Therapeutic drug for acute pulmonary disorder

Abstract

This therapeutic drug for acute pulmonary disorder comprises, as an active ingredient, extracellular vesicles derived from airway epithelial cells.

Description

Acute lung injury treatment

[0001] The present invention relates to a therapeutic agent for acute lung injury.

[0002] Acute lung injury (ALI), also known as acute respiratory distress syndrome (ARDS), is a respiratory disease with a poor prognosis and a potentially fatal respiratory failure induced by various factors.

[0003] The inventors have previously found that extracellular vesicles derived from airway epithelial cells are effective as a therapeutic agent for pulmonary fibrosis (see Patent Document 1).

[0004] International Publication No. 2020 / 209304

[0005] However, there is no fundamental therapeutic agent for ALI yet, and the development of such a therapeutic agent is urgently needed. Therefore, an object of the present invention is to provide a therapeutic agent for acute lung injury.

[0006] The present invention includes the following aspects. [1] A therapeutic agent for acute lung injury, comprising extracellular vesicles derived from airway epithelial cells as an active ingredient. [2] The therapeutic agent for acute lung injury according to [1], wherein the extracellular vesicles derived from airway epithelial cells express ANXA1 (annexin A1). [3] The therapeutic agent for acute lung injury according to [1], wherein the extracellular vesicles derived from airway epithelial cells contain at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p. [4] The therapeutic agent for acute lung injury according to [1], which is administered via the respiratory tract. [5] A composition for treating acute lung injury, comprising the therapeutic agent for acute lung injury according to any one of [1] to [4] and a pharmaceutically acceptable excipient. [6] The composition for treating acute lung injury according to [5], further comprising a recombinant ANXA1 protein. [7] The composition for treating acute lung injury according to [5], further comprising an FPR2 (formyl peptide receptor 2) agonist. [8] The composition for treating acute lung injury according to [5], further comprising at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p. [9] A quality evaluation kit for a therapeutic drug for acute lung injury according to any one of [1] to [4], comprising an anti-ANXA1 antibody and / or a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or an amplification product thereof.

[10] A method for evaluating the quality of a therapeutic agent for acute lung injury according to any one of [1] to [4], comprising confirming the expression of ANXA1 and / or at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in the extracellular vesicles derived from the airway epithelial cells.

[11] A method for predicting prognosis, comprising measuring in vitro the expression level of at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in a sample derived from a patient with acute lung injury, and evaluating the prognosis for the patient.

[12] A kit for predicting the prognosis of patients with acute lung injury, comprising a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or its amplification product.

[0007] The present invention contributes to the treatment of acute lung injury.

