WO2024117232A1 - Complexe immunogène et composition pharmaceutique - Google Patents

Complexe immunogène et composition pharmaceutique Download PDF

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WO2024117232A1
WO2024117232A1 PCT/JP2023/042983 JP2023042983W WO2024117232A1 WO 2024117232 A1 WO2024117232 A1 WO 2024117232A1 JP 2023042983 W JP2023042983 W JP 2023042983W WO 2024117232 A1 WO2024117232 A1 WO 2024117232A1
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ova
mdo
antigen
administration
mannoprotein
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PCT/JP2023/042983
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Japanese (ja)
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健 森
佳樹 片山
大輔 村上
順怡 李
耕二 長谷
大輔 ▲高▼橋
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国立大学法人九州大学
慶應義塾
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to immunogenic conjugates and pharmaceutical compositions.
  • This application claims priority based on Japanese Patent Application No. 2022-191916, filed on November 30, 2022, the contents of which are incorporated herein by reference.
  • Allergies are inflammatory diseases in which an excessive immune response occurs against specific allergen proteins.
  • the current radical treatment for allergies is desensitization therapy, which involves continuously administering small amounts of allergens and using dendritic cells (DCs) to induce regulatory T cells (Tregs) and establish immune tolerance.
  • DCs dendritic cells
  • Tregs regulatory T cells
  • current drugs use the allergens as is, and there is a risk that the allergens will bind to IgE antibodies, causing a severe allergic reaction. Therefore, current drugs can only be administered in small amounts, which causes treatment to take a long time. If it were possible to deliver large amounts of allergens to DCs while avoiding binding to IgE antibodies, these problems could be solved.
  • Non-Patent Document 1 As a ligand for MR, mannoprotein (MAN) derived from budding yeast, which contains highly branched mannose polymers and shows high affinity for MR, is considered to be effective.
  • MAN mannoprotein derived from budding yeast, which contains highly branched mannose polymers and shows high affinity for MR, is considered to be effective.
  • Patent Document 1 describes an immunogenic complex in which MAN is bound to an allergen via a reactive dialdehyde.
  • the present invention aims to provide an immunogenic complex that has low reactivity to IgE antibodies and is highly capable of inducing tolerant DCs, and a pharmaceutical composition containing the immunogenic complex.
  • the present invention includes the following aspects.
  • An immunogenic complex comprising an antigen and a mannoprotein, wherein the antigen and the mannoprotein are bound by a disulfide bond.
  • the immunogenic complex according to [1] wherein the immunogenic complex forms a nanoparticle comprising a shell and a core, the shell comprising the mannan chain of the mannoprotein, and the core comprising the antigen.
  • the antigen is an allergen or an autoantigen.
  • the immunomodulator is at least one selected from the group consisting of a retinoic acid receptor agonist, a retinoid X receptor agonist, a vitamin D receptor agonist, an aromatic hydrocarbon receptor agonist, a histone deacetylase inhibitor, and an mTOR inhibitor.
  • a pharmaceutical composition comprising the immunogenic complex according to any one of [1] to [6].
  • the present invention provides an immunogenic complex that has low reactivity to IgE antibodies and high ability to induce tolerant DCs, and a pharmaceutical composition containing the immunogenic complex.
  • An example of a method for preparing an immunogenic complex of one embodiment is shown.
  • An example of a method for preparing an immunogenic complex of one embodiment is shown.
  • the results of evaluating the effect of heating temperature and heating time on the particle size of disulfide-crosslinked MAN-OVA nanoparticles (MDO) are shown. "65°C”, "75°C”, and "85°C” indicate heating temperatures.
  • OVA ovalbumin; 5 min: MDO (heated for 5 minutes); 15 min: MDO (heated for 15 minutes); 25 min: MDO (heated for 25 minutes); 60 min: MDO (heated for 60 minutes).
  • OVA dNP disulfide cross-linked OVA nanoparticles
  • MDO(5) MDO with 5 mg/mL MAN
  • MDO(10) MDO with 10 mg/mL MAN
  • MDO(15) MDO with 15 mg/mL MAN
  • MDO(20) MDO with 20 mg/mL MAN.
  • MDO-15 w/o H 2 O 2 MDO with 15 mg/mL MAN (without hydrogen peroxide treatment); MDO-15 w H 2 O 2 : MDO with 15 mg/mL MAN (with hydrogen peroxide treatment).
  • SDS treatment SDS + DDT treatment.
  • OVA ovalbumin
  • Man mannoprotein
  • OVA dNP disulfide-crosslinked OVA nanoparticles
  • MO(5) MAN-OVA nanoparticles (MO) with 5 mg/mL MAN
  • MDO(5) MDO with 10 mg/mL MAN
  • MO(15) MO with 15 mg/mL MAN
  • OVA ovalbumin
  • OVA dNP disulfide-crosslinked OVA nanoparticles
  • MDO disulfide-crosslinked MAN-OVA nanoparticles
  • MGO glutaraldehyde-crosslinked MAN-OVA nanoparticles.
  • Fluorescence microscopy images evaluating the uptake of each nanoparticle into DCs are shown.
  • the results of evaluating antigen presentation by bone marrow dendritic cells (BMDCs) stimulated with each nanoparticle, based on IL-2 production, are shown.
  • the results of evaluating IL-10 production from bone marrow dendritic cells (BMDCs) stimulated with each type of nanoparticle are shown.
  • ATRA reductase RALDH
  • MDO (1:1): mixed with MP:OVA 1:1
  • MDO (3:1): mixed with MP:OVA 3:1
  • MDO (4:1): mixed with MP:OVA 4:1.
  • the results of evaluating the reactivity of each nanoparticle with anti-OVA antibody are shown.
  • the administration schedule of the experiment evaluating the risk of anaphylactic shock due to administration of each nanoparticle is shown below.
  • Alum Alum.
  • the results of evaluating the change in rectal temperature after administration of each nanoparticle as an index of anaphylactic shock are shown.
  • 1 shows the administration schedule for an experiment evaluating the preventive effect of each nanoparticle on airway allergic reaction by pre-oral administration.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-oral administration of each nanoparticle are shown.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-oral administration of each nanoparticle are shown.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-oral administration of each nanoparticle are shown.
  • 1 shows the administration schedule for an experiment evaluating the preventive effect of each nanoparticle on airway allergic reaction by pre-subcutaneous administration.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-subcutaneous administration of each nanoparticle are shown.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-subcutaneous administration of each nanoparticle are shown.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-subcutaneous administration of each nanoparticle are shown.
  • the results of evaluating the preventive effect of airway allergic reactions by pre-subcutaneous administration of each nanoparticle are shown. 1 shows the administration schedule for an experiment evaluating the therapeutic effect of each nanoparticle on airway allergic reaction by post-oral administration.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-oral administration are shown.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-oral administration are shown.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-oral administration are shown.
