WO2018133602A1 - 用于疾病治疗的胞外体制剂及其应用 - Google Patents

用于疾病治疗的胞外体制剂及其应用 Download PDF

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WO2018133602A1
WO2018133602A1 PCT/CN2017/116608 CN2017116608W WO2018133602A1 WO 2018133602 A1 WO2018133602 A1 WO 2018133602A1 CN 2017116608 W CN2017116608 W CN 2017116608W WO 2018133602 A1 WO2018133602 A1 WO 2018133602A1
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extracellular
extracellular body
ige
preparation
cells
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PCT/CN2017/116608
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French (fr)
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李莉
彭霞
谢国钢
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李莉
彭霞
谢国钢
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Priority to US16/479,561 priority Critical patent/US11759423B2/en
Publication of WO2018133602A1 publication Critical patent/WO2018133602A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • the present invention relates to the field of biomedicine, and in particular to an extracellular preparation for the treatment of diseases and uses thereof.
  • Allergic diseases are classic IgE-mediated diseases.
  • the World Allergy Organization (WAO) epidemiological survey of allergic diseases in 30 countries showed that about 22% of the population were affected by allergic diseases, and the number of cases increased year by year.
  • the mechanism is: the first time allergens enter the body to stimulate B lymphocytes to secrete specific IgE, which binds to the IgE high affinity receptor Fc ⁇ RI ⁇ present on mast cells and basophils (Fc ⁇ RI is composed of ⁇ , ⁇ , 2 ⁇
  • the tetramer of the subunit, the extracellular end of the alpha chain is the binding region of IgE), and the allergen can bind to the IgE on the surface of mast cells and basophils when it enters the body again, resulting in adjacent Fc ⁇ RI cross-linking.
  • inflammatory mediators histamines, leukotrienes, prostaglandins
  • cytokines and chemokines leading to increased vascular permeability, telangiectasia, smooth muscle contraction, glandular Increased body secretion, etc.
  • local or systemic allergic reactions including allergic rhinitis, asthma, conjunctivitis, eczema, food allergies, drug allergies.
  • An autoimmune disease is a disease caused by the body's immune response to its own antigen, which causes damage to its own tissues. It involves many organs and tissues and covers hundreds of diseases, such as rheumatoid arthritis (RA). Systemic lupus erythematosus (SLE), polymyositis/dermatomyositis, scleroderma, Sjogren's syndrome (SS), etc. As scientists become more aware of the immune system, increase detection methods, and improve diagnostic levels, more and more autoimmune diseases are discovered. According to the statistics of the Association for Autoimmune Diseases, there are about 147 to 250,000 patients with autoimmune diseases in the United States, up to 8% of the total population. It is the third major disease after cardiovascular disease and cancer. In recent years, due to people’s living environment and The change in living habits has increased the incidence of autoimmune diseases year by year.
  • autoantibodies are one of the important features of autoimmune diseases.
  • Autoimmune antibodies exist in most autoimmune patients.
  • the positive rate of anti-ds-DNA antibody in SLE patients exceeds 75%, and the positive rate of anti-SSA antibodies in SS patients is also more than 75%.
  • These antibodies may participate in the development and progression of autoimmune diseases by: 1 interacting with cell surface receptors, altering the structure of the receptor or blocking the binding of the receptor to the corresponding ligand; 2 forming immune complexes, depositing in blood vessels Causes tissue damage; 3 induces cell lysis by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • IgE-type autoantibodies also play an important pathogenic role in certain autoimmune diseases.
  • serum IgE anti-dsDNA autoantibodies are associated with the severity of the disease in patients, which activates plasmacytoid dendritic cells (pDC), which secrete large amounts of IFN- ⁇ , thereby aggravating the injury response in SLE patients.
  • pDC plasmacytoid dendritic cells
  • IgE anti-ANA, anti-dsDNA, anti-BP230 and BP180 autoantibodies are also found in patients with RA, CSU and bullous pemphigoid (BP), which are closely related to the development of the disease.
  • Cardiovascular disease also known as circulatory disease, is a group of heart and vascular diseases, including coronary heart disease, cerebrovascular disease, peripheral peripheral arterial vascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, and pulmonary embolism. Cardiovascular disease is the leading cause of death worldwide. According to statistics, in 2012, 17.5 million people worldwide died of cardiovascular disease, accounting for 31% of the total number of deaths worldwide. Among them, 7.4 million people died of coronary heart disease and 6.7 million died. In the stroke.
  • Coronary heart disease is the leading cause of death in cardiovascular disease, accounting for more than 40% of cardiovascular deaths.
  • Coronary heart disease is a coronary artery angiogenic atherosclerotic lesion that causes stenosis or obstruction of the vascular lumen, causing heart disease caused by myocardial ischemia, hypoxia or necrosis, including asymptomatic myocardial ischemia (occult coronary heart disease), angina pectoris, Five clinical types of myocardial infarction, ischemic heart failure (ischemic heart disease) and sudden death.
  • the traditional view is that coronary heart disease is a simple lipid deposition disease. New research shows that coronary heart disease is closely related to inflammation.
  • IgE allergic inflammation-related molecules
  • mast cells are deposited in plaques and participate in the development of coronary heart disease. Serum IgE levels were elevated in patients with coronary heart disease, and IgE and its receptor Fc ⁇ RI levels were also increased in plaques, and IgE levels were positively correlated with arterial stenosis in patients with coronary heart disease.
  • IgE can promote the interaction of its high-affinity receptor Fc ⁇ RI with TLR4 of other cells (macrophages and endothelial cells), thereby promoting the release of inflammatory molecules from macrophages, and inducing macrophages, endothelial cells, and smooth muscle cells. Apoptosis accelerates atherosclerotic plaque formation.
  • IgE-mediated diseases are mainly allergic diseases, and the treatment of allergic diseases is still based on symptomatic treatment, including antihistamines, mast cell stabilizers (such as sodium cromoglycate, sodium hydroxypropionate, etc.) and hormones.
  • Class immunosuppressive agents such as prednisone, dexamethasone, etc.
  • drugs can only temporarily relieve allergic symptoms, once the drug is easy to relapse, long-term medication should consider its side effects.
  • the treatment strategy for coronary heart disease mainly includes drug treatment (nitrate, calcium blocker, antiplatelet, anticoagulant or thrombolytic), interventional therapy and surgical treatment.
  • drug treatment nanorate, calcium blocker, antiplatelet, anticoagulant or thrombolytic
  • PCI interventional therapy
  • CABG coronary artery bypass grafting
  • a first aspect of the invention provides an extracellular preparation comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the content (v/v) of the extracellular body in the preparation is 0.001 to 30%, preferably 0.05 to 15%, more preferably 0.1 to 10%, according to the preparation. Total volume meter.
  • the concentration of total protein in the extracellular body is from 0.01 to 10 mg/mL, preferably from 0.05 to 5 mg/mL, more preferably from 0.5 to 3.5 mg/mL; optimally from 1 to 3 mg. /mL.
  • the formulation is a liquid formulation, and/or a lyophilized powder formulation.
  • the dosage form of the formulation includes an injection, a spray, and/or a paint.
  • the extracellular body contains an Fc ⁇ RI protein located on the outer surface.
