Immunogenic complex and method for producing same, and pharmaceutical composition

Abstract

This immunogenic complex comprises gelatin particles and an antigen supported on the gelatin particles, wherein the antigen is a denatured antigenic protein. The antigen may be a thermally denatured antigenic protein, may be encapsulated in the gelatin particles, and may be an allergen or a self-antigen.

Description

Immunogenic complex, method for producing the same, and pharmaceutical composition

[0001] The present invention relates to an immunogenic complex, a method for producing the same, and a pharmaceutical composition. This application claims priority based on Japanese Patent Application No. 2025-018233 filed in Japan on February 6, 2025, and incorporates its content herein by reference.

[0002] Conventionally, desensitization therapy in which an allergen such as a protein is administered to patients with allergic diseases is known. In desensitization therapy, the allergen is administered little by little to present the antigen to dendritic cells and induce regulatory T cells (Regulatory T cell: Treg cells), thereby establishing immune tolerance.

[0003] For example, Patent Document 1 discloses a technique for isolating and generating a protein contained in cedar pollen and using it as an allergen in desensitization therapy.

[0004] Republished WO00 / 068262

[0005] However, in the drugs conventionally used in desensitization therapy, since the allergen is used as it is, it may cause an allergic reaction as a side effect by binding to IgE antibodies in the patient's body.

[0006] The present invention has been made in view of such circumstances, and an object thereof is to provide a technique capable of improving safety as compared with the prior art in improving allergic diseases or autoimmune diseases by desensitization therapy.

[0007] In order to solve the above problems, one aspect of the present invention includes the following aspects. [1] An immunogenic complex comprising gelatin particles and an antigen supported on the gelatin particles, wherein the antigen is a denatured antigen protein.

[0008] [2] The immunogenic complex according to [1], wherein the antigen is a heat-denatured antigen protein.

[0009] [3] The immunogenic complex according to [1] or [2], wherein at least a part of the gelatin constituting the gelatin particles is arranged to fit the antigen.

[0010] [4] An immunogenic complex according to any one of [1] to [3], wherein the antigen is encapsulated within the gelatin particles.

[0011] [5] The immunogenic complex according to any one of [1] to [4], wherein the antigen is an allergen or an autoantigen.

[0012] [6] The immunogenic complex according to any one of [1] to [5], wherein the antigen is one or more antigen proteins selected from the group consisting of antigen proteins contained in pollen, antigen proteins contained in food, and antigen proteins contained in mites.

[0013] A pharmaceutical composition comprising an immunogenic complex described in any one of [7] [1] to [6].

[0014] [8] The pharmaceutical composition according to [7] for treating or preventing allergies or autoimmune diseases.

[0015] [9] A method for producing an immunogenic complex, comprising: step 1 of forming gelatin particles; step 2 of mixing the gelatin particles formed in step 1 with an antigen protein to form a complex; and step 3 of heating the complex to thermally denature the antigen protein.

[0016]

[10] The method for producing an immunogenic complex according to [9], wherein in step 3, the complex is heated to 25°C or higher.

[0017] According to the present invention, it is possible to provide a technology that can improve safety compared to conventional methods when treating allergic diseases or autoimmune diseases through desensitization therapy.

[0018] Figure 1 is a schematic diagram showing a preferred example of a method for producing an immunogenic complex using the core cellvation method. Figure 2 is a graph showing the particle size distribution of gelatin particles encapsulating ovalbumin. Figure 3 is a graph showing the particle size distribution of gelatin particles encapsulating egg white or skim milk. Figure 4 is a graph showing the particle size distribution of gelatin particles encapsulating Cry j1 and Cry j2. Figure 5 is a scanning electron microscope (SEM) image of gelatin particles encapsulating ovalbumin. Figure 6 is an electrophoresis image showing the result of subjecting gelatin particles encapsulating egg white to SDS-PAGE. Figure 7 is an electrophoresis image showing the result of subjecting gelatin particles encapsulating skim milk to SDS-PAGE. Figure 8 is an electrophoresis image showing the result of subjecting gelatin particles encapsulating Cry j1 and Cry j2 to SDS-PAGE. Figure 9 is a graph showing the relationship between the degradation amount and time when gelatin particles not containing an antigen protein and particles obtained by heating gelatin particles not containing an antigen protein are each treated with collagenase. Figure 10 is a graph showing the relationship between the ovalbumin release amount and time when gelatin particles encapsulating ovalbumin and gelatin particles encapsulating denatured ovalbumin are each treated with collagenase. Figure 11 is an image of a fluorescence microscope showing the uptake of ovalbumin, G ＯＶＡ , or hG ＯＶＡ by bone marrow dendritic cells. Figure 12 is a graph showing the result of measuring the uptake amount of ovalbumin, G ＯＶＡ , or hG ＯＶＡ by bone marrow dendritic cells by fluorescence intensity using flow cytometry. Figure 13 is a graph showing the production amount of IL-2 from OT-II CD4 ＋ T cells co-cultured with bone marrow dendritic cells treated with PBS, ovalbumin, G ＯＶＡ , or hG ＯＶＡ for 4 hours or 24 hours. Figure 14 is a graph showing the change in rectal temperature when ovalbumin, G ＯＶＡ , or hG ＯＶＡ is intraperitoneally administered to an allergic model mouse. Figure 15 is a graph showing the result of subjecting gelatin particles encapsulating ovalbumin, G ＯＶＡ , or hG ＯＶＡThis graph shows the results of evaluating the binding affinity of obalbumin-specific antibodies to G. Figure 16 shows BALB / c mice bound to mouse serum albumin, obalbumin, and G. ＯＶＡ , or hG ＯＶＡ This graph shows the results of measuring the amount of anti-ovalbumin IgG antibody bound to a sample of serum collected on day 28 after subcutaneous administration of the drug once a week for four weeks, using the ELISA method. Figure 17 shows the results of measuring the amount of mouse serum albumin, ovalbumin, and G2 in BALB / c mice. ＯＶＡ , or hG ＯＶＡ Figure 18 is an image showing the results of evaluating the retention of gelatin particles using Cy7 labeling. Figure 19 is a graph showing the changes in fluorescence intensity when evaluating the retention of gelatin particles using Cy7 labeling. Figure 20 is a graph showing the amounts of anti-ovalbumin IgE antibody, anti-ovalbumin IgG2a antibody, and anti-ovalbumin IgG1 antibody in serum after intranasal administration of ovalbumin following immune tolerance induction in a mouse allergic asthma model. Figure 21 is a graph showing macrophages, neutrophils, eosinophils, and their total cell count in BALF after intranasal administration of ovalbumin following immune tolerance induction in a mouse allergic asthma model. Figure 22 is a graph showing the amount of cytokines in the BALF supernatant after intranasal administration of ovalbumin following induction of immune tolerance in a mouse allergic asthma model.