  • 1 shows the administration schedule for an experiment evaluating the therapeutic effect of each nanoparticle on airway allergic reaction by post-subcutaneous administration.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-subcutaneous administration are shown.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-subcutaneous administration are shown.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-subcutaneous administration are shown.
  • the results of evaluating the therapeutic effect of each nanoparticle on airway allergic reaction after post-subcutaneous administration are shown.
  • the results of evaluating the inhibition of mononuclear cell infiltration by administration of each nanoparticle are shown.
  • 1 shows the administration schedule for an experiment evaluating the inhibitory effect of pre-oral administration of each nanoparticle on type IV allergic response.
  • the results of evaluating the inhibitory effect of pre-oral administration of each nanoparticle on type IV allergic response are shown.
  • the figures show the results of evaluating the effect of the OVA content on the particle size of disulfide-crosslinked HSA (human serum albumin)-OVA nanoparticles (HO): HO-0: HO with 0% OVA content; HO-5: HO with 5% OVA content; HO-10: HO with 10% OVA content; HO-20: HO with 20% OVA content; HO-50: HO with 50% OVA content.
  • Non-reduced SDS treatment; reduced: SDS + DDT treatment.
  • HO-5 HO with 5% OVA content
  • HO-10 HO with 10% OVA content
  • HO-20 HO with 20% OVA content
  • HO-50 HO with 50% OVA content
  • MHO-10 10 mg/mL disulfide-crosslinked MAN-HSA-OVA nanoparticles (MHO) of MAN.
  • MHO-0 0 mg/mL MHO of MAN
  • MHO-5 5 mg/mL MHO of MAN
  • MHO-7.5 7.5 mg/mL MHO of MAN
  • MHO-10 10 mg/mL MHO of MAN.
  • Fluorescence microscopy images evaluating the uptake of each nanoparticle into DCs are shown.
  • the results of evaluating the change in DC phenotype due to stimulation with each nanoparticle are shown below: (a) MHC class II (IA b ) expression; (b) CD80 expression; (c) CD86 expression; (d) PD-L1 expression.
  • MHO-10-R MHO-10 + rapamycin.
  • the antigen presentation of BMDCs stimulated with each nanoparticle was evaluated based on IL-2 production.
  • the results of evaluating the reactivity of each nanoparticle with anti-OVA antibody are shown. 1 shows the results of a basophil activation test in peripheral blood from healthy subjects.
  • the peripheral blood from healthy subjects after each stimulation was stained with CRTH2 (CD294)-FITC, CD203c-PE, and CD3-PC7, and analyzed by a flow cytometer.
  • the figures show the results of a basophil activation test in peripheral blood from patients with Japanese cedar pollen allergy.
  • the figures show the results of staining peripheral blood from healthy subjects with CRTH2 (CD294)-FITC, CD203c-PE, and CD3-PC7 after each stimulation and analyzing the blood using a flow cytometer.
  • 1 shows the results of a basophil activation test in peripheral blood from healthy subjects and from patients with cedar pollen allergy.
  • the term “comprises” means that it may contain components other than the target component.
  • the term “consists of” means that it does not contain components other than the target component.
  • the term “consists essentially of” means that it does not contain components other than the target component in a form that exerts a special function (such as a form that completely loses the effect of the invention). In this specification, when “comprises,” it includes aspects that "consist of” and aspects that “consist essentially of.”
  • a numerical range expressed using " ⁇ ” means that the range includes the numbers written before and after " ⁇ " as the lower and upper limits.
  • a first aspect of the present disclosure is an immunogenic complex.
  • the immunogenic complex of this aspect includes an antigen and a mannoprotein.
  • the antigen and the mannoprotein are bound by a disulfide bond.
  • Antigen refers to any substance capable of inducing an immune response, such as humoral and cellular immune responses, in a target organism.
  • An antigen may be any substance capable of inducing proliferation, activation, maturation, etc. of immune cells when in contact with immune cells, or any substance capable of inducing cytokine production, antibody production, etc. from immune cells.
  • the antigen is a protein or peptide.
  • An antigen may be a protein that may be an allergen, a protein of an infectious agent (such as a virus, bacteria, fungus, etc.), a protein derived from a neoplastic cell (such as a tumor cell), a peptide or fragment of said protein, or a recombinant protein of said protein.
  • An antigen may be an autoantigen.
  • the antigen is an allergen or an autoantigen.
  • Autoantigen refers to a substance possessed by an individual itself that may be an antigen.
  • allergen refers to a substance that can cause an allergy in an individual.
  • An allergen is a substance that is recognized as a foreign substance by an individual's immune system and causes an immune response, primarily the production of immunoglobulin E (IgE).
  • allergens include pollen allergens, arthropod-derived allergens, food-derived allergens, and allergens present in insect saliva, nails, or needles.
  • the allergen is a pollen allergen.
  • pollen allergens include allergens derived from cedar pollen, cypress pollen, white birch pollen, rice pollen, ragweed pollen, mugwort pollen, and Japanese knotweed pollen.
  • Antigens may be used alone or in combination of two or more types.
  • Mannoproteins are glycoproteins composed of mannan and protein. Mannan is a polysaccharide whose main component is mannose. Mannoproteins are known in nature as one of the components of the cell walls of microorganisms, particularly yeast. For example, in yeast, mannoproteins exist in a structure in which a polypeptide chain is bound to the end of ⁇ -mannan.
  • mannoproteins are composed of an internal chain portion N-glycosidically linked to an asparagine residue of a polypeptide, a polymeric mannan chain portion on the outside of the internal chain portion, and an oligosaccharide chain portion O-glycosidically linked to a serine or threonine residue of a peptide.
  • Mannoproteins may be obtained from natural sources or by known chemical synthesis methods. Mannoproteins may be obtained, for example, from yeast.
  • yeast include Saccharomyces, Pichia, and Candida, with Saccharomyces yeasts being preferred.
  • Saccharomyces yeasts include S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. cerevisiae, S. chevalieri, S. dairenensis, S. S. ellipsoides, S. eubayanus, S. exigus, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, S.
  • yeast is S. cerevisiae.
  • the method for obtaining mannoprotein is not particularly limited, and known methods can be used.
  • Methods for obtaining mannoprotein from yeast include, for example, a method that utilizes the solubility of mannoprotein in water. Examples include a method of extracting mannoprotein from yeast with hot water, and a method of extracting mannoprotein from yeast with alkali.
  • mannoprotein can be obtained by extracting mannoprotein from yeast with hot water, followed by precipitation with cetablon (hexadecyltrimethylammonium bromide). Commercially available mannoprotein may be used.
  • the antigen and the mannoprotein are bound by a disulfide bond.
  • a thiol group (-SH) of the antigen and a thiol group of the polypeptide chain of the mannoprotein are bound by a disulfide bond to form a nanoparticle.
  • the antigen is a protein or peptide
  • the thiol group may be a thiol group of a cysteine residue of the protein or peptide.
  • the thiol group of the mannoprotein may be a thiol group of a cysteine residue of the polypeptide chain of the mannoprotein.
