EVs expressing the CD63-HA-NA fusion protein was equal to or greater than that of the mixture of EVs. Since immune induction was confirmed for both HA and NA with CD63-HA-NA, it became clear that the antigen is sufficient for immune induction whether it is expressed inside or outside the EV.

[0100] Example 3 Experimental Method A DNA construct was designed to express full-length CD63 (SEQ ID NO: 1) fused to its C-terminus with (i) influenza H1N1 hemagglutinin (with the transmembrane region from 529-549aa of the amino acid sequence of SEQ ID NO: 2 deleted) and (ii) nucleoprotein (SEQ ID NO: 8), SARS-CoV-2 nucleocapid (SEQ ID NO: 9), or the RBD of SARS-CoV-2 spike protein (314-536aa of SEQ ID NO: 10). In addition, a DNA construct was designed to express full-length CD63 (SEQ ID NO: 1) fused to its C-terminus with (iii) SARS-CoV-2 nucleocapid (314-536aa of SEQ ID NO: 9) and (iv) SARS-CoV-2 spike protein (with the transmembrane region from 1214-1234aa of the amino acid sequence of SEQ ID NO: 10 deleted) or the above RBD. These genes were synthesized using a vector builder after codon optimization. A common flexible linker, the GS linker {(GGGGS)3} (SEQ ID NO: 5), was inserted between each gene. The fusion genes were designed to be expressed downstream of the CMV promoter. The resulting fusion genes were named CD63-HA-NP, CD63-HA-N, CD63-HA-RBD, and CD63-S-N, CD63-N-RBD, respectively, and the amino acid sequences of the fusion proteins encoded by these fusion genes were as shown in SEQ ID NOs: 11-15.

[0101]

[0102] EVs expressing each fusion protein were prepared as in Example 1. EV(HA / NP) expresses CD63-HA-NP, EV(HA / N) expresses CD63-HA-N, EV(N / S-RBD) expresses CD63-N-RBD, EV(S / N) expresses CD63-S-N, and EV(HA / S-RBD) is an extracellular vesicle that expresses CD63-HA-RBD. The obtained EVs were immunized into mice as in Example 1. Next, to evaluate the cellular immune response, splenocytes (2×10 ６ / well) were prepared and incubated in complete RPMI-1640 medium containing 5 μg / ml of HA antigen, NP antigen, S antigen, or N antigen (all from Sinobiological) or in complete RPMI-1640 medium without antigen as a control group. After 24 hours of incubation, the culture supernatant was collected, and the IL-13 concentration in the supernatant was measured by ELISA (R&D Systems) according to the manufacturer's protocol.

[0103] Results As shown in Figures 5 to 8, specific T cell immunity against each of the two different antigens carried by any of the EVs was induced. That is, by preparing and administering one type of EV expressing multiple antigens, an immune activation effect comparable to that of preparing and administering EVs expressing each antigen separately can be achieved. In addition, this strategy of presenting multiple different antigens on the EV surface as one fusion protein is considered beneficial in that the expression ratios of multiple different antigens tend to be constant.

[0104] Inactivated vaccines against pathogens have been developed and are in use. However, some pathogens have a high mutation rate, and the strains that circulate may differ depending on the year, season, or time of year. Because inactivated vaccines take time to manufacture, if they are produced after an outbreak occurs, the vaccine will not be able to contain the outbreak in time. Therefore, vaccines are manufactured for strains that are selected based on predictions of which strains will circulate, using data from around the world. This prediction can take one to two months. However, the prediction may be wrong, reducing the effectiveness of the vaccine. In addition, even if an inactivated vaccine is effective against a specific strain, it may not be sufficiently effective against different strains or newly emerging strains. Furthermore, inactivated vaccines require adjuvants to enhance the immune response, but side effects from adjuvants can be a problem. Moreover, inactivated vaccines have a weak ability to induce cellular immunity. While inactivated vaccines use chicken eggs, this requires preparing a large quantity of eggs, which demands considerable effort and time. Furthermore, it takes approximately two weeks for the eggs inoculated with the virus to mature, and another several weeks to a month is needed for virus replication and collection. Inactivated vaccines require several more weeks for the virus inactivation and purification processes.

[0105] In contrast, the EV vaccine of the present invention enables the induction of potent antigen-specific antibodies and cellular immunity with a small dose (for example, 1 / 100th the dose of an inactivated vaccine). The EV vaccine of the present invention does not require an adjuvant. Furthermore, the EV vaccine of the present invention can be manufactured simply by designing a fusion protein, expressing it in cells, and purifying the EV released from the cells (for example, in 1-2 weeks). Therefore, extremely rapid vaccine development and manufacturing are possible, solving at least one or all of the above-mentioned problems of inactivated vaccines, and it may be effective as a measure against epidemics and pandemics.

Claims

1. An immunogenic fusion protein comprising an extracellular vesicle marker protein or extracellular vesicle transition signal and multiple immunogenic peptides.

2. The fusion protein according to claim 1, wherein, if the immunogenic peptide is derived from a protein having a transmembrane domain, the transmembrane domain is deleted in the immunogenic peptide.

3. An extracellular vesicle expressing the fusion protein described in claim 1 or 2 on its surface.

4. An immunogenic composition comprising the extracellular vesicles described in claim 3.

5. The immunogenic composition according to claim 4, which does not contain an adjuvant.

6. The immunogenic composition according to claim 4 or 5, for use in inducing specific antibodies against multiple immunogenic peptides and / or specific cellular immunity.

7. A protein, extracellular vesicle, or immunogenic composition according to any one of claims 1 to 6, wherein the plurality of immunogenic peptides are derived from a virus.

8. A protein, extracellular vesicle, or immunogenic composition according to any one of claims 1 to 7, wherein the plurality of immunogenic peptides comprise the full-length or partial-length neuraminidase of the influenza virus.

9. A protein, extracellular vesicle, or immunogenic composition according to any one of claims 1 to 8, wherein the plurality of immunogenic peptides comprise the full length or a portion thereof of hemagglutinin of the influenza virus.

10. A protein, extracellular vesicle, or immunogenic composition according to any one of claims 1 to 9, wherein antigen-specific antibodies against each of several immunogenic peptides are induced in vivo after administration to an individual, and the induced antibody titer for each peptide is at least twice the pre-induced titer.

11. The immunogenic composition according to any one of claims 1 to 10, wherein antigen-specific antibodies against each of several immunogenic peptides are induced in vivo after administration to an individual, and the amount of each induced antibody falls within the range of the mean value ± 25% of the total antibody amount.

12. A nucleic acid encoding the fusion protein according to claim 1 or 2.

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