[0019] In this specification, the term "comprise" means that it may include components other than the component being discussed. The term "consist of" means that it does not include components other than the component being discussed. The term "consistently of" means that it does not include components other than the component being discussed in a manner that performs a special function (such as a manner that completely eliminates the effect of the invention). In this specification, when "comprise" is used, it includes the "consist of" and "consistently of" manners.

[0020] In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0021] The immunogenic complex according to preferred embodiments of the present invention and its manufacturing method will be described in detail below, with reference to drawings as appropriate. However, the present invention is not limited to the following embodiments.

[0022] [Immunogenic complex] An immunogenic complex according to a preferred embodiment of the present invention comprises gelatin particles and an antigen supported on the gelatin particles, wherein the antigen is a denatured antigenic protein.

[0023] In the immunogenic complex of this embodiment, gelatin particles function as a delivery means (carrier) for delivering antigens to cells. Because gelatin has low immunogenicity, side effects from the delivery means can be minimized. Note that the immunogenic complex of this embodiment, and the pharmaceutical composition containing said immunogenic complex, should not be used in patients with gelatin allergies.

[0024] In the immunogenic complex, the antigen protein is supported on porous gelatin particles. Therefore, the immunogenic complex has sustained release properties for the antigen protein. In this embodiment, the antigen is a denatured antigen protein, and its three-dimensional structure has changed from the original antigen protein before denaturation. Therefore, even after the antigen protein is released from the gelatin, basophils and mast cells prevent recognition of the antigen protein by IgE antibodies on their surface (in other words, the binding of IgE antibodies), and as a result, the IgE-driven Th2 reaction, i.e., the allergic reaction which is a side effect of the antigen protein, can be suppressed.

[0025] Furthermore, even if the antigen protein denatures from its expressed state and its three-dimensional structure changes, it does not affect the T cell epitope, thus maintaining the therapeutic effect of desensitization (immune tolerance). As will be detailed in the examples below, it is strongly suggested that using an immunogenic complex containing a denatured antigen protein may enhance the therapeutic effect of desensitization compared to using an undenatured antigen protein.

[0026] In addition, as will be detailed in the examples later, using denatured antigen proteins improves the uptake efficiency by dendritic cells compared to cases where undenatured antigen proteins are supported on gelatin particles or when naked antigen proteins are administered. This increases the antigen presentation efficiency, enhances the stimulating ability of regulatory T cells (Treg), and consequently increases IL-2 production, thereby improving the therapeutic effect on allergic reactions and autoimmune diseases.

[0027] The average particle size of the immunogenic complex (in other words, the average particle size of the gelatin particles carrying the antigen protein) is not particularly limited, but may be, for example, 1 nm to 5 μm, 100 nm to 5 μm, 100 to 1000 nm, 100 to 500 nm, 100 to 300 nm, 150 to 250 nm, or 170 to 230 nm. In particular, a size of around 200 nm is preferred as it is easily taken up by dendritic cells through endocytosis. The average particle size of the particles can be measured by a dynamic light scattering spectrophotometer. The particle size can be adjusted by the gelatin concentration and stirring speed during particle preparation.

[0028] The polydispersity (PDI) of the particle size of the immunogenic complex is not particularly limited, but may be, for example, 0.09 to 0.16 or 0.11 to 0.15. The polydispersity of the particle size of gelatin particles can be measured by a dynamic light scattering spectrophotometer.

[0029] Methods for atomizing gelatin include, for example, a method of discharging droplets of liquid containing molten gelatin into the atmosphere of a heated tube or drying chamber and drying them (air drop method), a method of discharging droplets of gelatin solution into a hydrophobic solvent and dispersing them (liquid drop method), a method of emulsifying a gelatin solution and dispersing fine droplets containing gelatin (liquid dispersion method), and a coacervation method by phase separation. Among these, the coacervation method described in detail in the embodiment of the [method for producing immunogenic complexes] can be suitably used.

[0030] The ratio (mass ratio) of gelatin to antigen in the immunogenic complex is not particularly limited, but for example, gelatin:antigen may be 3:4 to 50:1, 1:1 to 10:1, or 1:1 to 3:1.

[0031] <Gelatin Particles> The gelatin that makes up the gelatin particles is not particularly limited as long as it is gelatin derived from animal collagen protein. Gelatin particles can carry and encapsulate antigens of any type.

[0032] The average molecular weight of gelatin is not particularly limited, but from the viewpoint of facilitating the formation of particles that satisfy the above-mentioned average particle diameter conditions, it may be, for example, 1,000 to 120,000, 5,000 to 120,000, 50,000 to 120,000, or 80,000 to 110,000. In this specification, the average molecular weight is the weight-average molecular weight measured in accordance with the PAGI (Photographic and Gelatin Industries) method, 10th edition (2006).

[0033] When analyzed with an amino acid analyzer, gelatin preferably contains at least 300 glycine residues per 1000 amino acid residues on average, and also contains alanine and proline. This composition of gelatin facilitates the uptake of immunogenic complexes into cells.

[0034] The isoelectric point of gelatin is not particularly limited, but it may be, for example, pH 7 to 9, or 4.5 to 5.5.

[0035] It is preferable that the gelatin is arranged to fit the antigen (in other words, it fits the antigen). When the complex of gelatin particles and antigen is heated and denatured, as will be explained later in the embodiment of [Method for Producing Immunogenic Complexes], the gelatin constituting the gelatin particles refolds into a triple helix structure, at least partially, as it cools after heating. At this time, the gelatin refolds (rearranges) to fit the surface pattern (e.g., hydrophobicity, charge and / or shape) of the antigen (e.g., antigen protein) supported or encapsulated on the gelatin particles.

[0036] <Antigen> In this specification, "antigen" means any substance that can induce an immune response in the organism to which it is administered. An antigen may be any substance that, upon contact with immune cells, induces the proliferation, activation, and / or maturation of immune cells, or any substance that induces cytokine production and / or antibody production from immune cells.

[0037] The antigen is preferably an allergen or an autoantigen. An "autoantigen" is a substance that an individual possesses that can act as an antigen. An "allergen" is a substance that can cause allergies in an individual. Allergens are substances that are recognized as foreign by the individual's immune system and trigger an immune response, mainly the production of immunoglobulin E (IgE). Examples of allergens include pollen allergens, allergens derived from arthropods such as insects, and food allergens. The gelatin particles of the immunogenic complex may carry, for example, antigen proteins contained in these allergens.

[0038] Pollen allergens include, for example, those found in cedar pollen, cypress pollen, birch pollen, rice pollen, ragweed pollen, mugwort pollen, and Japanese hop pollen.

[0039] Examples of antigenic proteins contained in cedar pollen include Cry j1 and Cry j2.

[0040] Examples of arthropod-derived allergens include those found in mites, moths, or midges.

[0041] Food-derived allergens include, for example, allergens found in shrimp, crab, walnuts, wheat, buckwheat, eggs, milk (e.g., skim milk), peanuts, almonds, abalone, squid, salmon roe, oranges, cashews, kiwifruit, beef, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, and apples.