  • Disulfide bonds can be formed between thiol groups of the antigen; between a thiol group of the antigen and a thiol group of the mannoprotein; and between thiol groups of the mannoprotein.
  • the immunogenic complex of this embodiment may form nanoparticles by binding the antigen and the mannoprotein via a disulfide bond.
  • the size of the nanoparticles is not particularly limited, but may have an average particle diameter of about 10 to 2000 nm, for example. The average particle diameter tends to increase when the amount of mannoprotein used is increased.
  • the average particle diameter of the nanoparticles may be about 30 to 1000 nm, about 50 to 500 nm, or about 50 to 300 nm.
  • the average particle diameter of the nanoparticles can be measured using a dynamic light scattering spectrophotometer.
  • the particle diameter polydispersity (PDI) of the nanoparticles is not particularly limited, but may be, for example, about 0.1 to 0.8, or about 0.2 to 0.6.
  • the particle diameter polydispersity of the nanoparticles can be measured using a dynamic light scattering spectrophotometer.
  • the nanoparticle comprises a shell and a core.
  • the shell of the nanoparticle comprises mannan chains of a mannoprotein.
  • the core of the nanoparticle comprises an antigen.
  • the core may further comprise a polypeptide chain of a mannoprotein.
  • the core of the nanoparticle comprises an antigen and a polypeptide chain of a mannoprotein, which are linked by disulfide bonds to form the core (see FIG. 1).
  • the immunogenic complex of this embodiment may contain optional components in addition to the antigen and mannoprotein, such as matrix proteins, immunomodulators, and the like.
  • Microx protein refers to a protein other than the antigen of interest that forms an immunogenic complex together with the antigen and mannoprotein.
  • the matrix protein can be used, for example, when the cost of the antigen is high, to allow a small amount of antigen to form an immunogenic complex.
  • the matrix protein is not particularly limited. In order to prevent the induction of an unexpected immune response, it is preferable to use a protein derived from the same organism as the subject of administration of the immunogenic complex.
  • a human protein can be used as the matrix protein.
  • a specific example of the matrix protein is albumin.
  • human serum albumin can be preferably used as the matrix protein.
  • the matrix protein may be used alone or in combination of two or more kinds.
  • the matrix protein is present in the core of the nanoparticle and is bound to either or both of the antigen and the polypeptide chain of the mannoprotein by disulfide bonds to form the core together with them.
  • Immunomodulator means a drug that has the effect of modulating immune function.
  • the immunomodulator is an immunosuppressant that suppresses immune function.
  • 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 signal transduction system.
  • An example of a retinoic acid receptor agonist is all-trans-retinoic acid.
  • An example of a retinoid X receptor agonist is bexarotene.
  • An example of a vitamin D receptor agonist is vitamin D3.
  • An example of an aromatic hydrocarbon receptor agonist is 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester.
  • An example of a histone deacetylase inhibitor is suberoylanilide hydroxamic acid (SAHA).
  • An example of an mTOR inhibitor is rapamycin.
  • the immunomodulatory agent may be used alone or in combination of two or more kinds.
  • the immunomodulator is thought to be present in the core of the nanoparticle due to intermolecular forces, etc.
  • FIG. 1 An example of a method for producing an immunogenic complex according to an embodiment is shown in Fig. 1.
  • Fig. 1 an antigen and mannoprotein (MAN) are used as materials for the immunogenic complex.
  • Fig. 1 shows only one thiol group each of the antigen and mannoprotein, but each of the antigen and mannoprotein may have two or more thiol groups.
  • Figure 2 shows an example of a method for producing an immunogenic complex according to one embodiment.
  • an antigen, mannoprotein (MAN), and matrix protein are used as materials for the immunogenic complex.
  • the antigen, mannoprotein, and matrix protein are shown to have only one thiol group each, but each may have two or more thiol groups.
  • the method for producing the immunogenic complex may include, for example, a step of heating a solution in which an antigen and a mannoprotein are dissolved to form nanoparticles (hereinafter also referred to as “step (i)”), and a step of oxidizing the nanoparticles obtained in step (i) (hereinafter also referred to as “step (ii)").
  • the antigen and mannoprotein are dissolved in an appropriate buffer solution to prepare a solution containing the antigen and mannoprotein.
  • the buffer solution is not particularly limited, and can be one commonly used in the field of biochemistry.
  • the buffer solution include phosphate buffer (PB), phosphate buffered saline (PBS), acetate buffer, citrate buffer, citrate phosphate buffer, borate buffer, tartaric acid ornamental solution, Tris buffer, HEPES buffer, etc.
  • the buffer solution preferably contains a salt (e.g., sodium chloride).
  • the salt concentration include 10 to 100 mM, preferably 20 to 80 mM, more preferably 30 to 70 mM, and even more preferably 40 to 60 mM.
  • the pH of the buffer solution is not particularly limited, but is preferably near neutral, and preferably 6.0 to 8.0.
  • a specific example of the buffer solution is 10 mM phosphate buffer solution (pH 7.4) to which 50 mM NaCl has been added.
  • the mixing ratio of the antigen and the mannoprotein is not particularly limited. When the ratio of the mannoprotein is increased, the size of the nanoparticles tends to increase.
  • the mannoprotein concentration in the antigen/mannoprotein solution is not particularly limited, but may be, for example, 1 to 50 mg/mL, preferably 5 to 30 mg/mL, more preferably 5 to 20 mg/mL, and even more preferably 5 to 15 mg/mL.
  • the mannoprotein concentration is within the above-mentioned preferred range, the PDI tends to be small.
  • the antigen concentration in the antigen/mannoprotein solution is not particularly limited, but may be, for example, 1 to 50 mg/mL, preferably 1 to 10 mg/mL, and more preferably 1 to 5 mg/mL.
  • the matrix protein When a matrix protein is used, the matrix protein is dissolved in a buffer together with the antigen and mannoprotein to prepare a solution containing the antigen, mannoprotein and matrix protein (see FIG. 2).
  • the total mass of the antigen and matrix protein can be considered as the mass of the antigen mentioned above.
  • the ratio of the antigen to the total mass (100% by mass) of the antigen and the matrix protein is, for example, 1 to 90% by mass, and may 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.
  • the immunomodulator When an immunomodulator is used, the immunomodulator is dissolved in a buffer solution together with the antigen and mannoprotein to prepare a solution containing the antigen, mannoprotein and immunomodulator (and optionally the matrix protein).
  • the amount of immunomodulator used is not particularly limited and can be appropriately selected depending on the type of immunomodulator.
  • the concentration of rapamycin in the solution can be, for example, 1 to 100 ⁇ g/mL, or can be 5 to 50 ⁇ g/mL or 10 to 30 ⁇ g/mL.
  • the heat treatment temperature can be set appropriately depending on the denaturation temperature of the antigen (and matrix protein).