  • the source of the mast cells is selected from the group consisting of primary mast cells (eg, bone marrow-derived mast cells, umbilical cord-derived mast cells, tissue-derived mast cells), mast cell lines (LAD-2), Basophils, basophils (KU812), cells transfected with Fc ⁇ RI ⁇ (eg, pluripotent stem cells, mesenchymal stem cells, dendritic cells, Hela cells, 293T cells, HMC-1 cells, etc.) Any other primary cells and cell lines).
  • primary mast cells eg, bone marrow-derived mast cells, umbilical cord-derived mast cells, tissue-derived mast cells
  • LAD-2 mast cell lines
  • Basophils basophils
  • basophils basophils
  • cells transfected with Fc ⁇ RI ⁇ eg, pluripotent stem cells, mesenchymal stem cells, dendritic cells, Hela cells, 293T cells, HMC-1 cells, etc.
  • the pharmaceutically acceptable carrier is selected from the group consisting of phosphate buffer, raw A saline solution, a trehalose solution, or a combination thereof.
  • the formulation further contains albumin.
  • the albumin has a content (v/v) of 0.5 to 50%, preferably 2.5 to 25%, more preferably 5-15%, in the preparation.
  • the total volume of the formulation preferably 0.5 to 50%, preferably 2.5 to 25%, more preferably 5-15%.
  • the formulation contains 0.003-1% by weight, preferably 0.015-0.5% by weight, more preferably 0.03-0.35% by weight of extracellular body, based on the total weight of the formulation.
  • the formulation contains from 0.1 to 10% by weight, preferably from 0.5 to 5% by weight, more preferably from 1 to 3% by weight, of albumin, based on the total weight of the formulation.
  • the weight ratio of extracellular body to albumin in the preparation is 1-10:10-1, preferably 1-5:5-1, more preferably 1-2: 2-1.
  • a second aspect of the present invention provides an extracellular body comprising an Fc ⁇ RI protein located on an outer surface, the extracellular body having a size of 30-100 nm, and the extracellular body is cultured hypertrophy in vitro Cell secretion is formed and the Fc ⁇ RI protein is substantially in an unbound state.
  • the "substantially” means that ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90% ⁇ 95%, ⁇ 98% or almost 100% of the Fc ⁇ RI protein is in an unbound state.
  • the "in an unbound state” means that the Fc ⁇ RI protein does not form a "Fc ⁇ RI protein-IgE" complex with IgE.
  • the source of the mast cells is selected from the group consisting of primary mast cells (eg, bone marrow-derived mast cells, umbilical cord-derived mast cells, tissue-derived mast cells), mast cell lines (LAD-2), Basophils, basophils (KU812), cells transfected with Fc ⁇ RI ⁇ (eg, pluripotent stem cells, mesenchymal stem cells, dendritic cells, Hela cells, 293T cells, HMC-1 cells, etc.) Any other primary cells and cell lines).
  • primary mast cells eg, bone marrow-derived mast cells, umbilical cord-derived mast cells, tissue-derived mast cells
  • LAD-2 mast cell lines
  • Basophils basophils
  • basophils basophils
  • cells transfected with Fc ⁇ RI ⁇ eg, pluripotent stem cells, mesenchymal stem cells, dendritic cells, Hela cells, 293T cells, HMC-1 cells, etc.
  • a third aspect of the invention provides the use of the extracellular body of the second aspect of the invention for the preparation of a medicament for IgE-mediated related diseases.
  • a fourth aspect of the invention provides the use of a formulation of the first aspect of the invention for the manufacture of a medicament for the treatment of a disease associated with IgE.
  • the IgE-mediated related disease is selected from the group consisting of an allergic disease, an autoimmune disease, a cardiovascular disease, a nephrotic syndrome, or a combination thereof.
  • the allergic disease is selected from the group consisting of allergic asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, systemic allergic reaction, food allergy, drug allergy, or a combination thereof.
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus and rheumatoid Arthritis, bullous herpes zoster, scleroderma, mixed connective tissue disease, Hashimoto's thyroiditis, ulcerative colitis, or a combination thereof.
  • the cardiovascular disease is selected from the group consisting of acute myocardial infarction, stable angina pectoris, unstable angina pectoris, acute coronary syndrome, calcific aortic valve disease, or a combination thereof.
  • the medicament is further for one or more uses selected from the group consisting of:
  • the inhibiting the activation of mast cells comprises decreasing one or more indicators selected from the group consisting of: beta-hexosaminidase release rate, histamine release rate, serotonin, tryptase, guanidine Proteases, prostaglandins, leukotrienes, cytokines (IL-6, IL-4, IL-5).
  • a fifth aspect of the invention provides a method for inhibiting the activation of mast cells in vitro, comprising the steps of:
  • the mast cells are cultured to inhibit the activation of mast cells.
  • the concentration of the extracellular body is 0.011-21000 ⁇ g/mL, preferably 0.0210-1500 ⁇ g/mL, more preferably 0.250-0.6200 ⁇ g/mL.
  • the mast cells are from a human or a non-human mammal.
  • the non-human mammal is a rodent or primate, preferably a mouse, a rat, a guinea pig, a rabbit, and/or a monkey.
  • a sixth aspect of the invention provides a method for preparing an extracellular body, comprising the steps of:
  • an in vitro culture system comprising bone marrow-derived mast cells and a medium suitable for growth of said cells, and said culture system further comprising 0.1-5 mmol/L of L-glutamine, 0.1- 5 mmol/L sodium pyruvate, and 5-15% (v/v) fetal bovine serum without extracellular bodies;
  • the concentration of the bone marrow-derived mast cells is 1 ⁇ 10 5 - 1 ⁇ 10 7 /ml.
  • step (ii) the culture is carried out at 37 ⁇ 1 °C.
  • a seventh aspect of the invention provides a method of treating an IgE-mediated related disease, comprising the steps of:
  • the extracellular preparation of the first aspect of the invention is administered to a subject in need of treatment.
  • the subject comprises a human or a non-human mammal.
  • the subject comprises a rodent such as a mouse or a rat.
  • the extracellular preparation is administered in an amount of from 0.01 to 5 mg/kg, preferably from 0.1 to 2 mg/kg, more preferably from 0.2 to 1 mg/kg of body weight.
  • Figure 1 shows the A. electron microscope showing exosomes as a circular structure with a diameter of 50-80 nm;
  • B. Western blot shows exosomes expressing exosomes characteristic molecules TSG101 protein and CD81 protein, BMMC cells and their exocclusion Fc ⁇ RI was expressed in vivo, while Fc ⁇ RI was not expressed in P815 cells and their exosomes;
  • Figure 2 shows the dose-effect relationship between the exosomes dose and the relative fluorescence intensity (rFI) of BMMC.
  • the red line indicates that different concentrations of P815-exosome (not expressing Fc ⁇ RI) were pre-incubated with IgE for 2 h, then incubated with BMMC, and FITC-labeled anti-IgE antibody was added after washing.
  • Flow cytometry was used to detect the amount of IgE not bound to exosome; blue line indicates Different concentrations of BMMC-exosome (expressing Fc ⁇ RI) were pre-incubated with IgE for 2 h, and then the remaining amount of IgE was detected.
  • the green line indicates that different concentrations of BMMC-exosome, IgE and BMMC were simultaneously incubated, and FITC-labeled anti-IgE antibody was added for detection. The amount of IgE remaining.