[0042] The antigen may be one or more antigen proteins selected from the group consisting of antigen proteins contained in pollen, antigen proteins contained in food, and antigen proteins contained in dust mites. Examples of antigen proteins contained in pollen include those contained in the pollen allergens mentioned above. Examples of antigen proteins contained in food include those contained in the food-derived allergens mentioned above, and may also be proteins contained in egg white such as ovalbumin, or proteins contained in skim milk. For details, please refer to the examples described below.

[0043] The causes of antigen protein denaturation are not particularly limited, but examples include thermal denaturation and denaturation due to pH, with thermal denaturation being particularly preferred. Furthermore, the antigen protein may also be a glycoprotein.

[0044] An immunogenic complex may contain only one type of antigen protein, or it may contain multiple types of antigen proteins. As will be explained later, for example, by encapsulating multiple types of antigen proteins in gelatin and then denaturing them by heat, gelatin particles carrying denatured multiple types of antigen proteins can be easily produced.

[0045] In immunogenic complexes, the antigen protein may be encapsulated within gelatin particles.

[0046] <Optional Components> The immunogenic complex of this embodiment may contain optional components in addition to gelatin and antigen protein. Examples of optional components include matrix proteins and immunomodulators. The optional components may be encapsulated within the gelatin particles.

[0047] The amount of optional components other than gelatin and antigen protein is not particularly limited as long as they do not inhibit the desensitizing effect of the antigen protein. However, when the total mass of gelatin and antigen protein is 100 parts by mass, the amount of optional components may be, for example, 150 parts by mass or less, 100 parts by mass or less, 50 parts by mass or less, 30 parts by mass or less, 10 parts by mass or less, or 5 parts by mass or less.

[0048] (Matrix Proteins) "Matrix proteins" refer to proteins other than the target antigen that, together with the antigen and gelatin, form an immunogenic complex. Matrix proteins can be used, for example, to form an immunogenic complex with a small amount of antigen when the antigen is expensive.

[0049] The type of matrix protein is not particularly limited, but to prevent the induction of unexpected immune responses, it is preferable to use a matrix protein derived from the same organism as the target of the immunogenic complex or the pharmaceutical composition described later. For example, if the target of the immunogenic complex or the pharmaceutical composition described later is human, it is preferable to use a human protein as the matrix protein. A specific example of a matrix protein is albumin. When the target is human, human serum albumin can be suitably used as the matrix protein. A single matrix protein may be used, or two or more may be used in combination.

[0050] The ratio of antigen to the total mass (100% by mass) of antigen and matrix protein can be, for example, 1 to 90% by mass, and may also be 3 to 80% by mass, 5 to 70% by mass, 10 to 60% by mass, 15 to 50% by mass, 15 to 40% by mass, or 20 to 40% by mass.

[0051] If the gelatin particles of the immunogenic complex contain a matrix protein, it is preferable that the matrix protein is encapsulated within the gelatin.

[0052] (Immunomodulators) An "immunomodulator" refers to a drug that modulates immune function. An immunomodulator may also be an immunosuppressant that suppresses immune function. Examples of immunomodulators include retinoic acid receptor agonists, retinoid X receptor agonists, vitamin D receptor agonists, aromatic hydrocarbon receptor agonists, histone deacetylase inhibitors, and mTOR inhibitors. An "agonist" is a compound that binds to a receptor and activates an intracellular signaling pathway.

[0053] Examples of retinoic acid receptor agonists include all-trans-retinoic acid. Examples of retinoid X receptor agonists include bexarotene. Examples of vitamin D receptor agonists include vitamin D3. Examples of aromatic hydrocarbon receptor agonists include 2-(1'H-indole-3'-carbonyl)-thiazole-4-carbonoxylic acid methyl ester. Examples of histone deacetylase inhibitors include suberoylanilide hydroxamic acid (SAHA). Examples of mTOR inhibitors include rapamycin. Immunomodulators may be used individually or in combination of two or more.

[0054] When particulate immunogenic complexes contain immunomodulators, the immunomodulators are thought to be encapsulated within the gelatin by intermolecular forces, etc.

[0055] According to this embodiment, since the antigen protein is supported on gelatin particles and is also denatured, the occurrence of allergic reactions, which are a side effect, can be suppressed, thus improving safety compared to immunogenic substances used in conventional desensitization therapy.

[0056] [Method for Producing Immunogenic Complexes] A preferred embodiment of the present invention provides a method for producing immunogenic complexes, comprising: step 1 forming gelatin particles; step 2 mixing the gelatin particles formed in step 1 with an antigen protein to form a complex; and step 3 heating the complex to thermally denature the antigen protein. According to this embodiment, the immunogenic complexes detailed in the above-described embodiment of [Immunogenic Complexes] can be produced.

[0057] In the manufacturing method of this embodiment, gelatin particles are formed in step 1 using the coacervation method, as will be explained below. However, in step 1, gelatin particles may also be formed by other methods, such as the air drop method, the liquid drop method, or the liquid dispersion method.

[0058] Figure 1 is a schematic diagram showing a preferred example of a method for producing immunogenic complexes using the coacervation method. In each figure, unless otherwise specified, "G" refers to gelatin particles that do not contain antigen proteins. 抗原 " " refers to gelatin particles containing undenatured antigen proteins, while "hG" refers to gelatin particles that do not contain antigen proteins and have been heated. 抗原 " refers to gelatin particles containing an antigen protein that have been heated. In other words, the subscript indicates the type of antigen protein supported on the gelatin, and "h" indicates that it has been heated. Figure 1 shows compounds used in the production of immunogenic complexes, their amounts, concentrations, and processing methods, but these are preferred examples and the present invention is not limited to these.

[0059] In the manufacturing method of this embodiment, first, the gelatin is atomized by mixing the gelatin solution with an organic solvent and separating the phases (step 1). The gelatin solution is a solution obtained by dissolving gelatin in water or a buffer solution.

[0060] Gelatin can be obtained from animal collagen proteins (for example, collagen proteins derived from bovine bones, cowhide, pigskin, pig tendons, fish scales, and fish meat) by known methods, such as acid treatment or alkali treatment followed by extraction with hot water, but commercially available gelatin may also be used.

[0061] Examples of buffer solutions include, but are not limited to, phosphate buffer, phosphate-buffered saline, acetate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0062] The type of organic solvent is not particularly limited as long as it can granulate gelatin through phase separation with water, but examples include alcohols such as acetone, ethanol, 1-propanol, 2-propanol, and 1-butanol. In this case, the higher the gelatin concentration in the gelatin aqueous solution, the larger the average particle size of the gelatin particles tends to be.

[0063] The gelatin concentration in the gelatin aqueous solution before mixing with the organic solvent is not particularly limited, but may be, for example, 5 to 100 mg / mL, 5 to 50 mg / mL, 5 to 20 mg / mL, or 8 to 15 mg / mL.

[0064] The amount of organic solvent mixed in is not particularly limited as long as it can granulate the gelatin, but for example, it may be 2 to 50 times the amount (by volume) of the gelatin aqueous solution, or it may be 2 to 10 times the amount.