  • the heat treatment temperature can be set to a temperature at which the antigen (and matrix protein) denatures. By setting the heat treatment temperature to a temperature at which the antigen (and matrix protein) denatures, nanoparticles are stably formed. In addition, reactivity of the antigen with the antibody is suppressed.
  • Specific examples of heat treatment temperatures include 60 to 90°C, with 65 to 90°C being preferred, 70 to 90°C being more preferred, and 75 to 85°C being even more preferred.
  • the heat treatment time can be set to a time sufficient for denaturing the antigen (and matrix protein) at the heat treatment temperature.
  • the heat treatment time can be, for example, 1 minute or more, preferably 5 minutes or more, more preferably 10 minutes or more, even more preferably 15 minutes or more, and particularly preferably 20 minutes or more.
  • the treatment time may be 30 minutes or more, or may be 60 minutes or more.
  • the upper limit of the treatment time is not particularly limited, but can be, for example, 100 minutes or less, or 80 minutes or less.
  • step (i) the antigen and mannoprotein, and optionally the matrix protein and the immunomodulator, are aggregated in the solution.
  • Pre-NP nanoparticle
  • the mixture may be cooled using an ice bath or the like. Cooling can stop the formation of nanoparticles.
  • the oxidation treatment of the nanoparticles (Pre-NP) can be carried out using an oxidizing agent.
  • the oxidizing agent include hydrogen peroxide, oxidized glutathione (GSSG), potassium nitrate, perchlorate, hypochlorite, nitric acid, permanganate, ozone, etc.
  • the oxidizing agent is hydrogen peroxide.
  • the oxidizing agent may be used alone or in combination of two or more kinds.
  • the amount of the oxidizing agent used can be appropriately set depending on the type of the oxidizing agent.
  • the oxidizing agent is hydrogen peroxide, it can be added to the solution obtained in step (i) so that the final concentration becomes, for example, 0.1 to 1 mass %.
  • the reaction conditions for the oxidation reaction may be any conditions that allow the thiol groups in the nanoparticles to form disulfide bonds.
  • the reaction temperature is, for example, 0 to 30°C, preferably 1 to 20°C, more preferably 2 to 10°C, and even more preferably 3 to 5°C.
  • the reaction time is, for example, 1 hour or more, preferably 5 hours or more, more preferably 8 hours or more, even more preferably 10 hours or more, and particularly preferably 12 hours or more.
  • the upper limit of the treatment time is not particularly limited, but can be, for example, 30 hours or less, or 20 hours or less.
  • step (ii) disulfide bonds are formed between the thiol groups in the nanoparticles. This makes it possible to obtain nanoparticles of an immunogenic complex in which the antigen and mannoprotein are bound by disulfide bonds.
  • nanoparticles of the immunogenic complex may be collected by ultrafiltration or the like.
  • ultrafiltration By performing ultrafiltration, free antigens, mannoproteins, oxidizing agents, matrix proteins, immunomodulators, etc. can be removed.
  • an ultrafiltration membrane with a molecular weight cutoff of 100 kDa can be used.
  • the recovered nanoparticles of the immunogenic complex may be freeze-dried and stored.
  • the nanoparticles may be suspended in a 1% by weight glucose solution and freeze-dried.
  • the immunogenic complex has a shape in which the antigen is covered with mannan chains of mannoprotein. Therefore, it is easily taken up into dendritic cells by the mannose receptor (MR) and CD209 of DC.
  • the immunogenic complex taken up into DC is cleaved within the DC as the disulfide bonds are reduced to thiol groups. This releases the antigen from the immunogenic complex.
  • the antigen released into the DC cell is degraded within the DC cell to a peptide of about 15 to 30 amino acid residues. This peptide is presented by MHC molecules as a T cell epitope. This induces tolerant DC.
  • the immunogenic complex of this embodiment has low reactivity with antigen-specific antibodies because the antigen is not exposed. Furthermore, by thermally denaturing the antigen during the preparation of the immunogenic complex, the reactivity with antigen-specific antibodies is further reduced. This suppresses binding between the antigen and IgE antibodies. Therefore, with the immunogenic complex of this embodiment, it is possible to administer many antigens to the subject while avoiding binding with IgE antibodies.
  • a second aspect of the present disclosure is a pharmaceutical composition.
  • the pharmaceutical composition of this aspect contains the immunogenic complex of the first aspect.
  • the pharmaceutical composition of this aspect contains the immunogenic complex of the first aspect as an active ingredient.
  • the pharmaceutical composition of this embodiment may contain an optional component in addition to the immunogenic complex of the first embodiment.
  • the optional component may include, for example, a pharma- ceutically acceptable carrier.
  • pharmaceutical-ceutically acceptable carrier refers to a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the subject to which it is administered.
  • not substantially toxic refers to a carrier that does not show toxicity to the subject to which it is administered at a dose that is normally used.
  • the pharma-ceutically acceptable carrier is a carrier that does not inhibit the immunogenicity of the immunogenic complex of the first embodiment and does not show substantial toxicity to the subject to which it is administered.
  • the pharma-ceutically acceptable carrier includes any known pharma-ceutically acceptable component that is typically considered to be an inactive component.
  • the pharma-ceutically acceptable carrier is not particularly limited, but examples thereof include solvents, diluents, vehicles, excipients, flow enhancers, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, and fillers.
  • the pharma- ceutical acceptable carrier may be used alone or in combination of two or more kinds.
  • the pharmaceutical composition of this embodiment may contain, for example, a buffer solution as a solvent. Examples of the buffer solution include those mentioned above.
  • the pharmaceutical composition may contain other ingredients in addition to the above ingredients.
  • the other ingredients are not particularly limited, and any ingredient commonly used in the pharmaceutical field may be used without any particular restriction.
  • examples of the other ingredients include pharmaceutical additives other than those mentioned above.
  • pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. These ingredients may be used alone or in combination of two or more kinds.
  • the pharmaceutical composition may contain an active ingredient other than the immunogenic complex of the first aspect.
  • the active ingredient include, but are not limited to, antiviral agents, antibiotics, anti-inflammatory agents, antipyretics, analgesics, etc.
  • One type of active ingredient may be used alone, or two or more types may be used in combination.
  • the pharmaceutical composition may contain an adjuvant.
  • adjuvants include, but are not limited to, aluminum hydroxide, calcium phosphate, monophosphoryl lipid A, chitosan, etc.
  • One type of adjuvant may be used alone, or two or more types may be used in combination.
  • the dosage form of the pharmaceutical composition is not particularly limited, and may be any dosage form commonly used as a pharmaceutical preparation.
  • the pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation.
  • Oral preparations include, for example, tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions, etc.
  • Parenteral preparations include, for example, injections, suppositories, nasal drops, enteral preparations, inhalants, etc.
  • Pharmaceutical compositions of these dosage forms may be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).
  • the route of administration of the pharmaceutical composition of this embodiment is not particularly limited, and may be oral or parenteral.