  • Figure 3 shows the effect of BMMC-exosome on IgE (panel A), histamine (panel B) and leukocytes and their differential counts (panel C) in alveolar lavage fluid of allergic asthma mice.
  • group a showed sensitized and stimulated control mice;
  • group bd was sensitized and challenged by OVA,
  • group b was given saline treatment,
  • group c was treated with exosome for one month, and group d was given exosome for treatment for 2 months, Group given exosome treatment for March
  • Figure 4 shows the effect of BMMC-exosome on inflammatory cell infiltration in lung tissue of allergic asthma mice.
  • Mouse lung tissue was fixed by formaldehyde, embedded in paraffin, sectioned, HE stained, a indicates lung tissue of control mice that were not sensitized and challenged; b indicates lung tissue of allergic mice treated with saline, and c indicates exosome
  • group d was given exosome for 2 months of allergic mouse lung tissue, and e was given exosome for 3 months of allergic mouse lung tissue.
  • the present inventors have unexpectedly discovered that mast cells cultured under specific conditions in vitro secrete specific exosomes containing a very abundant surface-based Fc ⁇ RI protein, and The Fc ⁇ RI protein described is essentially in an unbound state.
  • the Fc ⁇ RI protein-containing extracellular body can competitively bind to IgE, thereby significantly inhibiting the binding of IgE to mast cells, reducing the binding rate of IgE to mast cells, thereby extremely effectively eliminating serum IgE.
  • experiments have shown that the Fc ⁇ RI protein-containing extracellular bodies of the present invention unexpectedly inhibit the activation of mast cells and treat IgE-mediated related diseases such as allergic asthma. On the basis of this, the inventors completed the present invention.
  • extracellular body As used herein, the terms “extracellular body”, “Fc ⁇ RI-exosome” are used interchangeably and are meant to be derived from the secretion of in vitro cultured mast cells (preferably bone marrow-derived mast cells) containing the outer surface. The extracellular body of the Fc ⁇ RI protein.
  • Extracellular bodies are membrane vesicles secreted by cells that carry certain surface molecules and contents of proteins (proteins, RNA, etc.) and are important mediators of signal transmission between cells.
  • the extracellular body is widely distributed, has high biological activity and low immunogenicity, and is a microcosm of the cells from which it is derived.
  • the use of extracellular bodies as carriers to deliver target molecules and drugs has become a hot topic in biotherapy and vaccine development in recent years.
  • mast cell-derived extracellular bodies carry various membrane receptors, ligands, adhesion molecules, costimulatory signaling molecules, and nucleic acid molecules (miRNAs and microRNAs) such as Fc ⁇ RI and KIT, which may be mast cells and other cells.
  • membrane receptors ligands, adhesion molecules, costimulatory signaling molecules, and nucleic acid molecules (miRNAs and microRNAs)
  • Fc ⁇ RI and KIT nucleic acid molecules
  • the Fc ⁇ RI carried by the mast cell-derived extracellular body neutralizes the serum IgE, blocking the surface of IgE and mast cells and basophils.
  • the binding of IgE high-affinity receptor Fc ⁇ RI, inhibiting mast cell activation and alleviating the progression of the disease may be an effective way to treat IgE-related diseases.
  • the extracellular preparation of the present invention contains a safe and effective amount of an extracellular body together with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the extracellular body is 30-100 nm in size and the extracellular body is secreted by cultured mast cells in vitro.
  • the formulation of the invention may also contain a safe and effective amount of albumin, Albumin has the effect of stabilizing the formulation.
  • Albumin has the effect of stabilizing the formulation. Studies by the present inventors have shown that the addition of a certain proportion of albumin can significantly improve the therapeutic effect and stability of the Fc ⁇ RI-extrasome of the present invention.
  • the pharmaceutical composition of the present invention can be formulated into a liquid preparation which can be prepared by a conventional method, and the liquid preparation is preferably produced under aseptic conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram body weight to about 50 milligrams per kilogram body weight per day, from about 5 micrograms per kilogram body weight to about 10 milligrams per kilogram body weight, and about 10 micrograms per kilogram body weight to about 5 weight percent. Mg/kg body weight.
  • the formulations of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the medicament is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably.
  • the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • Fc ⁇ RI cells primary cultured mast cells, basophils, mast cell lines, basophils, and other cells transfected with Fc ⁇ RI
  • the resulting extracellular body is isolated from the culture system by in vitro culture for a period of time (e.g., 1-5 days, preferably 1.5-3 days).
  • the preparation or pharmaceutical composition of the present invention can be obtained by mixing the prepared Fc ⁇ RI-exosome with a pharmaceutically acceptable excipient.
  • a preferred formulation is a liquid Fc ⁇ RI-extracellular preparation.
  • a method of preparing an extracellular preparation comprises the steps of:
  • the obtained extracellular body is added to a medically acceptable solvent in a volume fraction of 0.1-30% (V/V), and the extracellular body content is 0.03-10 mg/mL, thereby obtaining extracellular Body preparation.
  • the surface prepared by the invention comprises an extracellular body (Fc ⁇ RI-exosome) of unbound Fc ⁇ RI ⁇ , which can be Effectively neutralizes free IgE in the body, reduces IgE binding to mast cell membrane receptors, reduces mast cell degranulation, inhibits allergic reactions, and gradually reduces the number of mast cells and IgE receptors, thereby achieving the goal of treating allergic and IgE-related diseases.
  • Fc ⁇ RI-exosome extracellular body of unbound Fc ⁇ RI ⁇
  • the extracellular body of the present invention or an extracellular preparation containing an extracellular body (or a corresponding drug) neutralizes free IgE in the body, reduces binding of IgE to mast cell membrane receptors, thereby reducing mast cell degranulation and inhibiting allergy.
  • the reaction aims to treat allergic and IgE-related diseases (such as allergic diseases (such as allergic asthma), autoimmune diseases, cardiovascular diseases).
  • the Fc ⁇ RI-extrasome of the present invention is obtained by extracting from a cell culture supernatant, is easy to prepare, can be derived from autologous cells, and is non-immunogenic.
  • the Fc ⁇ RI-extrasome of the present invention neutralizes serum free IgE, and reduces IgE binding to Fc ⁇ RI-positive cells, including mast cells, basophils, macrophages, etc., and can be used for IgE-mediated diseases, Including allergies, autoimmune diseases, cardiovascular diseases, etc.
  • the present invention discloses for the first time an extracellular preparation containing Fc ⁇ RI-exosome which can effectively neutralize serum free IgE and reduce binding of IgE to Fc ⁇ RI-positive cells, thereby treating IgE-mediated related diseases.
  • the present invention discloses for the first time an extracellular preparation containing Fc ⁇ RI-exosome, which can also reduce the expression level of Fc ⁇ RI on the cell surface of mast cells, basophils, dendritic cells and the like, and reduce hypertrophy. Activation of cells to treat IgE-mediated related diseases.
  • BMMC normal mouse bone marrow-derived mast cells
  • Bone marrow cells were extracted from the tibia and femur of mice (purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.), induced by interleukin 3 (IL-3) and stem cell factor (SCF) for 4 weeks.
  • IL-3 interleukin 3
  • SCF stem cell factor
  • the fetal bovine serum was taken, and the extracellular body was removed by ultracentrifugation (100,000 g for 90 min) to obtain serum for removing the extracellular body.