[0065] It is preferable to obtain a dispersion of gelatin particles by phase separation of gelatin, and then crosslink the gelatin particles. Crosslinking prevents the dissolution of gelatin when immersed in water after drying, and also suppresses the dissolution of gelatin when the antigen protein is later denatured by heat. Crosslinking may be performed using a crosslinking agent, or it may be self-crosslinking without the use of a crosslinking agent.

[0066] The crosslinking agent may be a compound having multiple functional groups that form chemical bonds with, for example, hydroxyl groups, carboxyl groups, amino groups, thiol groups, and imidazole groups. Examples of such crosslinking agents include glutaraldehyde, paraformaldehyde, water-soluble carbodiimides including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate (CMC), compounds having two or more epoxy groups including ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, and glycerol polyglycidyl ether, and propylene oxide. Among these, glutaraldehyde and EDC are preferred, and glutaraldehyde is more preferred, from the viewpoint of further increasing reactivity. Alternatively, glutamine and lysine may be crosslinked with transglutaminase.

[0067] Examples of self-crosslinking include heating or crosslinking by irradiation with electron beams or ultraviolet light.

[0068] If the aldehyde is used to form particles in step 1, it is preferable to block (capp) the aldehyde groups by mixing with glycine after crosslinking the gelatin. Then, stirring is continued to vaporize the organic solvent.

[0069] Next, the gelatin particles are washed with pure water and dried. Drying methods include, but are not limited to, freeze-drying. After drying the gelatin particles, the gelatin particles are mixed with the antigen protein and incubated to support and encapsulate the antigen protein in the gelatin particles (step 2). In the lower part of Figure 1, labeled "G", an example of an antigen protein, ovalbumin (OVA), is shown to be supported on the gelatin.

[0070] When using optional components such as immunomodulators or matrix proteins, they may be mixed with the antigen protein and gelatin particles in step 2, or they may be dissolved together with the gelatin and formed into particles in step 1.

[0071] Finally, the complex of gelatin particles and antigen protein obtained in this way is heated to denature it (step 3). This yields an immunogenic complex. By heating the complex of gelatin particles and antigen protein, the binding of IgE antibodies can be suppressed as described above, and the antigen protein can be associated with gelatin. In the lower part of Figure 1, "hG" shows how ovalbumin (OVA), an example of an antigen protein, is supported on gelatin in a denatured state.

[0072] The heating temperature in step 3 is not particularly limited as long as it can denature the antigen protein, but may be, for example, 25°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, or 80°C or higher. The upper limit of the heating temperature in step 3 is not particularly limited, but may be, for example, 95°C or lower, 90°C or lower, or 85°C or lower. The upper and lower limits of the heating temperature mentioned above can be combined arbitrarily.

[0073] The heating time in step 3 can be set appropriately according to the type of antigen protein and the heating temperature conditions described above, but for example, it can be 1 minute or more, preferably 5 minutes or more, it may be 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, or 60 minutes or more. There is no particular upper limit to the heating time, but for example it can be 100 minutes or less, or 80 minutes or less. The upper and lower limits of the heating time can be combined arbitrarily.

[0074] After heating in step 3, as the temperature decreases, the gelatin refolds into a triple helix structure, at least partially. At this time, the gelatin refolds (rearranges) to fit the surface pattern (e.g., hydrophobicity, charge, and / or shape) of the antigen protein encapsulated within the gelatin particles.

[0075] According to the manufacturing method of this embodiment, the antigen protein can be firmly supported on the gelatin particles by heating the complex of gelatin particles and antigen protein.

[0076] Furthermore, when multiple types of antigen proteins are heat-denatured before being supported or encapsulated in gelatin particles, the denaturation temperature differs for each type of antigen protein. As a result, some antigen proteins precipitate without being encapsulated in the gelatin particles, making it difficult to support or encapsulate multiple types at once. However, in the manufacturing method of this embodiment, the antigen proteins are heat-denatured after being supported or encapsulated in the gelatin particles, making it easy to support and encapsulate multiple types of antigen proteins in the gelatin particles.

[0077] [Pharmaceutical Composition] A pharmaceutical composition according to a preferred embodiment of the present invention contains an immunogenic complex. This immunogenic complex is the same as the immunogenic complex described in detail in the [Immunogenic Complex] embodiment. The pharmaceutical composition contains the immunogenic complex as an active ingredient. The pharmaceutical composition of this embodiment may be a pharmaceutical product or a quasi-drug.

[0078] The pharmaceutical composition of this embodiment may contain optional components in addition to the immunogenic complex detailed in the [Immunogenic Complex] embodiment. Examples of optional components include pharmaceutically acceptable carriers.

[0079] A "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the target of administration. "Substantial toxicity" means that the ingredient does not exhibit toxicity to the target of administration at the doses normally used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the immunogenicity of the immunogenic complex and does not exhibit substantial toxicity to the target of administration. A pharmaceutically acceptable carrier includes all known pharmaceutically acceptable ingredients that are typically considered inactive ingredients. A pharmaceutically acceptable carrier is not particularly limited, but examples include solvents, diluents, vehicles, excipients, flow promoters, binders, granulators, dispersants, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. A pharmaceutically acceptable carrier may be used alone or in combination of two or more types.

[0080] The pharmaceutical composition may include, for example, a buffer as a solvent. Examples of buffers include the types of buffers described above.

[0081] The pharmaceutical composition may contain, in addition to the above-mentioned components, other components besides the carrier. These other components are not particularly limited and can be those commonly used in the pharmaceutical field without any restrictions. Examples of other components include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives such as antioxidants, chelating agents, flavoring and odor-correcting agents, sweeteners, thickeners, buffering agents, and coloring agents. These other components may be used individually or in combination of two or more.

[0082] The pharmaceutical composition may contain active ingredients other than immunogenic complexes. Examples of active ingredients include, but are not limited to, antibiotics, anti-inflammatory agents, antipyretics, and analgesics.

[0083] The pharmaceutical composition may contain an adjuvant. Examples of adjuvants include, but are not limited to, aluminum hydroxide, calcium phosphate, monophosphoryl lipid A, and chitosan. The adjuvant may be used alone or in combination of two or more types.

[0084] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions, etc. Examples of parenteral preparations include injections, suppositories, nasal sprays, enteral preparations, ointments, creams, topical solutions, inhalants, etc. Pharmaceutical compositions of these dosage forms can be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).

[0085] The administration route of the pharmaceutical composition of this embodiment is not particularly limited and may be oral or parenteral. Parenteral administration includes sublingual administration, intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration, with sublingual and subcutaneous administration being preferred among parenteral administrations. In particular, when the immunogenic complex contains antigenic proteins contained in pollen and / or antigenic proteins contained in mites, the pharmaceutical composition is preferably a subcutaneous, sublingual, or oral preparation. Furthermore, when the immunogenic complex contains antigenic proteins contained in food, the pharmaceutical composition is preferably an oral preparation.