  • parenteral administration include sublingual administration, intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration.
  • the pharmaceutical composition can be administered in a therapeutically effective amount of the immunogenic complex of the first aspect.
  • therapeutically effective amount means an amount of a drug effective for treating or preventing a target disease.
  • a therapeutically effective amount of an immunogenic complex can be an amount effective for inducing tolerant DCs.
  • the therapeutically effective amount may be appropriately determined depending on the symptoms, body weight, age, and sex of the patient, as well as the dosage form and administration method of the pharmaceutical composition.
  • the pharmaceutical composition can be administered in a single dose of 0.001 to 1000 mg of antigen per kg of the body weight of the subject. The dose may be 0.005 to 500 mg/kg, 0.01 to 300 mg/kg, 0.02 to 200 mg/kg, or 0.03 to 100 mg/kg.
  • the pharmaceutical composition may contain a therapeutically effective amount of the immunogenic complex per unit dosage form.
  • the content of the immunogenic complex in the pharmaceutical composition may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.
  • the pharmaceutical composition may be administered in a single dose or repeatedly.
  • the administration interval may be appropriately determined depending on the symptoms, weight, age, and sex of the patient, as well as the dosage form of the pharmaceutical composition and the administration method.
  • the administration interval may be, for example, every few hours, 2-3 times a day, once a day, once every 2-3 days, once a week, once a month, once every few months, etc.
  • the subject to which the pharmaceutical composition is administered is not particularly limited.
  • the subject to which the pharmaceutical composition is administered is preferably a mammal, and may be a human or a non-human mammal.
  • non-human mammals include primates (monkeys, gorillas, chimpanzees, marmosets, etc.), rodents (mice, rats, guinea pigs, hamsters, etc.), pets (dogs, cats, rabbits, ferrets, etc.), and livestock (cows, pigs, horses, goats, sheep, etc.).
  • the pharmaceutical composition can be used for treating or preventing allergies or autoimmune diseases.
  • the allergies or autoimmune diseases to which the pharmaceutical composition can be applied are not particularly limited. Examples of allergies include, but are not limited to, hay fever, food allergies, dust mite allergies, atopic dermatitis, allergic asthma, allergic rhinitis, allergic urticaria, and the like. Examples of autoimmune diseases include, but are not limited to, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, vasculitis, and the like.
  • the disclosure provides a method of treating or preventing allergy or an autoimmune disease comprising administering to a subject an immunogenic conjugate of the first aspect.
  • the immunogenic conjugate may be administered in a therapeutically effective amount.
  • the disclosure provides an immunogenic conjugate of the first aspect for treating or preventing an allergy or autoimmune disease.
  • the disclosure provides the use of the immunogenic complex of the first aspect in the manufacture of a pharmaceutical composition for treating or preventing an allergy or an autoimmune disease.
  • the disclosure provides the use of the immunogenic conjugate of the first aspect for treating or preventing allergy or an autoimmune disease.
  • Ovalbumin Ovalbumin
  • MAN mannoprotein
  • ConA concanavalin A
  • Tris tris(hydroxymethyl)aminomethane
  • LPS lipopolysaccharide
  • Hydrogen peroxide H2O2 , 30 wt% in water
  • dithiothreitol DTT
  • sulfuric acid 98%, H2SO4
  • calcium chloride CaCl2
  • magnesium chloride hexahydrate MgCl2 ⁇ 6H2O
  • sodium dodecyl sulfate SDS
  • glycine sodium chloride
  • NaCl NaCl
  • Mildform 10N 10% formalin neutral buffer
  • CBB solution for protein assay 4% paraformaldehyde solution (PFA), disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Nacalai Tesque, Inc.
  • D-glucose was purchased from Tokyo Chemical Industry Co., Ltd.
  • Biotin anti-mouse IgE antibody, biotin anti-mouse IgG1 antibody, and biotin anti-mouse IgG2a antibody were purchased from BioLegend.
  • NPs Mannan-Coated OVA Nanoparticles
  • PB phosphate buffer
  • OVA and MAN were mixed at different concentrations to obtain working solutions. In the following tests, the concentrations of OVA and MAN are expressed using the final concentrations in the working solutions.
  • Protein nanoparticles (NPs) were prepared by heating 1 mL of the working solution at 65°C, 75°C, or 85°C. The heating times were 0, 5, 15, 25, or 60 minutes, respectively. The NP formation was stopped by cooling in an ice bath.
  • MAN-OVA NPs (MO) without disulfide bonds.
  • H 2 O 2 was added to the MO solution up to 0.3 wt %, and the mixture was reacted overnight at 4°C to form intermolecular disulfide bonds.
  • Free OVA, MAN, and H2O2 were removed by ultrafiltration (100 kDa Amicon, Merck Millipore) to recover disulfide-crosslinked MAN-OVA NPs (MDO).
  • MDO was freeze-dried in a 1 wt% glucose solution to ensure long-term storage.
  • 20 mg/mL OVA and 20 mg/mL MAN were mixed in equal amounts and conjugated with glutaraldehyde (final concentration 25 mM) to prepare a MAN-OVA conjugate (MGO).
  • Nanoparticle size and zeta potential The particle size, particle size distribution and zeta potential of the nanoparticles were measured at 25° C. using a dynamic light scattering spectrophotometer (DLS, Zetasizer Pro ZSU3200).
  • OVA and MAN OVA concentration were measured using a protein assay CBB solution based on the Bradford method.
  • OVA standard solution was serially diluted 2-fold, and a standard curve was created by linear regression. Samples were mixed with CBB solution and incubated at room temperature for 10 minutes. Absorbance was measured at 595 nm using a plate reader (Infinite200PRO M Plex, Tecan).
  • a phenol-sulfuric acid assay was used to measure MAN concentration.
  • a standard curve was prepared by gradient dilution of MAN. 5% (w/v) phenol was mixed with the sample, and H2SO4 was added and mixed well. After incubation at room temperature for 40 minutes, the absorbance was measured at 490 nm.
  • ConA agglutination assay was performed as previously reported (Cui, Z.; J Control Release 2002, 81 (1-2), 173-184.). Briefly, ConA was dissolved in phosphate-buffered saline (PBS, 10 mM, pH 7.4), and 5 mM CaCl2 and 5 mM MgCl2 were added to obtain a 1 mg/mL ConA solution. 40 ⁇ L of sample was mixed with 200 ⁇ L of ConA solution. Turbidity was measured at 36 nm for 300 seconds.
  • PBS phosphate-buffered saline
  • ⁇ Mouse> C57BL/6J mice (7-8 weeks old, male) and BALB/c mice (6-8 weeks old, female) were purchased from Kyudou Co., Ltd.
  • OT-II Tg/wt,Ly5.1/Ly5.1 mice were fed CE-2 (Kyudou Co., Ltd.) and had access to bedding and water.
  • Cage changing was performed in a laminar flow hood. Mice were housed under a 12-hour light/12-hour dark cycle. Animal experiments were performed in accordance with the guidelines of the Kyushu University Animal Care and Management Committee.