[0008] (A) Cryo-transmission electron microscopy image of purified HBEC-EVs using ultracentrifugation. (B) Results of nanoparticle tracking analysis showing particle size of purified HBEC-EVs. The vertical axis of the graph represents the number of EV particles (×10 8) / mL, and the horizontal axis represents the particle size (nm) of EVs. (A) Photograph of a gel showing the results of electrophoresis. The left lane contains a ladder standard, and the right lane contains total RNA from HBEC-EVs. (B) Analysis of HBEC-EVs using a bioanalyzer. The y-axis of the electropherogram displays signal intensity in arbitrary fluorescence units (FU), and the x-axis displays RNA size in nucleotides (nt). This figure shows the results of a multiplexed bead-based flow cytometry assay to detect HBEC-EV surface signatures. A multiplexed population of 37 capture beads coated with dye-conjugated antibodies was incubated with three different HBEC-EV samples. This graph shows the results of analyzing the mean fluorescence intensity (MFI) of each marker. ND: Not detected. Western blot results of HBECs and HBEC-EVs for CD9, CD63, CD81, MHC-2, and β-actin. Cytokine secretion in THP-1 cells 24 hours after administration of LPS (1 μg / ml) and HBEC-EVs (10 μg / ml). THP-1 cells were treated with 100 nM PMA for 24 hours prior to the experiment. ****P <0.0001, ***P <0.001. NS; not significant. Cytokine secretion in HBECS 8 hours after administration of poly(I:C) (250 ng / ml) and HBEC-EVs (10 μg / ml). THP-1 cells were treated with 100 nM PMA for 24 hours prior to the experiment. ***P <0.001, **P <0.01, *P <0.05. NS; not significant. (A) miRNA expression profile in HBEC-EVs obtained through miRNA-seq analysis (GSE156572). The top 10 expressed miRNAs in HBEC-EVs were ranked in descending order of expression. (B) Gene ontology analysis results from DIANA-mirPath v3.0 analysis. Based on the 10 most abundant miRNAs in HBEC-EVs, 12 important immune response pathways are listed among the top 50 pathways.This graph shows IL-6 mRNA expression in HBECs 8 hours after exposure to poly(I:C) (250 ng / ml) and transfection with each pre-miRNA. ****P <0.0001, ***P <0.001, **P <0.01, *P <0.05. NS; not significant. This graph shows IL-6 mRNA expression in THP-1 cells 24 hours after exposure to LPS (1 μg / ml) and transfection with each pre-miRNA. THP-1 cells were treated with 100 nM PMA for 24 hours before the experiment. ****P <0.0001, ***P <0.001, **P <0.01, *P <0.05. NS; not significant. This graph shows the results of GO analysis of proteins co-expressed in two different HBEC-EVs using the DAVID 2021 Cellular Component_ALL. This graph shows the results of GO analysis of proteins co-expressed in two different HBEC-EVs using DAVID 2021 Biological Process_Direct. (A) Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in HBECs treated with HBEC-EVs in the presence of poly(I:C) (250 ng / ml). (B) ELISA results showing the levels of IL-6 protein in HBECs treated with HBEC-EVs in the presence of poly(I:C) (250 ng / ml). (A) Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in THP-1 cells treated with HBEC-EVs in the presence of LPS (1 μg / ml). (B) ELISA results showing the levels of IL-6 protein in THP-1 cells treated with HBEC-EVs in the presence of LPS (1 μg / ml). THP-1 cells were treated with 100 nM PMA for 24 hours before the experiment. Table 1 shows the top 10 miRNAs shown to directly target NF-κB pathway-related genes in humans and mice. Western blot results of HBECs and HBEC-EVs for ANXA1, CD9, and β-actin.Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in HBECs treated with recombinant ANXA1 (rANXA1) in the presence of poly(I:C) (250 ng / ml). Expression of FPR1, FPR2, and FPR3 mRNA was examined in HBECs 24 hours after poly(I:C) exposure (250 ng / ml). ***P <0.001, **P <0.01. NS; not significant. Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in HBECs treated with HBEC-EVs in the presence of poly(I:C) (250 ng / ml) and WRW4 (10 μM). Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in type II alveolar epithelial cells (ATII) treated with HBEC-EVs in the presence of poly(I:C) (250 ng / ml) and WRW4 (10 μM). Figure 1 shows a schematic protocol for intratracheal (it) HBEC-EVs treatment in a mouse model of ALI induced by both LPS (intraperitoneal: ip) and poly(I:C) (it). Photographs of BALF samples from ALI model mice. Graphs showing the total cell and neutrophil counts in BALF from ALI model mice (untreated n=12, ALI_PBS n=15, ALI_HBEC-EVs n=15). ****P <0.0001, ***P <0.001, **P <0.01). HE staining images of representative lung sections from each group of treated mice. Scale bar, 100 μm. Graph showing pulmonary inflammation scores from HE staining of lung sections from each group of treated mice (Untreated n=8, ALI_PBS n=12, ALI_HBEC-EV n=12). ****P<0.0001, **P<0.01. Graph showing IL-6 and TNF-α protein concentrations measured by ELISA in BALF from each group of treated mice (Untreated n=8, ALI_PBS n=7, ALI_HBEC-EVs n=10). ****P<0.0001, **P<0.01, *P<0.05.Immunohistochemical staining of FPR2 in representative lung sections from a non-treated group and a mouse model of ALI induced by both LPS and poly(I:C). The open arrow indicates a cluster of FPR2-positive macrophages, and the closed arrow indicates an FPR2-positive alveolar epithelial cell. Scale bar, 100 μm. Western blot results showing the levels of p-NF-κB, NF-κB, and β-actin in the lungs of mice from each treatment group. This figure represents a potential therapeutic strategy for ALI through immune modulation by HBEC-EVs.

[0009] [Therapeutic Agent for Acute Lung Injury] In one embodiment, the present invention provides a therapeutic agent for acute lung injury, which comprises extracellular vesicles (EVs) derived from airway epithelial cells as an active ingredient.

[0010] In this embodiment, extracellular vesicles (hereinafter also referred to as EVs) refer to vesicles secreted from cells. EVs also include vesicles such as exosomes, microvesicles, and apoptotic bodies. The surface of EVs contains lipids and proteins derived from the cell membrane, and the interior contains intracellular substances such as nucleic acids and proteins.

