Exosome preparation for treating disease and application thereof

Abstract

Provided are an exosome preparation formed by secretion by a mast cell cultured in vitro, a preparation method of an exosome therein, an exosome containing FcεRI protein on an outer surface thereof and in a substantially unbound state, and uses of the exosome preparation or exosome in a method for inhibiting mast cell activation in vitro and in preparing a drug for treating an IgE-mediated disease.

Claims

1. A method of treating an IgE-mediated related disease, comprising: administering an exosome formulation to a subject in need of treatment, wherein: the exosome formulation comprises an exosome with a size of 30-100 nm and a pharmaceutically acceptable carrier; the exosome is formed by the secretion of cultured mast cells in vitro; and the exosome contains an FcεRI protein located on the outer surface, wherein the IgE-mediated related disease is an allergic disease, and wherein the exosome is obtained by induction of 4 weeks with a combination of interleukin 3 (IL-3) and stem cell factor (SCF).

2. A method of treating an IgE-mediated related disease, comprising: administering an exosome of to a subject in need of treatment, wherein: the exosome comprises an FcεRI protein located on an outer surface; the exosome has a size of 30-100 nm; the exosome is formed by the secretion of cultured mast cells in vitro; and the FcεRI protein is substantially in an unbound state, wherein the IgE-mediated related disease is an allergic disease, and wherein the exosome is obtained by induction of 4 weeks with a combination of IL-3 and SCF.

Description

DESCRIPTION OF FIGURE

(1) FIG. 1 shows: A. the exosome is a circular structure with a diameter of 50-80 nm under electron microscope; and B. Western blot shows the exosome expressing exosome characteristic molecule TSG101 protein and CD81 protein, and FcεRI was expressed on the BMMC cell and the exosome thereof, while FcεRI was not expressed on the P815 cell and the exosome thereof.

(2) FIG. 2 shows the dose-effect relationship between the dose of the exosome and the relative fluorescence intensity (rFI) of BMMC. The red line shows the amount of IgE not bound to exosome detected by flow cytometry after different concentrations of P815-exosome (not expressing FcεRI) were pre-incubated with IgE for 2 h, then incubated with BMMC, and FITC-labeled anti-IgE antibody was added after washing. The blue line shows the remaining amount of IgE detected after different concentrations of BMMC-exosome (expressing FcεRI) were pre-incubated with IgE for 2 h. The green line shows the remaining amount of IgE detected after different concentrations of BMMC-exosome, IgE and BMMC were simultaneously incubated, and FITC-labeled anti-IgE antibody was added.

(3) FIG. 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. Among them, group a represents the control mice that is not sensitized and challenged; the mice in groups b-d are sensitized and challenged by OVA, wherein group b is given saline treatment, group c is given exosome for the treatment of one month, and group d is given exosome for the treatment of 2 months, group e is given exosome for the treatment of 3 months.

(4) FIG. 4 shows the effect of BMMC-exosome on inflammatory cell infiltration in lung tissue of mice with allergic asthma. The lung tissue in mice is fixed by formaldehyde, embedded in paraffin, sectioned, HE stained. a represents lung tissue of control mice that is not sensitized and challenged; b represents lung tissue of allergic mice treated with saline, and c represents the lung tissue of allergic mice given exosome treatment for one month, and group d represents the lung tissue of allergic mice given exosome treatment for 2 months, and e represents the lung tissue of allergic mice given exosome treatment for 3 months.

DETAILED DESCRIPTION

(5) After an extensive and in-depth study, the present inventors have unexpectedly discovered that the mast cell cultured under specific conditions in vitro can secrete the specific exosome containing a very abundant FcεRI protein located on the surface, and the FcεRI protein is essentially in an unbound state. The FcεRI protein-containing exosome can competitively bind to IgE, thereby significantly inhibiting the binding of IgE to the mast cell, reducing the binding rate of IgE to the mast cell, thereby extremely effectively eliminating serum IgE. Furthermore, experiments have shown that the FcεRI protein-containing exosome of the present invention can also unexpectedly inhibit the activation of the mast cell and treat IgE-mediated related diseases such as allergic asthma. On this basis, the inventors complete the present invention.