  • Mouse bone marrow-derived mast cells and commercially available 1640 culture medium (with a final concentration of 1 mmol/L glutamine, a final concentration of 1 mmol/L sodium pyruvate, and containing 10% of the serum containing exosome)
  • the cells were mixed and adjusted to a concentration of 1 ⁇ 10 6 /ml, and cultured at 37° C. for 48 hours; the culture solution was collected, centrifuged at 300 g for 10 min, the supernatant was retained, the cell pellet was removed, and the cell debris was removed; the supernatant was centrifuged at 16500 g for 20 min, and the supernatant was retained.
  • the cell pellet was removed, and the cell debris and apoptotic bodies were removed.
  • the supernatant was centrifuged at 120,000 g for 90 min, the supernatant was removed, and the pellet was collected to obtain an extracellular body, resuspended in PBS buffer, and the protein concentration was measured by a BCA kit.
  • Fc ⁇ RI-exosome (BMMC-extrasome) was prepared according to the methods of Example 1 and Example 2 with BMMC (expressing Fc ⁇ RI) and P815 cells (without expressing Fc ⁇ RI) (purchased from ATCC) as the source cells of the extracellular body. Formulation and Fc ⁇ RI(-)-exosome (P815-exosome) preparation.
  • the extracellular body was incubated with IgE for 2 h before being added to BMMC, and the other group was incubated with BMMC-extrasome and IgE simultaneously into BMMC. After incubation with each cell, the cells were washed and added with fluorescein. Isothiocyanate, FITC) labeled anti-IgE antibody, flow cytometry to detect fluorescence on the surface of BMMC.
  • Isothiocyanate, FITC labeled anti-IgE antibody
  • Fc ⁇ RI-exosome (BMMC-exosome) preparation was prepared according to Example 1 and Example 2, and BMMC-exosome (100 ⁇ g) was incubated with IgE for different time (0.5, 1, 2, 4 h), and then added.
  • BMMC-exosome 100 ⁇ g was incubated with IgE for different time (0.5, 1, 2, 4 h), and then added.
  • sensitization was carried out for 1 h at 37 ° C; after washing, DNP-HSA (0.5 ⁇ g/ml) was added to stimulate, and the cell supernatant (A) was collected, the cell pellet was lysed and the lysate was collected, and the supernatant (B) was obtained by centrifugation.
  • An Fc ⁇ RI-exosome (BMMC-exosome) preparation was prepared as in Example 1 and Example 2.
  • mice were sensitized by intraperitoneal injection of pre-mixed OVA (20 ⁇ g) plus adjuvant aluminum hydroxide (4 mg) on days 0, 7, and 14 and started with 1% OVA on day 21. The solution was inhaled, 30 min/time/day, and continuously inhaled for 5 days.
  • mice purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • group A control group
  • group B asthma group
  • group C treatment group
  • group D treatment of the March group
  • group E treatment group
  • the control group was sensitized and stimulated with normal saline
  • the asthma group was sensitized and challenged with OVA
  • the treatment group was inhaled with 1% OVA from day 32, 30 min/time/day, twice a week, weekly stimulation.
  • 40 ⁇ g/150 ⁇ L of BMMC-extrasome was injected into the tail vein for the first 30min, and the total injection was 4 times.
  • the treatment group in the February group and the treatment group was the same as the treatment group in January, except that the injection times were 8 and 12 times respectively. ;
  • Airway High Reactivity was detected after the last challenge of each group of mice; serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected to detect the number of inflammatory cells, inflammatory mediators, and lung tissue. Pathological changes.