  • BMDCs Myeloid Dendritic Cells
  • BMDCs were first negatively selected from bone marrow cells with biotin anti-mouse F4/80 and streptavidin nanobeads, and then positively selected with anti-mouse CD11c nanobeads.
  • OVA-rhodamine was used to prepare fluorescently labeled NPs.
  • BMDCs (2 ⁇ 10 5 cells/well) were seeded in 12-well plates and incubated with rhodamine-labeled OVA, OVA dNPs (disulfide-crosslinked OVA nanoparticles), MDO, and MGO for different times.
  • MAN was added to the medium at a final concentration of 1 mg/mL 30 min before sample addition. Dead cells were stained with SYTOX TM Green (Thermo Fisher Scientific).
  • MFI mean fluorescence intensity
  • BMDCs were seeded in a 96-well plate (5 ⁇ 10 4 cells/well) and pulsed with PBS, OVA, MDO, or MGO for 3 hours. Then, BMDCs and OT-II CD4 + T cells (2.5 ⁇ 10 5 cells/well) were cocultured for 24 hours.
  • OT-II CD4 + T cells were isolated from the spleen of OT-II mice using a Mouse CD4 T Cell Isolation Kit (Biolegend). The IL-2 concentration in the culture medium was measured using an IL-2 Mouse ELISA Kit (Thermo Fisher Scientific).
  • mice were sensitized with 200 ⁇ L PBS containing 10 ⁇ g OVA and 1 mg alum by intraperitoneal injection three times at 7-day intervals. Seven days after the last sensitization, the mice were sacrificed and peripheral blood was collected by retro-orbital bleeding. Serum was obtained from the peripheral blood.
  • An indirect immunoassay (indirect-ELISA) protocol was used. 100 ⁇ L of OVA, OVA-dNP, MDO, and MGO (as 100 ⁇ g/mL of OVA) were coated onto a 96-well plate at 4°C for 24 hours. After blocking with ELISA-ELISPOT diluent, serially diluted sera were added to the plate and incubated overnight at 4°C. Biotin anti-mouse IgE, biotin anti-mouse IgG1, and biotin anti-mouse IgG2a antibodies were added to the plate, respectively. After incubation, streptavidin-HRP was added, and TMB solution was used as a substrate. Finally, the reaction was stopped, and the optical density (OD) of each well was measured at 450 nm.
  • mice were sensitized by intraperitoneal injection of 200 ⁇ L PBS containing 10 ⁇ g OVA and 1 mg alum on days 0 and 14. On day 28, mice were intraperitoneally injected with 200 ⁇ g OVA, OVA dNP, MDO, and MGO (containing 200 ⁇ g OVA). After intraperitoneal injection, rectal temperature was measured every 10 min using a thermometer (Physitemp PTM1, Muromachi Kikai Co., Ltd.).
  • mice received (a) oral administration of PBS, OVA, MDO and MGO (containing 0.5 mg OVA) on days 0-4 and 7-11, or (b) subcutaneous administration of PBS, OVA, MDO and MGO (containing 20 ⁇ g OVA) on days 0, 2, 4, 7, 9 and 11.
  • mice were sensitized by intraperitoneal injection of 10 ⁇ g OVA and 1 mg alum.
  • mice were intranasally administered 25 ⁇ g OVA.
  • mice were sacrificed and bronchoalveolar lavage fluid (BAL), peripheral blood and lung tissue were collected.
  • BAL bronchoalveolar lavage fluid
  • mice were first sensitizing mice with intraperitoneal injections of 10 ⁇ g OVA and 1 mg alum on days 0 and 7. Next, mice received (a) oral administration of PBS, OVA, MDO, and MGO (containing 0.5 mg OVA) on days 14-18 and 21-25, or (b) subcutaneous administration of PBS, OVA, MDO, and MGO (containing 20 ⁇ g OVA) on days 14, 16, 18, 21, 23, and 25. Finally, mice were intranasally challenged with 25 ⁇ g OVA on days 32-35. Mice were sacrificed on day 36, and bronchoalveolar lavage fluid (BAL), peripheral blood, and lung tissue were collected.
  • BAL bronchoalveolar lavage fluid
  • cytokines in BAL fluid IL-4, IL-5, IL-6, IL-10, IL-13, and IFN- ⁇ in the BAL fluid were detected using a BD Cytometric Bead Array (BD Life Sciences-Biosciences), followed by measurement using a flow cytometer (CyroFLEX-S, Beckman Coulter Inc.).
  • thiol groups were not detected in native OVA (0 min), but after heating, thiol groups appeared on the OVA surface and became detectable.
  • the free thiol group concentration of NPs not treated with H2O2 was significantly higher than that of dNPs treated with H2O2 (Fig . 6), indicating the formation of disulfide bonds in dNPs.
  • the addition of MAN reduced the detectable thiol groups, which may be due to the inhibition of the reaction of DTNB with SH by the coating of MAN.
  • ConA is a tetrameric lectin with four binding sites for ⁇ -mannose.
  • the turbidity of OVA dNP did not change significantly.
  • the turbidity of MDO increased significantly, and the turbidity increased with increasing MAN content. This suggested that a large amount of mannan was present on the surface of MDO-20. Due to the unimodal distribution and high MAN content of MDO-15, MDO-15 was used in subsequent experiments.
  • BMDCs ⁇ Induction of regulatory T cells by MDO>
  • T cell stimulating ability of BMDCs was measured using OT-II CD4 + T cells that have a specific TCR that recognizes the OVA peptide/I-AB complex and secrete IL-2 in response to it.
  • BMDCs were pulsed with OVA, MDO, and MGO for 3 hours, respectively, and then co-cultured with OT-II CD4 + T cells for 24 hours, and the secreted IL-2 concentration in the medium was measured using an ELISA kit.
  • MDO treatment significantly increased the IL-2 concentration compared to OVA treatment and MGO treatment ( Figure 11).
  • MDO treatment increased the IL-10 concentration compared to OVA treatment and MGO treatment ( Figure 12).
  • MDO treatment increased the activity of ATRA reductase (RALDH) compared to OVA treatment ( Figure 13).
  • MGO and MDO treatments increased the proportion of regulatory T cells compared to OVA treatment ( Figure 14).
  • mice were administered two routes of administration (oral and subcutaneous).
  • oral administration mice were administered PBS, 500 ⁇ g OVA, or 500 ⁇ g OVA as MDO or MGO on days 0-4 and 7-11.
  • Mice were sensitized by subcutaneous injection of OVA and alum on days 14 and 21, and finally administered OVA intranasally on days 28-31 ( Figure 18).
  • serum, bronchoalveolar lavage fluid (BAL), and lungs were collected.
  • Anti-OVA IgE levels were reduced by OVA pre-treatment and more strongly by MDO and MGO pre-treatment ( Figure 19).