(6) As used herein, the terms “exosome”, “FcεRI-exosome” can be used interchangeably and refer to an exosome containing an FcεRI protein located on the outer surface which is secreted by the mast cell cultured in vitro (preferably, the bone marrow-derived mast cell).

(7) Exosome

(8) Exosomes (exosome or vesicular body) are the membrane vesicles secreted by cells that carries certain surface molecules and contents of metrocytes (proteins, RNA, etc.) and are important mediators of signal transmission between cells. The exosome is widely distributed and has high biological activity and low immunogenicity, and is a microcosm of the cells from which it is derived. The use of the exosomes as carriers to deliver target molecules and drugs has become a hot topic in biotherapy and vaccine development in recent years.

(9) The study has shown that mast cell-derived exosome carries various membrane receptors, ligands, adhesion molecules, costimulatory signaling molecules, and nucleic acid molecules (miRNAs and microRNAs) such as FcεRI and KIT, which may be an important way for mast cells to interact with other cells and membrane proteins.

(10) In the present invention, based on the low immunogenicity of the exosome of the autologous mast cell, the FcεRI carried by the mast cell-derived exosome neutralizes the serum IgE, blocking the binding of IgE to IgE high affinity receptor FcεRI on the surface of mast cells and basophils, inhibiting the activation of the mast cell and alleviating the progression of the disease, which may be an effective way to treat IgE-related diseases.

(11) Exosome Formulation

(12) The exosome formulation of the present invention contains a safe and effective amount of an exosome together with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be matched to the mode of administration.

(13) In a preferred embodiment, the exosome is 30-100 nm in size and the exosome is secreted by cultured mast cells in vitro.

(14) In a preferred embodiment, the formulation of the present invention may also contain a safe and effective amount of albumin which has the effect of stabilizing the formulation. Studies by the inventors have shown that the addition of a certain proportion of albumin can significantly improve the therapeutic effect and stability of the FcεRI-exosome of the present invention.

(15) The pharmaceutical composition of the present invention can be formulated into a liquid formulation which can be prepared by a conventional method, and the liquid formulation is preferably prepared 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 milligrams per kilogram body weight. In addition, the formulation of the present invention may also be used with other therapeutic agents.

(16) When a formulation of the present invention is used, 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. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are all within the skill of a skilled physician.

(17) Preparation Method of FcεRI-Exosome and the Formulation

(18) In the present invention, cells expressing FcεRI on the surface (primary cultured mast cells, basophils, mast cell lines, basophils, other cells expressing FcεRI through transfection) are used as a source of the exosome. The formed exosome 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).

(19) In the present invention, the formulation or pharmaceutical composition of the present invention can be obtained by mixing the prepared FcεRI-exosome with a pharmaceutically acceptable excipient.

(20) In the present invention, a preferred formulation is a liquid FcεRI-exosome formulation.

(21) Typically, a method of preparing an exosome formulation comprises the steps of:

(22) 1) preparing FcεRI-exosome by ultracentrifugation or Fibercell™ 3D hollow fiber cell culture system;

(23) 2) adding albumin to a medically acceptable solvent, the volume ratio of which is 0.5-50%;

(24) 3) adding the obtained exosome into a medically acceptable solvent in a volume ratio of 0.1-30% (V/V), and the content of the exosome is 0.03-10 mg/mL, thereby obtaining the exosome formulation.

(25) Usage

(26) The exosome containing unbound FcεRIα on the surface (FcεRI-exosome) prepared by the present invention can effectively neutralize free IgE in vivo, reduce binding of IgE to mast cell membrane receptor, thereby reducing mast cell degranulation and inhibiting allergic reaction, which can gradually reduce the number of mast cells and the expression of IgE receptors, thereby achieving the purpose of treating allergies and IgE-related diseases.

(27) Typically, an exosome or an exosome formulation (or a corresponding drug) containing the exosome of the present invention can neutralize free IgE in vivo, reduce the binding of IgE to mast cell membrane receptors, thereby reducing mast cell degranulation and inhibiting allergy, thereby achieving the purpose of treating allergies and IgE-related diseases (such as allergic diseases (such as allergic asthma), autoimmune diseases, cardiovascular diseases).