  • BMMC-extracellular body can effectively improve AHR in asthmatic mice and reduce serum OVA-specific IgE (OVA-specific IgE, OVA-sIgE) in asthmatic mice.
  • OVA-specific IgE OVA-sIgE
  • Level the number of inflammatory cells in BALF and the levels of histamine and Th2 cytokines (IL-4, IL-5 and IL-13), increased secretion of anti-inflammatory factors (IL-10 and IFN- ⁇ ), and reduced inflammatory cells in lung tissue Infiltration, mucus secretion and proliferation of airway smooth muscle.
  • IL-4, IL-5 and IL-13 histamine and Th2 cytokines
  • IL-10 and IFN- ⁇ anti-inflammatory factors
  • reduced inflammatory cells in lung tissue Infiltration, mucus secretion and proliferation of airway smooth muscle.
  • the effect of BMMC-exosome inhibition of allergic reactions increases with prolonged treatment time.

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Abstract

提供了一种由体外经培养的肥大细胞分泌形成的胞外体制剂及其中胞外体的制备方法，含有位于外表面的基本上处于未结合状态的FcεRI蛋白的胞外体，以及上述胞外体制剂或胞外体在体外抑制肥大细胞活化的方法和制备治疗IgE介导的相关疾病的药物的用途。

Description

用于疾病治疗的胞外体制剂及其应用 技术领域
本发明涉及生物医药领域，具体地，涉及一种用于疾病治疗的胞外体制剂及其应用。
背景技术
过敏性疾病是经典的IgE介导的疾病。世界变态反应组织(WAO)对30个国家过敏性疾病的流行病学调查显示：约22％人群受到过敏性疾病的干扰，发病人数逐年上升。其机理为：过敏原初次进入机体刺激B淋巴细胞分泌特异性IgE，后者与肥大细胞和嗜碱性粒细胞上存在的IgE高亲和力受体FcεRIα结合(FcεRI是由α、β、2个γ亚基组成的四聚体，α链的胞外端是IgE的结合区域)，过敏原再次进入机体时可结合在肥大细胞和嗜碱性粒细胞表面的IgE上，导致相邻的FcεRI交联，引起肥大细胞和嗜碱性粒细胞活化，释放炎症介质(组胺、白三烯、前列腺素)、细胞因子和趋化因子等，导致血管通透性增加、毛细血管扩张、平滑肌收缩、腺体分泌增加等，引起局部或全身过敏反应症状，包括过敏性鼻炎、哮喘、结膜炎、湿疹、食物过敏、药物过敏等。
自身免疫性疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，该疾病累及多种器官和组织，涵盖的疾病种类多达上百种，如类风湿关节炎(RA)、系统性红斑狼疮(SLE)、多发性肌炎/皮肌炎、硬皮病、干燥综合征(SS)等。随着科学家对免疫系统认识的深入、检测手段的增加以及诊断水平的提高，越来越多的自身免疫病被发现。据自身免疫病协会统计，美国约有1470-2350万自身免疫性疾病患者，高达总人口的8％，是位于心血管疾病和肿瘤之后的第三类主要疾病，近年来，由于人们生存环境和生活习惯的改变，自身免疫病发病率逐年上升。
自身抗体的产生是自身免疫病重要的特征之一。大部分自身免疫病患者体内都存在自身抗体，如SLE患者抗ds-DNA抗体阳性率超过75％，SS患者抗SSA抗体阳性率也达75％以上。这些抗体能可能通过以下途径参与自身免疫病的发生和发展：①与细胞表面受体作用，改变受体的结构或阻断受体与相应配体的结合；②形成免疫复合物，沉积在血管处引起组织损伤；③通过补体依赖的细胞毒性(CDC)、抗体依赖的细胞介导的细胞毒性作用(ADCC)诱导细胞裂解。
传统的观点认为，参与这些疾病的抗体多数为IgG型。近年来研究发现，IgE型的自身抗体在某些自身免疫性疾病也发挥了重要的致病作用。在SLE患者中，血清IgE型抗dsDNA自身抗体与患者的疾病严重程度相关，该抗体能激活浆细胞样树突状细胞(pDC)，使其分泌大量的IFN-γ，从而加重SLE患者损伤反应。此外，RA、CSU和大疱性类天疱疮(BP)患者中也逐渐发现IgE型抗ANA、抗dsDNA、抗BP230和BP180自身抗体，这些抗体均与疾病的发生发展密切相关。
心血管疾病又称为循环系统疾病，是一组心脏和血管疾病，包括冠心病、脑血管疾病、周围末梢动脉血管疾病、风湿性心脏病、先天性心脏病、深静脉血栓和肺栓塞等。心血管疾病是全球范围造成死亡的最主要原因，据统计2012年全球有1750万人死于心血管疾病，占全球死亡总数的31％，其中估计740万人死于冠心病，670万人死于中风。
冠心病是心血管疾病中最主要的致死类型，约占心血管疾病死亡人数中的40％以上。冠心病是冠状动脉血管发生动脉粥样硬化病变而引起血管腔狭窄或阻塞，造成心肌缺血、缺氧或坏死而导致的心脏病，包括无症状心肌缺血(隐匿性冠心病)、心绞痛、心肌梗死、缺血性心力衰竭(缺血性心脏病)和猝死5种临床类型。传统的观点认为冠心病一种单纯的脂质沉积性疾病，新的研究表明冠心病与炎症密切相关，多种炎症因子和炎症细胞(巨噬细胞、中性粒细胞、肥大细胞等)聚集在斑块处。最新的研究发现，过敏性炎症相关的分子(IgE)和细胞(肥大细胞)沉积在斑块处，参与冠心病的发生发展。冠心病患者血清IgE水平增高，斑块处的IgE及其受体FcεRI水平也增加，且IgE水平与冠心病患者的动脉狭窄程度呈正相关。进一步的研究表明，IgE能促进其高亲和力受体FcεRI与其他细胞(巨噬细胞和内皮细胞)的TLR4的相互作用，从而促进巨噬细胞释放炎症分子，诱导巨噬细胞、内皮细胞、平滑肌细胞凋亡，加速动脉粥样硬化斑块形成。
目前IgE介导的疾病主要是过敏性疾病，对于过敏性疾病的治疗手段仍以对症治疗为主，包括抗组胺药物、肥大细胞稳定剂(如色甘酸钠、色羟丙钠等)和激素类(即免疫抑制剂，如强的松、地塞米松等)药物。这些药物只能暂时缓解过敏症状，一旦停药容易复发，长期服药要考虑其毒副作用。
自身免疫病的治疗也尚缺乏理想的方法。目前的治疗策略包括抗炎(皮质激素、水杨酸制剂、合成的前列腺素抑制剂)、免疫抑制(环孢素A、FK-506)、 对症治疗(I型糖尿病患者使用胰岛素、甲状腺功能亢进者用抗甲状腺药物，病情严重者采用血浆置换疗法。这些方法只能缓解疾病症状，难以根治疾病。
冠心病的治疗策略主要包括药物治疗(硝酸酯类、钙离子阻滞剂、抗血小板、抗凝药物或溶栓剂)、介入治疗和手术治疗。尽管不同的治疗手段取得了较显著的疗效，但目前的药物治疗对病变严重的心肌缺血效果有限，而介入治疗(PCI)和心脏冠脉搭桥(CABG)治疗后手术部位或冠状动脉其他部位可发生再狭窄，均不能根治冠心病。因此寻找新的治疗途径，已经成为临床研究的重要课题。
因此，本领域迫切需要开发一种可有效治疗IgE介导的相关疾病的新的治疗途径。
发明内容
本发明的目的在于提供一种可有效治疗IgE介导的相关疾病的新的治疗途径。
本发明第一方面提供了一种胞外体制剂，包括：
胞外体，所述胞外体的大小为30-100nm，所述胞外体由体外经培养的肥大细胞分泌形成的；和
药学上可接受的载体。
在另一优选例中，所述制剂中，所述胞外体的含量(v/v)为0.001-30％，较佳地0.05-15％，更佳地0.1-10％，按所述制剂的总体积计。
在另一优选例中，所述胞外体中的总蛋白的浓度为0.01-10mg/mL，较佳地0.05-5mg/mL，更佳地0.5-3.5mg/mL；最佳地1-3mg/mL。
在另一优选例中，所述制剂为液态制剂、和/或冻干粉制剂。
在另一优选例中，所述制剂的剂型包括注射剂、喷剂、和/或涂剂。