[0086] The pharmaceutical composition can administer a therapeutically effective amount of the immunogenic complex. "Therapeutic effective amount" refers to the amount of drug effective for treating or preventing the target disease. For example, the therapeutically effective amount of the immunogenic complex may be an amount effective in inducing tolerant dendritic cells. The therapeutically effective amount should be appropriately determined based on the type of antigen protein, the patient's symptoms, weight, age, and sex, as well as the dosage form and method of administration of the pharmaceutical composition. For example, the pharmaceutical composition may contain 0.001 to 1000 mg of the antigen protein per kg of body weight of the recipient as a single dose.

[0087] The pharmaceutical composition may contain, for example, an immunogenic complex in a proportion of 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

[0088] The pharmaceutical composition may be administered as a single dose or as a repeated dose. In the case of repeated administration, the administration interval should be appropriately determined based on the type of antigen protein, the patient's symptoms, weight, age, and sex, as well as the dosage form and method of administration of the pharmaceutical composition. For example, the administration interval may be every few hours, two to three times a day, once a day, once every two to three days, once a week, once a month, once every few months, etc.

[0089] The target recipients of the pharmaceutical composition are not particularly limited, but may be, for example, humans or other mammals. Examples of other mammals include primates (monkeys, gorillas, chimpanzees, marmosets, etc.), rodents (mice, rats, guinea pigs, hamsters, etc.), rabbits, hedgehogs, dogs, cats, horses, cattle, pigs, goats, sheep, etc.

[0090] The pharmaceutical composition can be used to treat or prevent allergic or autoimmune diseases.

[0091] Examples of allergies covered include, but are not limited to, hay fever, food allergies, dust mite allergies, atopic dermatitis, allergic asthma, allergic rhinitis, and allergic urticaria.

[0092] Autoimmune diseases include, but are not limited to, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, Sjögren's syndrome, and vasculitis.

[0093] [Other Embodiments] In another embodiment, the present invention provides an immunogenic complex for the treatment or prevention of allergies or autoimmune diseases. The immunogenic complex is similar to that described in the Embodiment of [Immunogenic Complex].

[0094] In another embodiment, the present invention provides the use of an immunogenic complex for the manufacture of a pharmaceutical composition for the treatment or prevention of allergies or autoimmune diseases. The immunogenic complex is similar to that detailed in the [Immunogenic Complex] embodiment.

[0095] In another embodiment, the present invention provides the use of an immunogenic complex for the treatment or prevention of allergies or autoimmune diseases. The immunogenic complex is similar to those detailed in the [Immunogenic Complex] embodiment.

[0096] In another embodiment, the present invention provides a method for treating an allergic disease, comprising administering an effective amount of an immunogenic complex to a patient in need of treatment. The immunogenic complex is the same as that detailed in the embodiment of [Immunogenic Complex].

[0097] The present invention is not limited to the embodiments described above, and it goes without saying that various modifications are possible within the scope of the invention as described in the claims, and these modifications are also included within the scope of the present invention.

[0098] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0099] [Materials and Methods] <Preparation of immunogenic complex using coacervation method> First, gelatin powder was added to ultrapure water at 40°C and stirred for 2 hours to dissolve, preparing a 12.5 mg / mL gelatin aqueous solution. The gelatin used (isoelectric point = pH 9.0, weight-average molecular weight = 99000 Nitta Gelatin) was produced by acid treatment of pigskin. Next, 5 mL of 1 M (mol / L) acetone was added to 1.25 mL of the gelatin aqueous solution and stirred at 40°C to atomize the gelatin by phase separation (Step 1).

[0100] Subsequently, 20 μL of an 8.28 mM 25 wt% glutaraldehyde aqueous solution (Fujifilm Wako Pure Chemical Industries) was added, and the mixture was stirred at 40°C for 6 hours to chemically crosslink the gelatin. Then, 2 mL of a 0.5 M glycine aqueous solution (Fujifilm Wako Pure Chemical Industries) was added to block the unreacted aldehyde groups of glutaraldehyde.

[0101] Next, the solution was stirred overnight at 40°C to vaporize any remaining acetone. To recover the gelatin particles, the dispersion was centrifuged at 25°C for 10 minutes at 16,000 × g (or 20 minutes at 100,000 × g) and resuspended in ultrapure water. The centrifugation and resuspension process was repeated three times to purify the gelatin particles.

[0102] Subsequently, 10 μL of egg white ovalbumin (40 mg / mL, Grade V, Sigma-Aldrich) was added to 1 mg of lyophilized gelatin particles, and the mixture was incubated overnight at 37°C to support and encapsulate ovalbumin as an example of an antigen protein on the gelatin particles (Step 2). After incubation, the resulting gelatin particle-ovalbumin complex was dispersed in PBS solution and heated at 85°C for 1 hour (Step 3) to prepare denatured ovalbumin-containing gelatin particles (immunogenic complexes) (hG ＯＶＡ ).

[0103] In addition, instead of ovalbumin, a system was developed in which Cry j1 and Cry j2, antigen proteins derived from cedar pollen, were mixed and supported and encapsulated in gelatin particles (hG Ｃｒｙ　Ｊ ) and a system in which egg white is supported and encapsulated in gelatin particles instead of ovalbumin (hG ＥＷ ) and a system in which skim milk (Fujifilm Wako Pure Chemical Industries) is supported and encapsulated in gelatin particles instead of ovalbumin (hG Ｍｉｌｋ A separate system was also established in which each antigen protein was not denatured (in other words, step 3 was not performed). In each system, the amount of antigen protein etc. encapsulated in the gelatin particles was the same as that of ovalbumin described above. For Cry j1 and Cry j2, 10 μL of Cry j1 and Cry j2 (0.5 mg / mL, biodynamics) were mixed with 1 mg of lyophilized gelatin particles and then supported and encapsulated in the gelatin particles.

[0104] <Preparation of Bone Marrow Dendritic Cells (BMDCs)> BMDCs were prepared from bone marrow cells of C57BL / 6J mice. First, bone marrow cells from the femur and tibia were cultured in Advanced RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 2 mM GlutaMAX, 10 mM HEPES buffer, penicillin (100 units / mL), streptomycin (100 μg / mL), 10% thermally inactivated FBS, 50 μM β-mercaptoethanol, 20 ng / mL recombinant mouse GM-CSF, and 10 ng / mL recombinant mouse IL-4. After 3 days, additional medium was gently added to the cells. On day 5, half of the medium was replaced with fresh medium. On day 7, cells that were loosely adhered to the non-adherent cells were collected. Subsequently, bone marrow dendritic cells were separated by magnetic sorting. To induce BMDC maturation, 100 ng / mL of LPS was added to the BMDC culture medium.

[0105] <Evaluation of gelatin particle uptake by bone marrow dendritic cells> Isolated bone marrow dendritic cells (5 × 10) were observed using a fluorescence microscope. ４ Cells (per well) were treated with gelatin particles from each system containing 10 μg / mL rhodamine-labeled OVA for 4 or 24 hours. Bone marrow dendritic cells were washed with PBS to remove excess gelatin particles and fixed with 4% PFA. The nuclei of the bone marrow dendritic cells were stained with 10% Hoechst. Subsequently, intracellular uptake of gelatin particles by bone marrow dendritic cells was observed using a fluorescence microscope (BZ-X800, KEYENCE).