  • Anti-OVA IgG2a levels were significantly increased by MDO treatment, whereas IgG1 levels were not different (Figure 19).
  • Pre-administration of OVA, MDO, and MGO suppressed the increase in eosinophils and neutrophils and reduced the total cell count in the BAL fluid, but no significant difference was observed in the number of macrophage cells ( Figure 20).
  • the levels of IL-4, IL-5, IL-6, and IL-13 mediated by Th2 cells in the BAL fluid were significantly reduced by MDO pre-administration and MGO pre-administration compared to PBS treatment ( Figure 21).
  • Pre-administration of OVA also reduced Th2 cytokines, but not as significantly as MDO pre-administration and MGO pre-administration.
  • mice were given 20 ⁇ g of PBS or OVA, or 20 ⁇ g of OVA as MDO or MGO on days 0, 2, 4, 7, 9, and 11.
  • the subsequent course was the same as for oral administration ( Figure 22).
  • Pre-administration of OVA, pre-administration of MDO, and pre-administration of MGO showed similar IgE production inhibitory effects (Figure 23).
  • IgG2a levels increased with MDO pre-administration, but IgG1 levels did not differ among all groups.
  • IgG2a/IgE and IgG2a/IgG1 values indicated that MDO pre-administration and MGO pre-administration showed superior IgE inhibitory effects ( Figure 23).
  • mice were sensitized with OVA and alum on days 0 and 7 and orally administered PBS, 20 ⁇ g OVA, or 500 ⁇ g OVA as MDO or MGO on days 14-18 and 21-25 (Figure 26).
  • Nasal administration of OVA was performed on days 32-35.
  • Anti-OVA IgE levels were decreased after OVA post-administration and MDO post-administration ( Figure 27). There was a tendency for a decrease after MGO post-administration, but there was no statistical difference (Figure 27).
  • mice were administered PBS, 20 ⁇ g OVA, or 20 ⁇ g OVA as MDO or MGO on days 14, 16, 18, 21, 23, and 25 (Figure 30).
  • MDO post-administration reduced anti-OVA IgE production ( Figure 31).
  • BAL fluid neutrophil, eosinophil, and total cell counts were reduced by MDO post-administration ( Figure 32).
  • IL-4, IL-5, and IL-6 were reduced by MDO post-administration (Figure 33).
  • Mononuclear cell infiltration was reduced by OVA pre-administration and MGO post-administration, but more dramatically by MDO post-administration (Figure 34: subcutaneous treatment).
  • HSA Human serum albumin
  • OVA ovalbumin
  • MAN mannoprotein
  • ConA concanavalin A
  • LPS lipopolysaccharide
  • Hydrogen peroxide H 2 O 2 , 30 wt % in water
  • sulfuric acid (98%, H 2 SO 4 )
  • calcium chloride CaCl 2
  • magnesium chloride hexahydrate MgCl 2 ⁇ 6H 2 O
  • sodium chloride NaCl
  • DTNB 2,2'-dinitro-5,5'-dithiodibenzoic acid
  • 5(6)-Carboxytetramethylrhodamine N-succinimidyl ester HNS-Rho was purchased from Thermo Fisher Scientific.
  • NuPAGE sample reducing agent (10x), NuPAGE LDS sample buffer (4x), ELISA-ELISPOT diluent (5x), TMB (3,3',5,5'-tetramethylbenzidine) solution (1x), and HRP-conjugated Streptavidin (horseradish peroxidase) were purchased from Invitrogen.
  • QuickBlue staining solution was purchased from Bio Dynamics Institute Co., Ltd.
  • 4% paraformaldehyde solution (PFA), disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Nacalai Tesque, Inc.
  • Biotin anti-mouse IgE antibody, biotin anti-mouse IgG1 antibody, and biotin anti-mouse IgG2a antibody were purchased from BioLegend.
  • HSA NPs were prepared under various conditions [buffer (10 mM phosphate buffer containing 0 mM, 50 mM or 135 mM NaCl (pH 7.4)]; heating temperature (65°C, 75°C or 85°C); heating time (5 min, 10 min, 30 min or 60 min)]. HSA was dissolved in the buffer and left at 4°C overnight. The HSA dispersion was filtered through a 0.22 ⁇ m membrane to remove aggregates. 1 mL of the HSA solution was heated in a water bath and immediately cooled in an ice bath.
  • H2O2 was added to the NP solution to a final concentration of 0.3 wt%, and the solution was kept at 4°C overnight to form disulfide bonds inside the NPs.
  • Free HSA and H2O2 were removed by ultrafiltration (100 kDa Amicon, Merck Millipore).
  • HSA-OVA NP(HO) HSA-OVA NP(HO)
  • rapamycin 10 ⁇ L of rapamycin (1 mg/mL) was added to 1 mL of the mixed solution of MAN, HSA, and OVA.
  • the buffer used for dissolving HSA, OVA, and MAN was 10 mM PB + 50 mM NaCl (pH 7.4).
  • the mixed solution was heated at 85°C for 60 minutes. After cooling the mixed solution, H 2 O 2 was added, and the solution was left to stand overnight at 4°C, and the NP solution was collected. Ultrafiltration was performed using a 100 kDa filter to collect the NP.
  • Rapamycin was quantified by a validated high performance liquid chromatography (HPLC) method using an Inertsil C8 column (5 ⁇ m, 4.6 mm ⁇ 150 mm) with an Elite Lachrom L-2455 diode array detector (Hitachi). The mobile phase was changed from 70% methanol and 30% Milli-Q water to 90% methanol and 10% Milli-Q water within 20 min. The flow rate was set at 1.0 mL/min, injection volume was 20 ⁇ L, and column oven temperature was set at 25° C. Rapamycin was detected using a photodiode array detector at a wavelength of 277 nm.
  • HPLC high performance liquid chromatography
  • BMDC phenotype analysis BMDCs were seeded in 12-well plates (3 ⁇ 10 6 cells/well), stimulated with 100 ng/mL LPS for 24 hours, and treated with PBS, OVA, HO-20, MHO-10, or MHO-10-R (MHO-10 encapsulated with rapamycin) for 24 hours. BMDCs were collected, and nonspecific binding of immunoglobulins to Fc receptors was inhibited with anti-mouse CD16/32 antibody. BMDCs were stained with anti-mouse I-Ab-FITC, CD86-APC, CD80-BV421, and propidium iodide (PI) (Biolegend). Subsequent analysis was performed by flow cytometry.
  • PI propidium iodide
  • NP preparation conditions The optimal conditions for preparing NPs from HSA, a matrix protein, were screened. HSA is neutral and negatively charged, so HSA aggregates are stably dispersed. The HSA solution was heated at a specific temperature (65-85°C) for up to 60 minutes. Then, H2O2 was added to crosslink the disulfide bonds. The size of HSA NPs increased with heating temperature (data not shown). This is thought to be due to the progression of denaturation with temperature. HSA is known to denature at 65-80°C. Since HSA is expected to be completely denatured and aggregation promoted at 85°C, 85°C was selected as the preparation temperature. When sodium chloride is added, the aggregate size increases, possibly due to the suppression of electrostatic repulsion. 50 mM NaCl was selected because excess sodium chloride destabilizes NPs.