在另一优选例中，所述胞外体含有位于外表面的FcεRI蛋白。
在另一优选例中，所述肥大细胞的来源选自下组：原代肥大细胞(如骨髓来源肥大细胞、脐血来源肥大细胞、组织来源肥大细胞)、肥大细胞系(LAD-2)、嗜碱性粒细胞、嗜碱性粒细胞系(KU812)、通过转染表达FcεRIα的细胞(如多能干细胞、间充质干细胞、树突状细胞、Hela细胞、293T细胞、HMC-1细胞等其他任何原代细胞及细胞系)。
在另一优选例中，所述药学上可接受的载体选自下组：磷酸盐缓冲液、生 理盐水、海藻糖溶液、或其组合。
在另一优选例中，所述制剂还含有白蛋白。
在另一优选例中，所述制剂中，所述白蛋白的含量(v/v)为0.5-50％，较佳地，2.5-25％，更佳地，5-15％，以所述制剂的总体积计。
在另一优选例中，所述制剂含有0.003-1wt％，较佳地，0.015-0.5wt％，更佳地0.03-0.35wt％的胞外体，以所述制剂的总重计。
在另一优选例中，所述制剂含有0.1-10wt％，较佳地，0.5-5wt％，更佳地1-3wt％的白蛋白，以所述制剂的总重计。
在另一优选例中，所述制剂中，胞外体与白蛋白的重量比为1-10：10-1，较佳地，1-5：5-1，更佳地，1-2：2-1。
本发明第二方面提供了一种胞外体，所述胞外体含有位于外表面的FcεRI蛋白，所述胞外体的大小为30-100nm，并且所述胞外体由体外经培养的肥大细胞分泌形成，并且所述的FcεRI蛋白基本上处于未结合状态。
在另一优选例中，所述的“基本上”指≥60％、≥70％、≥80％、≥90％≥95％、≥98％或几乎100％的FcεRI蛋白处于未结合状态。
在另一优选例中，所述的“处于未结合状态”指FcεRI蛋白未与IgE形成“FcεRI蛋白-IgE”复合物。
在另一优选例中，所述肥大细胞的来源选自下组：原代肥大细胞(如骨髓来源肥大细胞、脐血来源肥大细胞、组织来源肥大细胞)、肥大细胞系(LAD-2)、嗜碱性粒细胞、嗜碱性粒细胞系(KU812)、通过转染表达FcεRIα的细胞(如多能干细胞、间充质干细胞、树突状细胞、Hela细胞、293T细胞、HMC-1细胞等其他任何原代细胞及细胞系)。
本发明第三方面提供了一种本发明第二方面所述的胞外体的用途，用于制备IgE介导的相关疾病的药物。
本发明第四方面提供了一种本发明第一方面所述制剂的用途，用于制备治疗IgE介导的相关疾病的药物。
在另一优选例中，所述IgE介导的相关疾病选自下组：过敏性疾病、自身免疫病、心血管疾病、肾病综合征、或其组合。
在另一优选例中，所述过敏性疾病选自下组：过敏性哮喘、过敏性鼻炎、过敏性皮炎、过敏性结膜炎、全身过敏反应、食物过敏、药物过敏、或其组合。
在另一优选例中，所述自身免疫疾病选自下组：系统性红斑狼疮、类风湿 性关节炎、大疱性类天疱疹、硬皮病、混合性结缔组织病、桥本氏甲状腺炎、溃疡性结肠炎、或其组合。在另一优选例中，所述心血管疾病选自下组：急性心肌梗死、稳定型心绞痛、不稳定型心绞痛、急性冠脉综合征、钙化性主动脉瓣疾病、或其组合。
在另一优选例中，所述药物还用于选自下组的一种或多种用途：
(i)中和血清游离的IgE；
(ii)抑制IgE与肥大细胞表面的FcεRI的结合；
(iii)抑制肥大细胞的活化。
在另一优选例中，所述抑制肥大细胞的活化包括降低选自下组的一种或多种指标：β-己糖苷酶释放率、组胺释放率、5-羟色胺、类胰蛋白酶、糜蛋白酶、前列腺素、白细胞三烯、细胞因子(IL-6、IL-4、IL-5)。
本发明第五方面提供了一种体外非治疗性的抑制肥大细胞的活化的方法，包括步骤：
在本发明第一方面所述的胞外体制剂或本发明第二方面所述的胞外体存在下，培养肥大细胞，从而抑制肥大细胞的活化。
在另一优选例中，所述胞外体的浓度为0.011-21000μg/mL，较佳地，0.0210-1500μg/mL，更佳地，0.250-0.6200μg/mL。
在另一优选例中，所述肥大细胞来自人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物为啮齿动物或灵长目动物，较佳地包括小鼠、大鼠、豚鼠、兔和/或猴。
本发明第六方面提供了一种胞外体的制法，包括步骤：
(i)提供一体外培养体系，所述培养体系含有骨髓来源肥大细胞和适合所述细胞生长的培养基，并且所述培养体系中还含有0.1-5mmol/L的L-谷氨酰胺，0.1-5mmol/L丙酮酸钠，以及5-15％(v/v)无胞外体的胎牛血清；
(ii)在适合生长的条件下，培养1-5天，获得培养产物；和
(iii)从所述培养产物中分离本发明第二方面所述的胞外体。
在另一优选例中，在步骤(i)的培养体系中，所述骨髓来源肥大细胞的浓度为1×105-1×107个/ml。
在另一优选例中，在步骤(ii)中，在37±1℃下培养。
本发明第七方面提供了一种治疗IgE介导的相关疾病的方法，包括步骤：
向给需要治疗的对象施用本发明第一方面所述的胞外体制剂。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述的对象包括啮齿动物，如小鼠、大鼠。
在另一优选例中，所述胞外体制剂的施用量为0.01-5mg/kg，较佳地为0.1-2mg/kg，更佳地为0.2-1mg/kg体重。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了A.电子显微镜下显示外泌体为直径50-80nm的圆形结构；B.Western blot显示外泌体表达外泌体特征性分子TSG101蛋白和CD81蛋白，BMMC细胞及其外泌体均表达FcεRI，而P815细胞及其外泌体均不表达FcεRI；
图2显示了exosomes剂量与BMMC相对荧光强度(rFI)的量效关系。红线表示不同浓度的P815-exosome(不表达FcεRI)与IgE预先孵育2h，再与BMMC孵育，洗涤后加入FITC标记的抗IgE抗体，流式细胞仪检测未与exosome结合的IgE量；蓝线表示不同浓度的BMMC-exosome(表达FcεRI)与IgE预先孵育2h后，再检测剩余的IgE量；绿线表示不同浓度的BMMC-exosome、IgE和BMMC同时孵育后，加入FITC标记的抗IgE抗体，检测剩余IgE量。
图3显示了BMMC-exosome对过敏性哮喘小鼠肺泡灌洗液中IgE(A图)、组胺(B图)和白细胞及其分类计数(C图)的影响。其中，a组表示未经致敏和激发的对照小鼠；b-d组小鼠经OVA致敏和激发，其中b组给予生理盐水治疗，c组给予exosome治疗一月，d组给予exosome治疗2月，e组给予exosome治疗3月
图4显示了BMMC-exosome对过敏性哮喘小鼠肺组织炎症细胞浸润的影响。小鼠肺组织经甲醛固定、石蜡包埋、切片、HE染色，a表示未经致敏和激发的对照小鼠的肺组织；b表示经生理盐水治疗的过敏小鼠的肺组织，c表示给予exosome治疗一月的过敏小鼠肺组织，d组给予exosome治疗2月的过敏小鼠肺组织，e表示给予exosome治疗3月的过敏小鼠肺组织。
具体实施方式
本发明人经过广泛而深入的研究，意外地发现，在体外特定条件下培养的肥大细胞，会分泌的特定的胞外体，所述胞外体含有非常丰富的位于表面的FcεRI蛋白，并且所述的FcεRI蛋白基本上处于未结合状态。所述的含FcεRI蛋白的胞外体可竞争性地与IgE结合，从而显著抑制IgE与肥大细胞的结合，降低IgE与肥大细胞的结合率，从而极其有效地清除血清IgE。此外，实验证明，本发明的含FcεRI蛋白的胞外体还出乎意料地抑制肥大细胞的活化，治疗IgE介导的相关疾病(如过敏性哮喘)。在此基础上，本发明人完成了本发明。
如本文所用，术语“胞外体”、“FcεRI-胞外体”可互换使用，均指由经体外培养的肥大细胞(较佳地，骨髓来源肥大细胞)分泌形成的含有位于外表面的FcεRI蛋白的胞外体。
胞外体
胞外体(外泌体或囊泡体)是由细胞分泌的膜小囊泡，其携带母细胞的某些表面分子及内容物(蛋白、RNA等)，是细胞间信号传递的重要媒介。胞外体分布广、生物活性高、免疫原性低、是其来源细胞的缩影。利用胞外体为载体传递靶分子和药物成为近几年生物治疗和疫苗研制关注的热点。
研究发现，肥大细胞来源的胞外体携带FcεRI和KIT等各种膜受体、配体、粘附分子、共刺激信号分子，及核酸分子(miRNA和microRNA)，可能是肥大细胞与其他细胞、膜蛋白相互作用的重要方式。