[0106] The amount of gelatin particles taken up intracellularly by bone marrow dendritic cells was determined by measuring the fluorescence intensity of rhodamine-labeled ovalbumin using flow cytometry (CyroFLEX-S, Beckman Coulter).

[0107] <Evaluation of IL-2 production by T cells that have received antigen presentation from bone marrow dendritic cells> Isolated bone marrow dendritic cells (5 × 10) ４ Cells (per well) were treated with PBS, ovalbumin, or gelatin particles of each system (10 μg / mL) for 4 hours. After washing twice with PBS, the isolated OT-II CD4 cells were collected. ＋ T cells (5 x 10 ５The cells were co-cultured with the culture supernatant for 24 hours. The amount of IL-2 in the resulting culture supernatant was measured using the IL-2 Mouse ELISA Kit.

[0108] <Evaluation of Anaphylactic Reaction> Female BALB / c mice (6-8 weeks old, n=6) were administered 10 μg of OVA and 1 mg of Imject Alum intraperitoneally on days 0 and 14 to create an allergy model mouse. Subsequently, on day 28, ovalbumin and "G," a gelatin particle containing undenatured ovalbumin, were administered. ＯＶＡ " or hG ＯＶＡ OVA (200 μg) was administered intraperitoneally, and the changes in rectal temperature were recorded. Rectal temperature was measured every 10 minutes using a PTM1 Portable Temperature Monitor (Physitemp) and a RET-3 thermocouple temperature sensor (shaft diameter 19 × 0.7 mm).

[0109] <Evaluation of binding affinity between gelatin particles and ovalbumin-specific antibody> BALB / c mice were intraperitoneally administered 10 μg of ovalbumin and 1 mg of Imject Alum once a week for 3 weeks. From day 28 to day 30, ovalbumin was administered intranasally to the mice. Seven days after the last ovalbumin administration, serum samples were collected, and the binding affinity between gelatin particles and ovalbumin-specific antibody was evaluated by ELISA. n=3.

[0110] In detail, 100 μL of ovalbumin and G in a medium-bound 96-well plate (BioLegend). ＯＶＡ , or hG ＯＶＡOvalbumin (200 μg / mL) was coated overnight in ELISA coating buffer (BioLegend) at 4°C. Nonspecific binding sites were blocked for 1 hour with ELISA assay diluent (200 μL / well, BioLegend). Serial dilutions of serum samples containing ovalbumin-specific antibody were added to the plate (100 μL / well) and incubated overnight at 4°C. Next, 100 μL / well of biotin anti-mouse antibody was added and incubated at room temperature for 1 hour, followed by incubation with Streptabidin-HRP for 30 minutes. Then, TMB solution (100 μL / well) was added and incubated for 15 minutes. Finally, stop solution was added to each well to terminate the reaction (100 μL / well). Absorbance was measured at 450 nm and 570 nm using a microplate reader (Infinite 200 PRO, Tecan).

[0111] <Evaluation of gelatin particle retention> To track gelatin particles at the injection site, Cy7 (Lumiprobe Limited) labeled ovalbumin was used. ＯＶＡ and hG ＯＶＡ It was incorporated into the BALB / c mouse with Ovalbumin and G ＯＶＡ or hG ＯＶＡ The retention of gelatin particles was evaluated by subcutaneously injecting 200 μg of ovalbumin and measuring the fluorescence intensity of Cy7-labeled OVA over 7 days using the IVIS Lumina Imaging System (Xenogen).

[0112] <Evaluation of therapeutic effects in a mouse model of allergic asthma> BALB / c mice were sensitized by intraperitoneal administration of 10 μg of ovalbumin emulsified with 1 mg of Imject Alum on days 0 and 7. The group administered PBS at this time was designated as the vehicle group. Subsequently, on days 14, 16, 18, 21, 23 and 25, PBS (vehicle and control), ovalbumin, and G were administered. ＯＶＡ , or hG ＯＶＡWe attempted to induce immune tolerance by subcutaneously injecting mice with ovalbumin (20 μg). The vehicle group, which received PBS, was also administered PBS at the same number of days as above. Subsequently, on days 32, 33, 34, and 35, mice were intranasally administered 25 μg of ovalbumin or PBS (vehicles and control). One day after the last intranasal administration, i.e., on day 36, serum and bronchoalveolar lavage fluid (BALF) were collected.

[0113] Ovalbumin-specific IgE, IgG1, and IgG2a in serum were analyzed by ELISA.

[0114] BALF cells were collected by tracheal cannulation with 1 mL of PBS and centrifuged at 500 g for 5 minutes at 4°C. Cytokines in the BALF supernatant were measured using a cytometry bead array with flow cytometry. BALF cells in the pellet were resuspended in 800 μL of PBS, counted using a hemocytometer, transferred to a slide glass using Cytospin 4 Centriflation (Thermo Fisher Scientific), and stained with Diff-Quick®. Macrophages, neutrophils, and eosinophils in BALF cells were identified by their morphological differences using a fluorescence microscope.

[0115] [Results] One-way ANOVA followed by Tukey's post-hoc test was used to determine the statistical significance of the experimental results. Unless otherwise noted, in each figure, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0116] <Preparation Results of Immunogenic Complexes Using Coacervation Method> Table 1 below shows information such as the average particle size of gelatin particles (immunogenic complexes) containing ovalbumin as the antigen protein, gelatin particles containing egg white, and gelatin particles containing skim milk. Table 2 below shows information such as the average particle size of gelatin particles containing Cry j1 and Cry j2 as antigen proteins. The Z potential and particle size distribution, described later, were measured using a dynamic light scattering spectrophotometer (DLS, Zetasizer Pro ZSU3200, Malvern).

[0117]

[0118] In Table 1 and thereafter, "G ＥＷ " refers to gelatin particles containing undenatured egg white (step 3 has not been performed), and "G Ｍｉｌｋ " means gelatin particles containing undenatured skim milk (step 3 not performed). Also, "hG ＯＶＡ " refers to gelatin particles containing denatured ovalbumin (more specifically, particles in which ovalbumin is encapsulated within gelatin particles and then heat-denatured; the same applies hereafter), and "hG ＥＷ " refers to gelatin particles containing denatured egg white, and "hG Ｍｉｌｋ " " refers to gelatin particles containing denatured skim milk. "OVA EE%" refers to the encapsulation rate (inclusion rate) of ovalbumin within the gelatin particles.

[0119]

[0120] In Table 2 and thereafter, "G Ｃｒｙ　Ｊ " refers to gelatin particles containing undenatured Cry j1 and Cry j2 (step 3 not performed), and "hG Ｃｒｙ　Ｊ " refers to gelatin particles containing denatured Cry j1 and Cry j2.