(28) The Main Advantages of the Present Invention Include:

(29) (1) The FcεRI-exosome of the present invention is obtained by extracting from a cell culture supernatant, which is easy to be prepared and can be derived from autologous cells, and is non-immunogenic.

(30) (2) The FcεRI-exosome of the present invention neutralizes the free IgE in the serum, and reduces the binding of IgE to FcεRI-positive cells, including mast cells, basophils, macrophages, etc., and can be used for IgE-mediated diseases, including allergies, autoimmune diseases, cardiovascular diseases, etc.

(31) (3) The present invention discloses for the first time an exosome formulation containing FcεRI-exosome, which can effectively neutralize free IgE in the serum and reduce the binding of IgE to FcεRI-positive cells, thereby treating IgE-mediated related diseases.

(32) (4) The present invention discloses for the first time an exosome formulation containing FcεRI-exosome, which can also reduce the expression level of FcεRI on the cell surface of mast cells, basophils, dendritic cells and the like, and reduce the activation of mast cells to treat IgE-mediated related diseases.

(33) The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only for illustrating the present invention and not intended to limit the scope of the present invention. The conditions of the experimental methods not specifically indicated in the following examples are usually in accordance with conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the Journal of Microbiology: An Experimental Handbook (edited by James Cappuccino and Natalie Sherman, Pearson Education Press) or the manufacturer's proposed conditions.

(34) Unless otherwise stated, the materials used in the examples are all commercially available products.

Example 1 Preparation of FcεRI-Exosome

(35) In this example, normal mouse bone marrow-derived mast cells (BMMC) were used as the source cells of the exosomes. Methods are shown as below: Bone marrow cells were extracted from the tibia and femur of mice (purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd.), induced by interleukin 3 (IL-3) and stem cell factor (SCF) for 4 weeks, thereby obtaining the murine bone marrow derived mast cells.

(36) The fetal bovine serum was taken, and the exosomes were removed by ultracentrifugation (100,000 g for 90 min) to obtain the serum without the exosomes.

(37) Mouse bone marrow-derived mast cells and commercially available 1640 medium (added with a final concentration of 1 mmol/L glutamine and a final concentration of 1 mmol/L sodium pyruvate, and containing 10% of the serum without the exosomes) were mixed, and the cell concentration was adjusted to be 1×10.sup.6/ml, and the mixture was cultured at 37° C. for 48 hours; the culture solution was collected, centrifuged at 300 g for 10 min, and the supernatant was retained, and 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, and the cell pellets were removed, and the cell debris and apoptotic bodies were removed; the supernatant was centrifuged at 120,000 g for 90 min, and the supernatant was removed, and the pellet was collected, thereby obtaining the exosome, which was then resuspended in PBS buffer, and the protein concentration was measured with BCA kit.

(38) The result is shown in FIG. 1. The results of electron microscopy show that the exosome is 30-100 nm and the total protein concentration is 3 mg/mL. In addition, almost 100% of the Fc ε RI protein is in an unbound state (i.e., the Fc ε RI protein is unbound).

Example 2 Preparation of FcεRI-Exosome Formulation

(39) 900 μL of physiological saline was added into 50 μL of 20% human albumin to a final mass concentration of 1% (wt/wt); and 50 μL of 4×10.sup.8 cell-extracted exosome was added to obtain an exosome formulation. The absolute amount of total protein in the exosome was 2 mg.

Example 3 Binding Ability of FcεRI-Exosome to IgE

(40) FcεRI-exosome (BMMC-extrasome) formulation and the FcεRI(−)-exosome (P815-exosome) formulation were prepared according to the methods of Example 1 and Example 2 using BMMC (FcεRI-expressing) and P815 cells (without expressing FcεRI) (purchased from ATCC) as the source cells of the exosomes.