在本发明中，基于自体来源的肥大细胞胞外体免疫原性低特性，利用肥大细胞来源的胞外体携带的FcεRI中和血清IgE，阻断IgE与肥大细胞和嗜碱性粒细胞表面的IgE高亲和力受体FcεRI结合，抑制肥大细胞活化，缓解疾病的进程，可能是治疗IgE相关疾病的一条有效途径。
胞外体制剂
本发明的胞外体制剂，含有安全有效量的胞外体以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。
在一优选实施方式中，所述胞外体的大小为30-100nm，并且所述胞外体由体外经培养的肥大细胞分泌形成。
在一优选实施方式中，本发明的制剂还可含有安全有效量的白蛋白，所述 白蛋白具有稳定制剂的作用。本发明人的研究表明，加入一定比例的白蛋白，可显著提高本发明FcεRI-胞外体的治疗效果和稳定性。
本发明的药物组合物可以被制成液态制剂，其制备可通过常规方法进行,液态制剂宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约50毫克/千克体重，约5微克/千克体重-约10毫克/千克体重，约10微克/千克体重-约5毫克/千克体重。此外，本发明制剂还可与其他治疗剂一起使用。
使用本发明的制剂时，是将安全有效量的药物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
FcεRI-胞外体以及制剂的制备方法
在本发明中，典型地，以表面表达FcεRI细胞(原代培养的肥大细胞、嗜碱性粒细胞、肥大细胞系、嗜碱性粒细胞系、转染表达FcεRI的其它细胞)作为胞外体的来源，通过体外培养一段时间(如1-5天，较佳地1.5-3天)后，从培养体系中分离所形成的胞外体。
在本发明中，将所述制备的FcεRI-胞外体与药学上可接受的赋形剂进行混合，就可制得本发明的制剂或药物组合物。
在本发明中，优选的制剂为液态的FcεRI-胞外体制剂。
典型地，一种胞外体制剂的制备方法包括以下步骤：
1)采用超速离心法或FibercellTM3D中空纤维细胞培养系统制备FcεRI-胞外体；
2)将白蛋白加入医学上可接受的溶剂中，其体积占比为0.5-50％；
3)再将获取的胞外体加入到医学上可接受的溶剂中，其体积占比为0.1-30％(V/V)，胞外体的含量为0.03-10mg/mL，从而获得胞外体制剂。
用途
本发明所制备的表面含未结合型FcεRIα的胞外体(FcεRI-胞外体)，可 有效中和体内游离的IgE，减少IgE与肥大细胞膜受体的结合进而减少肥大细胞脱颗粒，抑制过敏反应，逐渐使肥大细胞数量和IgE受体表达减少，从而达到治疗过敏及IgE相关疾病的目的。
典型地，本发明的胞外体或含有胞外体的胞外体制剂(或相应药物)可中和体内游离的IgE，减少IgE与肥大细胞膜受体的结合进而减少肥大细胞脱颗粒，抑制过敏反应，从而达到治疗过敏及IgE相关疾病(如过敏性疾病(如过敏性哮喘)、自身免疫病、心血管疾病)的目的。
本发明的主要优点包括：
(1)本发明的FcεRI-胞外体从细胞培养上清中提取获得，易于制备、可来源于自体细胞、无免疫原性。
(2)本发明的FcεRI-胞外体中和血清游离IgE，减少IgE与FcεRI阳性的细胞结合，包括肥大细胞、嗜碱性粒细胞、巨噬细胞等，可用于IgE介导的相关疾病，包括过敏、自身免疫病、心血管疾病等。
(3)本发明首次公开了一种含有FcεRI-胞外体的胞外体制剂，其可有效的中和血清游离IgE，减少IgE与FcεRI阳性的细胞结合，从而治疗IgE介导的相关疾病。
(4)本发明首次公开了一种含有FcεRI-胞外体的胞外体制剂，其还能减少肥大细胞、嗜碱性粒细胞、树突状细胞等细胞表面的FcεRI的表达水平，减少肥大细胞的活化，从而治疗IgE介导的相关疾病。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明说明书中所用的试剂和材料均为市售产品。
实施例1 FcεRI-胞外体的制备
在本实施例中，以正常小鼠骨髓来源肥大细胞(Bone marrow-derived mast cells,BMMC)作为胞外体的来源细胞。方法如下：
从小鼠(购自上海西普尔-必凯实验动物有限公司)胫骨和股骨提取骨髓细胞，经白介素3(interleukin 3,IL-3)和干细胞因子(stem cell factor,SCF)诱导4周，获得小鼠骨髓来源肥大细胞。
取胎牛血清，超高速离心(100000g离心90min)去除胞外体，从而获得除去胞外体的血清。
将小鼠骨髓来源肥大细胞与市售的1640培养液(其中补加终浓度1mmol/L谷氨酰胺、终浓度1mmol/L丙酮酸钠，并且含10％所述的除去胞外体的血清)混合，调整细胞浓度为1×106/ml，在37℃培养48小时；收集培养液，300g离心10min，保留上清，去除细胞沉淀，去细胞碎片；上清16500g离心20min，保留上清，去除细胞沉淀，去除细胞碎片及凋亡小体等成分；上清120000g离心90min，去除上清，收集沉淀，即获得胞外体，用PBS缓冲液重悬，用BCA试剂盒测蛋白浓度。
结果如图1所示。电子显微镜检测结果显示胞外体为30-100nm，总蛋白浓度为3mg/mL。此外，几乎100％的FcεRI蛋白处于未结合状态(即FcεRI蛋白为未结合型)。
实施例2 FcεRI-胞外体制剂的制备
取900μL生理盐水，加入50μL 20％人白蛋白，使其终质量浓度为1％(wt/wt)；再加入50μL 4×108细胞提取的胞外体(exosome)，获得胞外体制剂。胞外体中的总蛋白的绝对量为2mg。
实施例3 FcεRI-胞外体与IgE的结合能力
以BMMC(表达FcεRI)和P815细胞(不表达FcεRI)(购自ATCC)作为胞外体的来源细胞，按照实施例1和实施例2的方法制备FcεRI-胞外体(BMMC-胞外体)制剂和FcεRI(-)-胞外体(P815-胞外体)制剂。
胞外体预先与IgE孵育2h后再加入BMMC中孵育，另一组将BMMC-胞外体与IgE同时加入BMMC中孵育，各组与细胞孵育结束后，洗涤并加入异硫氰荧光素(fluorescein isothiocyanate,FITC)标记的抗IgE抗体，流式细胞仪检测BMMC表面的荧光。
结果如图2所示。研究结果表明，BMMC-胞外体能抑制IgE与BMMC的结合，且随着BMMC-胞外体剂量增加，IgE与BMMC的结合率不断降低，两者呈负相关； 而P815-胞外体对IgE与BMMC的结合率则无明显影响。
实施例4 FcεRI-胞外体抑制IgE介导的肥大细胞活化
采用β-己糖苷酶释放和组胺释放试验检测FcεRI-胞外体对肥大细胞活化的影响。
按照实施例1和实施例2制备FcεRI-胞外体(BMMC-胞外体)制剂，将BMMC-胞外体(100μg)与IgE孵育不同时间(0.5、1、2、4h)后，加入到BMMC悬液中，37℃孵育1h致敏；洗涤后加入DNP-HSA(0.5μg/ml)激发，收集细胞上清(A)，裂解细胞沉淀并收集裂解产物，离心获取上清(B)。检测β-己糖苷酶和组胺浓度，计算其释放率，释放率＝A浓度×A体积/(A浓度×A体积+B浓度×B体积)。
结果表明，与未经BMMC-胞外体作用组相比，BMMC-胞外体作用组肥大细胞的β-己糖苷酶和组胺释放率均下降，且随着两者孵育时间增加，β-己糖苷酶和组胺释放率下降。这说明BMMC-胞外体能抑制BMMC活化。
实施例5 FcεRI-胞外体减轻小鼠过敏性哮喘
按照实施例1和实施例2制备FcεRI-胞外体(BMMC-胞外体)制剂。
构建小鼠过敏性哮喘模型：于第0，7，14天予小鼠腹腔注射预先混合好的OVA(20μg)加佐剂氢氧化铝(4mg)混合液致敏，第21天开始用1％OVA溶液做雾化吸入，30min/次/天，连续吸入5天。
分组：BALB/c小鼠(购自上海西普尔-必凯实验动物有限公司)随机分成5组，分别为对照组(A组)，哮喘组(B组)，治疗1月组(C组)，治疗2月组(D组)，治疗3月组(E组)。其中对照组以生理盐水致敏和激发；哮喘组以OVA致敏和激发；治疗一月组从第32天起，用1％OVA雾化吸入，30min/次/天，每周两次，每周激发前30min尾静脉注射40μg/150μL的BMMC-胞外体，总共注射4次；治疗2月组和治疗3月组处理方法同治疗1月组，不同之处在于注射次数分别为8次和12次；
各组小鼠末次激发后检测气道高反应性(Airway High Reactivity,AHR)；收集血清、肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织，检测炎症细胞数目、炎症介质水平、肺组织病理变化。