[0121] Figure 2 is a graph showing the particle size distribution of gelatin particles containing ovalbumin. Figure 3 is a graph showing the particle size distribution of gelatin particles containing egg white or skim milk. Figure 4 is a graph showing the particle size distribution of gelatin particles containing Cry j1 and Cry j2. Figure 5 is a scanning electron microscope (SEM) image of gelatin particles containing ovalbumin. A Hitachi SU9000 scanning electron microscope was used.

[0122] As shown in Figures 2 to 4, the average particle size for all particles was approximately 200 nm, suggesting that the presence or absence of thermal denaturation after encapsulation of the antigen protein had little effect on particle size.

[0123] On the other hand, as shown in Figure 5, numerous gelatin particles were observed using a scanning electron microscope. The gelatin particles observed with the scanning electron microscope had a diameter of approximately 100 nm, suggesting that the gelatin particles shrunk during the drying process required for preparing the SEM sample.

[0124] Figure 6 shows the electrophoretic image of gelatin particles containing egg white subjected to SDS-PAGE. Figure 7 shows the electrophoretic image of gelatin particles containing skim milk subjected to SDS-PAGE.

[0125] In Figures 6 and 7 and thereafter, "gelatin" is the lane in which unparticulated gelatin was run, "EW" is the lane in which egg white was run, "Milk" is the lane in which skim milk was run, "collagenase" is the lane in which collagenase was run, and "supernatant" is the lane in which the supernatant after impregnating gelatin with antigen protein was run. In addition, the five lanes from the right in Figure 6, from left to right, are lanes in which gelatin, egg white, gelatin particles without antigen protein, gelatin particles containing undenatured egg white, and gelatin particles containing heat-denatured egg white were treated with collagenase, respectively. The five lanes from the right in Figure 7, from left to right, represent gelatin, skim milk, gelatin particles without antigen protein, gelatin particles containing undenatured skim milk, and gelatin particles containing heat-denatured skim milk, each treated with collagenase.

[0126] Egg white is composed of four types of proteins (ovotransferrin: 78 kDa, ovalbumin: 45 kDa, ovomucoid: 35 kDa, lysozyme: 17 kDa). Skim milk is composed of five types of proteins (casein fraction: 24 kDa, 23 kDa, 19 kDa, β-lactoglobulin: 18.4 kDa, α-lactalbumin: 14 kDa). Collagenase, which is used to break down the gelatin matrix, is composed of at least seven types of proteases ranging from 40 to 140 kDa.

[0127] As shown in Figures 6 and 7, no bands were observed in the lanes where the supernatants of gelatin particles containing egg white, denatured egg white, skim milk, or denatured skim milk were run. This suggests that heated gelatin particles and unheated gelatin particles can each contain almost all of the antigen proteins mixed in step 2.

[0128] Furthermore, as shown in Figure 6, a band was observed in the lane where the collagenase-treated material was run, indicating that collagenase treatment releases the encapsulated antigen protein. Additionally, as shown in Figure 7, no band similar to that observed in the lane where skim milk was run was observed in the lane where the skim milk was run, suggesting that the skim milk protein was completely degraded by collagenase.

[0129] Figure 8 shows the electrophoresis results of SDS-PAGE testing of gelatin particles containing Cry j1 and Cry j2. In Figure 8, the five lanes from the right are, from left to right, gelatin, Cry j1 and Cry j2, gelatin particles without antigen proteins, gelatin particles containing undenatured Cry j1 and Cry j2, and gelatin particles containing heat-denatured Cry j1 and Cry j2, respectively, all treated with collagenase. As shown in Figure 8, no bands were observed in the lanes where the supernatant was run when preparing gelatin particles containing Cry j1 and Cry j2, or denatured Cry j1 and Cry j2, was prepared. This suggests that the gelatin particles can contain almost all of the Cry j1 and Cry j2 mixed in step 2, or their denatured forms.

[0130] On the other hand, in systems where gelatin particles containing Cry j1 and Cry j2, or denatured Cry j1 and Cry j2, were treated with collagenase, the gelatin concentration was high, and therefore the gelatin could not be completely decomposed. As a result, no bands indicating the released Cry j1 and Cry j2 were observed.

[0131] Figure 9 is a graph showing the relationship between the amount of decomposition and time when gelatin particles without antigen protein and gelatin particles without antigen protein that have been heated are treated with collagenase. Figure 10 is a graph showing the relationship between the amount of ovalbumin released and time when gelatin particles containing ovalbumin and gelatin particles containing denatured ovalbumin are treated with collagenase. In Figures 9 and 10, the dashed lines extending vertically indicate the timing of collagenase addition.

[0132] As shown in Figures 9 and 10, when gelatin particles are heated (in other words, when step 3 is performed), the rate of degradation by collagenase slows down compared to the unheated case, suggesting that the rate of antigen protein release also slows down.

[0133] <Evaluation of gelatin particle uptake by bone marrow dendritic cells> Figure 11 shows the uptake of ovalbumin and G into bone marrow dendritic cells. ＯＶＡ , or hG ＯＶＡ This is a fluorescence microscope image showing the uptake of ovalbumin and G into bone marrow dendritic cells. Figure 12 shows ovalbumin and G into bone marrow dendritic cells. ＯＶＡ , or hG ＯＶＡ This graph shows the results of measuring the uptake amount by fluorescence intensity using flow cytometry.

[0134] As shown in Figures 11 and 12, G is better than bare ovalbumin. ＯＶＡ G ＯＶＡ hG ＯＶＡ The uptake of ovalbumin was higher in this group. This result suggests that encapsulating the antigen protein in gelatin particles and denaturing it by heat can improve its uptake by dendritic cells. It is possible that heating alters the surface structure of the gelatin, exposing hydrophobic regions and specific amino acid sequences, making them easier for dendritic cells to recognize and phagocytose.

[0135] <Evaluation results of IL-2 production by T cells that have received antigen presentation from bone marrow dendritic cells> Figure 13 shows PBS, ovalbumin, and G ＯＶＡ , or hG ＯＶＡ OT-II CD4 co-cultured with bone marrow dendritic cells treated with 4 or 24 hours. ＋ This graph shows the amount of IL-2 produced by T cells.

[0136] As shown in Figure 13, G is greater than bare ovalbumin. ＯＶＡ G ＯＶＡ hG ＯＶＡ In the system where dendritic cells were treated, the amount of IL-2 produced by T cells was increased. This result suggests that encapsulating antigen proteins in gelatin particles and denaturing them by heat can improve the efficiency of antigen presentation by dendritic cells and immune activation by T cells.

[0137] <Evaluation results of anaphylactic reaction> Figure 14 shows the results of ovalbumin and G in allergy model mice. ＯＶＡ , or hG ＯＶＡThis graph shows the change in rectal temperature when administered intraperitoneally. As shown in Figure 14, when naked ovalbumin was administered, the rectal temperature dropped significantly, indicating that an anaphylactic reaction occurred. In contrast, when ovalbumin was encapsulated in gelatin particles... ＯＶＡ , or hG ＯＶＡ When administered intraperitoneally, no decrease in rectal temperature was observed. However, when heat-denatured ovalumin was administered without gelatin ("heat-denatured OVA"), a decrease in rectal temperature was observed. These results suggest that anaphylactic reactions can be prevented by supporting or encapsulating the antigen protein in gelatin particles.