  • the effect of the amount of MAN added on the preparation conditions of HSA/OVA NPs was evaluated with a fixed HSA/OVA ratio of 4:1.
  • the particle size and PDI of the obtained NPs (MHO series) increased with increasing MAN concentration ( Figure 39, Tables 3 and 4).
  • the MAN content in MHO quantified by the phenol-sulfuric acid method increased with increasing MAN concentration, indicating that the incorporation of MAN into NPs is concentration-dependent (Tables 3 and 4). Since the mannan portion of MAN is hydrophilic, mannan coats the surface of the NPs.
  • MHO-10 was dispersed in a buffer solution (10 mM PB + 50 mM NaCl, pH 7.4) and storage stability at 4°C was confirmed. No size change was observed after storage for at least 28 days (data not shown).
  • rapamycin an immunosuppressant
  • HPLC analysis of the rapamycin remaining in the filtrate of the NP dispersion we confirmed that rapamycin had been incorporated almost quantitatively (data not shown).
  • NPs (MHCryj) were prepared by the above method using 4.5 mg/mL HSA, 0.5 mg/mL Cry j, and 10 mg/mL MAN. MHCryj was slightly larger than MHO-10, but showed a uniform size distribution ( Figure 41, Table 5). In the PAGE analysis of MHCryj, bands of Cry j and HSA were observed under reducing conditions ( Figure 42), and no bands were observed under non-reducing conditions.
  • NPs have the effect of inducing tolerant DCs.
  • BMCs stimulated with LPS expressed high levels of MHC class II (I-A b ), costimulatory factors CD80 and CD86, indicating that BMDCs were mature.
  • I-A b MHC class II
  • CD80 and CD86 costimulatory factors
  • MHO-10 and MHO-10-R have the ability to suppress the activation of BMDCs, and that MHO-10-R may induce tolerant DCs due to the effect of rapamycin.
  • MAN induces signal transduction via mannose receptor and DC-SIGN, causing activation of NF- ⁇ B. Rapamycin enhances activation of NF- ⁇ B through inhibition of mammalian target of rapamycin, which is a signal transduction pathway different from that of MAN. Therefore, MAN and rapamycin may synergistically induce tolerant DC.
  • ⁇ Reactivity to anti-OVA antibodies> The reactivity of HO and MHO with anti-OVA antibodies was examined by ELISA. Anti-OVA antibodies were obtained from the serum of mice in which OVA-specific allergy was induced. As shown in FIG. 47, HO-20 and MHO-10 had much lower reactivity with anti-OVA antibodies than OVA, and there was no significant difference between these two NPs. This result indicates that MHO induces a weak IgE-mediated allergic reaction. In allergy treatment, this indicates that MHO is safer than native antigens used in conventional antigen-specific immunotherapy.
  • Example 3 To evaluate whether Cry j NP induces an allergic reaction, a basophil activation test was performed using human peripheral blood.
  • MHCryj Materials and Methods ⁇ Preparation of MHCryj> NP (MHCryj) was prepared in the same manner as above using 4.75 mg/mL HSA, 0.25 mg/mL Cry j 1/2 (a mixture of Cry j 1 and Cry j 2), and 7.5 mg/mL MAN. HSA and Cry j 1/2 were dissolved in PB and left to stand overnight at 4°C. The solution was filtered through a 0.22 ⁇ m membrane to remove aggregates. The solution was then heated in a water bath (85°C, 10 minutes) and immediately cooled in an ice bath.
  • H 2 O 2 was added to the solution to a final concentration of 0.1 wt%, and the solution was kept at 4°C overnight to form disulfide bonds inside the NP.
  • Free HSA and H 2 O 2 were removed by ultrafiltration (100 kDa Amicon, Merck Millipore).
  • the size and PDI of the obtained MHCryj were as follows: Size: 123.6 ⁇ 1.6 (d.nm) PDI: 0.34 ⁇ 0.04
  • the basophil activation test was performed using an Allergenecity kit (BECKMAN COULTER, Tokyo, Japan) according to the protocol attached to the Allergenecity kit.
  • peripheral blood from healthy subjects and peripheral blood from patients with cedar pollen allergy were used, collected using blood collection tubes containing ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • Peripheral blood was stimulated using Cry j 1/2 or MHCryj as an allergen.
  • Cry j 1/2 was diluted with PBS so that the amount of Cry j 1/2 added per 100 ⁇ L of peripheral blood was 0.01 ⁇ g, 0.1 ⁇ g, or 1 ⁇ g, and 20 ⁇ L was added to 100 ⁇ L of peripheral blood.
  • MHCryj was diluted with PBS so that the amount of Cry j 1/2 added per 100 ⁇ L of peripheral blood was 0.01 ⁇ g, 0.1 ⁇ g, or 1 ⁇ g, and 20 ⁇ L was added to 100 ⁇ L of peripheral blood.
  • Anti-IgE antibody was used as a positive control.
  • PBS was used as a negative control.
  • the samples used to stimulate each peripheral blood are summarized in Table 6. The values in Table 6 indicate the amount of Cry j 1/2 added ( ⁇ g) per 100 ⁇ L of peripheral blood.
  • each peripheral blood sample was stained with CRTH2 (CD294)-FITC, CD203c-PE, and CD3-PC7, and analyzed by a flow cytometer. Basophil activation was evaluated based on the analysis results. The rate of basophil activation was determined by calculating the percentage of basophils with increased CD203c fluorescence intensity compared to the negative control (PBS) in the CRTH2 (CD294) and CD203c double positive basophil population in the CD3 negative mononuclear cell region.
  • PBS negative control
  • CD203c double positive basophil population in the CD3 negative mononuclear cell region.
  • the positive control increased the fluorescence intensity of CD203c compared to the negative control (PBS), and an increase in the proportion of activated basophils was confirmed.
  • stimulation with Cry j (0.01 ⁇ g, 0.1 ⁇ g, 1 ⁇ g) also increased the fluorescence intensity of CD203c compared to the negative control (PBS), and an increase in the proportion of activated basophils was confirmed ( Figures 48 and 50, right).
  • the present invention provides an immunogenic complex that has low reactivity to IgE antibodies and high ability to induce tolerant DCs, and a pharmaceutical composition containing the immunogenic complex.

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Abstract

L'invention concerne un complexe immunogène comprenant un antigène et une mannoprotéine, ledit antigène et ladite mannoprotéine étant liés par un pont disulfure. L'invention concerne également une composition pharmaceutique comprenant ledit complexe immunogène.
PCT/JP2023/042983 2022-11-30 2023-11-30 Complexe immunogène et composition pharmaceutique WO2024117232A1 (fr)

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