结果如图3和图4所示。研究结果表明，BMMC-胞外体能有效改善哮喘小鼠的AHR，减少哮喘小鼠血清OVA特异性IgE(OVA-specific IgE，OVA-sIgE) 水平，BALF中炎症细胞数量以及组胺和Th2细胞因子(IL-4、IL-5和IL-13)水平，增加抗炎因子(IL-10和IFN-γ)的分泌，减轻肺组织炎症细胞的浸润、黏液分泌及气道平滑肌的增生。且BMMC-胞外体抑制过敏反应的效应随着治疗时间的延长而增强。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种胞外体制剂，其特征在于，包括：
    胞外体，所述胞外体的大小为30-100nm，所述胞外体由体外经培养的肥大细胞分泌形成的；和
    药学上可接受的载体。
  2. 如权利要求1所述的制剂，其特征在于，所述制剂中，所述胞外体的含量(v/v)为0.001-30％，较佳地0.05-15％，更佳地0.1-10％，按所述制剂的总体积计。
  3. 如权利要求1所述的制剂，其特征在于，所述胞外体中的总蛋白的浓度为0.01-10mg/mL，较佳地0.05-5mg/mL，更佳地0.5-3.5mg/mL；最佳地1-3mg/mL。
  4. 一种胞外体，其特征在于，所述胞外体含有位于外表面的FcεRI蛋白，所述胞外体的大小为30-100nm，并且所述胞外体由体外经培养的肥大细胞分泌形成，并且所述的FcεRI蛋白基本上处于未结合状态。
  5. 一种权利要求4所述的胞外体的用途，其特征在于，用于制备IgE介导的相关疾病的药物。
  6. 一种权利要求1所述制剂的用途，其特征在于，用于制备治疗IgE介导的相关疾病的药物。
  7. 一种体外非治疗性的抑制肥大细胞的活化的方法，其特征在于，包括步骤：
    在权利要求1所述的胞外体制剂或权利要求4所述的胞外体存在下，培养肥大细胞，从而抑制肥大细胞的活化。
  8. 如权利要求7所述的方法，其特征在于，所述胞外体的浓度为0.011-21000μg/mL，较佳地，0.0210-1500μg/mL，更佳地，0.250-0.6200μg/mL。
  9. 一种胞外体的制备方法，其特征在于，包括步骤：
    (i)提供一体外培养体系，所述培养体系含有骨髓来源肥大细胞和适合所述细胞生长的培养基，并且所述培养体系中还含有0.1-5mmol/L的L-谷氨酰胺，0.1-5mmol/L丙酮酸钠，以及5-15％(v/v)无胞外体的胎牛血清；
    (ii)在适合生长的条件下，培养1-5天，获得培养产物；和
    (iii)从所述培养产物中分离权利要求2所述的胞外体。
  10. 如权利要求9所述的方法，其特征在于，在步骤(i)的培养体系中，所述骨髓来源肥大细胞的浓度为1×105-1×107个/ml。
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Publication number Priority date Publication date Assignee Title
CN112029705B (zh) * 2020-08-31 2021-10-08 中国医学科学院微循环研究所 促内皮细胞产外泌体的方法、外泌体制剂和应用
CN114854688B (zh) * 2022-04-27 2023-09-26 广州医科大学附属第一医院（广州呼吸中心） 一种提高肥大细胞外泌体及其膜表面FcεRI的表达量的方法及其应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL116436A (en) * 1995-12-18 2006-12-31 Yissum Res Dev Co Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND
JP4662708B2 (ja) * 2001-08-17 2011-03-30 エクソセラ・エルエルシー エクソゾームにタンパク質を標的化する方法および組成物
CN1274364C (zh) * 2004-11-18 2006-09-13 李莉 抗FcεRIα单克隆抗体Fab片段与雷公藤红素的交联体
CN107412755A (zh) * 2009-07-02 2017-12-01 Ith免疫治疗控股公司 基于外泌体的癌症治疗
WO2011034597A1 (en) * 2009-09-17 2011-03-24 Children's Medical Center Corporation Diagnostic and therapeutic uses of soluble fc-epsilon receptor i for ige-mediated disorders
CN104487569A (zh) * 2012-05-10 2015-04-01 生物材料细胞公司 间充质干细胞的成骨分化
CN104293738A (zh) * 2013-09-11 2015-01-21 李莉 一种抗人FcεRⅠα亚基单克隆抗体及其应用
AU2014342477B2 (en) * 2013-11-01 2019-08-01 Immunitybio, Inc. Tumoricidal and antimicrobial compositions and methods
CN105597088A (zh) * 2016-02-02 2016-05-25 广州赛莱拉干细胞科技股份有限公司 一种制剂、其制备方法及其应用
CN108324735B (zh) * 2017-01-20 2024-02-09 李莉 用于疾病治疗的胞外体制剂及其应用
WO2022149779A1 (ko) * 2021-01-11 2022-07-14 주식회사 엑소코바이오 과발현된 Fc 수용체 또는 이의 일부를 포함하는 엑소좀 및 이의 제조방법
CN114854688B (zh) * 2022-04-27 2023-09-26 广州医科大学附属第一医院（广州呼吸中心） 一种提高肥大细胞外泌体及其膜表面FcεRI的表达量的方法及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI, F ET AL.: "Mast Cell -Derived Exosomes Promote Th2 Cell Differentiation via 0X40L-0X40 Ligation", JOURNAL OF IMMUNOLOGY RESEARCH, vol. 2016, 15 March 2016 (2016-03-15), pages 1 - 10, XP055504526, ISSN: 2314-8861, DOI: doi:10.1155/2016/3623898 *
REN, YANLI ET AL.: "Mast Cell Negative Regulators and Allergic Diseases", CHINESE JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 7, no. 6, 25 November 2008 (2008-11-25), pages 485 - 487, ISSN: 1671-6205 *
XIAO, HUI ET AL.: "the Role of the Mast Cell Derived Exosome in Non-small Cell Lung Carcinomas", CDFD, 15 April 2016 (2016-04-15), pages E072 - 15, ISSN: 1674-022X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108324735A (zh) * 2017-01-20 2018-07-27 李莉 用于疾病治疗的胞外体制剂及其应用
CN108324735B (zh) * 2017-01-20 2024-02-09 李莉 用于疾病治疗的胞外体制剂及其应用

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