[0138] <Evaluation results of binding affinity between gelatin particles and ovalbumin-specific antibody> Figure 15 shows ovalbumin, G ＯＶＡ , or hG ＯＶＡ This graph shows the results of the evaluation of the binding affinity of ovalbumin-specific antibodies to [substance].

[0139] As shown in Figure 15, anti-OVA IgE antibody, anti-OVA IgG2a antibody, and anti-OVA IgG1 antibody are all more effective than ovalbumin. ＯＶＡ G ＯＶＡ hG ＯＶＡ It showed low binding affinity to the antigen protein. This result suggests that denaturing the antigen protein makes it difficult for antibodies produced by B cells to recognize it.

[0140] hG of anti-OVA IgE that induces anaphylactic reactions ＯＶＡ The fact that the amount of binding to ovalbumin was maintained to some extent suggests that the gelatin was not able to completely encapsulate ovalbumin, and that some of it was exposed on the surface of the gelatin particles.

[0141] In contrast to this, hG ＯＶＡ However, the binding of anti-OVA IgE is very slight, and ovalbumin and G ＯＶＡ Compared to other methods, this suggests that it can suppress the occurrence of anaphylactic reactions.

[0142] Figure 16 shows BALB / c mice receiving mouse serum albumin, ovalbumin, and G ＯＶＡ , or hGＯＶＡ This graph shows the results of measuring the amount of anti-ovalbumin IgG antibody bound to a sample of serum collected on day 28 after subcutaneous administration of the drug once a week for four weeks, using the ELISA method. Figure 17 shows the results of measuring the amount of mouse serum albumin, ovalbumin, and G2 in BALB / c mice. ＯＶＡ , or hG ＯＶＡ This graph shows the results of measuring the amount of anti-ovalbumin IgM antibody bound to serum collected on day 28 after subcutaneous administration of the drug once a week for four weeks, using the ELISA method.

[0143] As shown in Figures 16 and 17, G contains porcine-derived gelatin. ＯＶＡ and hG ＯＶＡ The substance exhibited negligible immunogenicity in mice, comparable to that of MSA (mouse serum albumin), a low immunogenicity indicator. This result suggests that gelatin particles have low immunogenicity and may be safe to use.

[0144] <Evaluation Results of Gelatin Particle Retention> Figure 18 is an image showing the results of evaluating gelatin particle retention using Cy7 labeling. Figure 19 is a graph showing the change in fluorescence intensity when gelatin particle retention is evaluated using Cy7 labeling.

[0145] As shown in Figures 18 and 19, the amount of naked ovalbumin injected subcutaneously decreased to around 20% after 24 hours and was almost completely gone by day 5. In contrast, the amount of G injected subcutaneously decreased. ＯＶＡ and hG ＯＶＡ The percentage remained at 35% after 24 hours and was well maintained until day 7, the final day of evaluation. This result suggests that loading antigen proteins onto gelatin particles may reduce the frequency of antigen administration in desensitization therapy.

[0146] <Evaluation of therapeutic effects in a mouse allergic asthma model> Figure 20 is a graph showing the amount of anti-ovalbumin IgE antibody, anti-ovalbumin IgG2a antibody, and anti-ovalbumin IgG1 antibody in the serum of a mouse allergic asthma model after intranasal administration of ovalbumin following induction of immune tolerance. As shown in Figure 20, G ＯＶＡ or hG ＯＶＡIt was suggested that administering [substance] could reduce IgE production. ＯＶＡ and hG ＯＶＡ It also reduced Th2-dependent anti-obvalvumin IgG1 levels, particularly hG ＯＶＡ G ＯＶＡ This suggests that it more effectively suppresses the Th2 reaction compared to the previous method.

[0147] On the other hand, no significant differences were observed between the groups regarding anti-ovalbumin IgG2a antibodies, which exhibit a Th1 reaction and compete with IgE antibodies to bind to allergens.

[0148] Figure 21 is a graph showing the number of macrophages, neutrophils, eosinophils, and their total cell count in BALF after intranasal administration of ovalbumin following induction of immune tolerance in a mouse allergic asthma model. As shown in Figure 21, G ＯＶＡ or hG ＯＶＡ Administering this drug suppresses eosinophil migration, especially hG ＯＶＡ This suggests that the effect tends to be high.

[0149] Figure 22 is a graph showing the amount of cytokines in the BALF supernatant after intranasal administration of ovalbumin following induction of immune tolerance in a mouse allergic asthma model.

[0150] As shown in Figure 22, G ＯＶＡ or hG ＯＶＡ Administration of [substance name] reduced the production of Th2 cytokines, specifically IL-4, IL-5, IL-13, and IL-6. ＯＶＡ The effect was particularly pronounced in the treatment group. IL-4 is known to play an important role in Th2 cell development and IgE production. IL-5 is known to be involved in eosinophil activation and recruitment. IL-13 is known to contribute to IgE synthesis. IL-6 is an inflammatory cytokine. On the other hand, no significant differences were observed between the groups for TNF-α and IL-10.

[0151] Based on the above results, G ＯＶＡand hG ＯＶＡ Both were suggested to have the potential to be used as therapeutic agents for treating allergic diseases. Also, its therapeutic effect was suggested to be particularly remarkable in hG ＯＶＡ It was suggested that it was particularly remarkable in hG

[0152] According to the present invention, an allergic reaction can be suppressed by administering an immunogenic complex containing gelatin particles carrying an antigen protein. Therefore, the present invention is industrially applicable.

Claims

1. An immunogenic complex comprising gelatin particles and an antigen supported on the gelatin particles, wherein the antigen is a denatured antigenic protein.

2. The immunogenic complex according to claim 1, wherein the antigen is a heat-denatured antigen protein.

3. The immunogenic complex according to claim 2, wherein at least a portion of the gelatin constituting the gelatin particles is arranged to fit the antigen.

4. The immunogenic complex according to claim 1, wherein the antigen is encapsulated within the gelatin particles.

5. The immunogenic complex according to claim 1, wherein the antigen is an allergen or an autoantigen.

6. The immunogenic complex according to claim 1, wherein the antigen is one or more antigen proteins selected from the group consisting of antigen proteins contained in pollen, antigen proteins contained in food, and antigen proteins contained in mites.

7. A pharmaceutical composition comprising the immunogenic complex described in any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7 for treating or preventing allergies or autoimmune diseases.

9. A method for producing an immunogenic complex, comprising: step 1 of forming gelatin particles; step 2 of mixing the gelatin particles formed in step 1 with an antigen protein to form a complex; and step 3 of heating the complex to thermally denature the antigen protein.

10. The method for producing an immunogenic complex according to claim 9, wherein in step 3, the complex is heated to 25°C or higher.

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