(41) The exosomes were incubated with IgE for 2 h and then was added to BMMC for incubation, and in the other group, BMMC-exosome and IgE were simultaneously added into BMMC for incubation. After incubation with each cells for each group, it was washed and the fluorescein isothiocyanate (FITC)-labeled anti-IgE antibody was added, and flow cytometry was used to detect the fluorescence of the surface of the BMMC.

(42) The result is shown in FIG. 2. The results show that BMMC-exosome can inhibit the binding of IgE to BMMC, and as the dose of BMMC-exosome increases, the binding rate of IgE to BMMC decreases, which is negatively correlated; however, P815-exosome has no significant effect on the binding rate of IgE and BMMC.

Example 4 FcεRI-Exosome Inhibits IgE-Mediated Mast Cell Activation

(43) The effect of FcεRI-exosome on mast cell activation was examined using β-hexosidase release and histamine release assays.

(44) FcεRI-exosome (BMMC-exo some) formulation was prepared according to Example 1 and Example 2, and BMMC-exosome (100 μg) was incubated with IgE for different time (0.5, 1, 2, 4 h), and then added to BMMC suspension, and incubated for 1 h at 37° C. for sensitization; after being washed, DNP-HSA (0.5 μg/ml) was added to stimulate, and the cell supernatant (A) was collected, and the cell pellet was lysed and the lysate was collected, and the supernatant (B) was obtained by centrifugation. The β-hexosidase and histamine concentrations were measured, and the release rate was calculated, and the release rate=A concentration×A volume/(A concentration×A volume+B concentration×B volume).

(45) The results show that the release rates of the β-hexosidase and histamine of mast cells in the BMMC-exosome group have decreased compared with the non-BMMC-exosome group, and with the increase of the incubation time, the release rates of the β-hexosidase and histamine have decreased, indicating that BMMC-exosome can inhibit the activation of BMMC.

Example 5 FcεRI-Exosome Alleviates Allergic Asthma in Mice

(46) An FcεRI-exosome (BMMC-exosome) formulation was prepared according to Example 1 and Example 2.

(47) Construction of a mouse model of allergic asthma: the mice were sensitized by intraperitoneal injection of pre-mixed OVA (20 μg) and the adjuvant aluminum hydroxide (4 mg) mixture on days 0, 7, and 14, and on the day 21, the 1% OVA solution was used for aerosol inhalation, 30 min/time/day, and continuous inhalation for 5 days.

(48) Grouping: BALB/c mice (purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd.) were randomly divided into 5 groups: control group (group A), asthma group (group B), treatment group for 1 month (group C), treatment group for 2 months (group D), and treatment group for 3 months (group E) respectively. Wherein in the control group, it was sensitized and stimulated with normal saline; in the asthma group, it was sensitized and stimulated with OVA; in the treatment group for one month, the inhalation was carried out with 1% OVA from day 32, 30 min/time/day, twice a week, and 40 μg/150 μL of BMMC-exosome was injected into the tail vein 30 minutes before the weekly stimulation for a total of 4 injections. In the treatment group for 2 months and the treatment group for 3 months, the treatment method was the same with that in the treatment group for 1 month, except that the number of injections was 8 and 12 times respectively.

(49) Airway High Reactivity (AHR) was detected after the last stimulation of each group of mice; serum, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to detect the number of inflammatory cells, the levels of inflammatory mediators, and pathological changes in lung tissue.

(50) The results are shown in FIGS. 3 and 4. The results show that BMMC-exosome can effectively improve AHR in asthmatic mice, reduce OVA-specific IgE (OVA-sIgE) levels in the serum of asthmatic mice, the number of inflammatory cells in BALF, and histamine and Th2 cytokines (IL-4, IL-5 and IL-13) levels, increase the secretion of anti-inflammatory factors (IL-10 and IFN-γ), and reduce the infiltration of inflammatory cells in the lung tissue, mucus secretion and proliferation of airway smooth muscle. Moreover, the effect of BMMC-exosome on the inhibition of allergic reactions increases with prolonged treatment time.

(51) All publications mentioned herein are incorporated by reference as if each individual document was cited as a reference, as in the present application. It should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications, equivalents of which falls in the scope of claims as defined in the appended claims.