[0011] Airway epithelial cell-derived EVs preferably express ANXA1 (annexin A1 (Gene ID: 301)). Gene IDs represent accession numbers in the Gene database (URL: http: / / www.ncbi.nlm.nih.gov / gene / ). ANXA1 plays an important role in regulating inflammation and immune responses. ANXA1 exerts its anti-inflammatory effects by binding to specific cellular receptors, such as FPR2, and inhibiting the activation of pro-inflammatory pathways, particularly NF-κB signaling. This regulatory role makes ANXA1 essential for maintaining immune balance and tissue protection. As described in the Examples section, ANXA1 was identified as a highly expressed protein from a pool of 751 EV surface proteins and 2558 ligands. In this study, we found that recombinant ANXA1 ligand significantly suppressed p-NF-κB expression in poly(I:C)-treated human bronchial epithelial cells (HBECs). The ANXA1 expressed in airway epithelial cell-derived EVs is not particularly limited, and ANXA1 may be artificially overexpressed in airway epithelial cell-derived EVs by genetic manipulation such as genome editing.

[0012] Airway epithelial cell-derived EVs preferably contain at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p. As described later in the Examples, when the top 10 highly expressed miRNAs encapsulated in HBEC-EVs were evaluated, it was found that these nine miRNAs significantly reduced IL-6 expression in cells treated with LPS or poly(I:C). The base sequences of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p are represented by SEQ ID NOs: 1 to 9, respectively. The miRNA contained in airway epithelial cell-derived EVs preferably contains at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, with two types being more preferred, three types being more preferred, four types being more preferred, five types being more preferred, six types being more preferred, seven types being even more preferred, and eight types being even more preferred, with a combination of nine types, namely miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, being most preferred.

[0013] EVs can be prepared, for example, by culturing airway epithelial cells in a medium and recovering EVs from the culture supernatant. The culture temperature is the same as that for mammalian cells, preferably 30°C to 40°C. The culture period is preferably 1 to 10 days, more preferably 1 to 5 days. Airway epithelial cells are cultured preferably for 1 to 5 days, more preferably 1 to 3 days.

[0014] Methods for recovering EVs from culture medium include ultracentrifugation, immunoprecipitation, gel filtration, ultrafiltration, polymer precipitation, HPLC, and FACS, with ultracentrifugation being preferred.Ultracentrifugation combines crude centrifugation with filtration through a 0.22 μm filter to recover EVs primarily composed of exosomes with a diameter of approximately 30-200 nm.

[0015] In this embodiment, the cells may be primary cultured cells, subcultured cells, or immortalized cultured cells. The species from which the cells are derived is not limited, but mammals are preferred. Examples of mammals include humans, chimpanzees, monkeys, rats, mice, pigs, cows, horses, sheep, goats, dogs, and cats, with humans and mice being preferred, and humans being more preferred.

[0016] The amount of EVs contained in the therapeutic agent of this embodiment can be appropriately determined taking into account various factors such as the subject's gender, weight, age, and symptoms. In the case of an inhalant, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of the active ingredient may be administered in a single inhalation. In the case of oral administration, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of the active ingredient may be administered per day. In the case of an injection, for example, 0.1 μg to 1 g, for example, 0.001 to 200 mg of the active ingredient may be administered per day. In the case of a suppository, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of the active ingredient may be administered per day. In the case of a topical skin preparation, for example, 1 μg to 10 g, for example, 0.01 to 2000 mg of the active ingredient may be administered per day.

[0017] The dosage form is not particularly limited and may be selected appropriately as needed. Generally, the dosage form may be oral, such as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs, or may be administered as injections, infusions, suppositories, inhalants, transmucosal absorbents, or sprays. Injections are administered intravenously, either alone or mixed with standard fluids such as glucose or amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally, as needed. Suppositories are administered rectally. Topical skin preparations are applied, patched, or sprayed onto the affected area. Inhalants are administered using devices such as nebulizers, metered-dose inhalers, and dry powder inhalers.

[0018] The therapeutic agent of this embodiment may be administered, for example, via the respiratory tract, inhalation, aerosol administration, injection, instillation, oral, transdermal, nasal, topical, vaginal, or rectal routes.

[0019] The frequency of administration may be three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, etc. The administration period may be one day, two days, three days, one week, two weeks, one month, six months, a year, or longer.

[0020] [Composition for treating acute lung injury] In one embodiment, the present invention provides a composition for treating acute lung injury, comprising the above-mentioned therapeutic agent for acute lung injury and a pharmaceutically acceptable additive.

[0021] The composition for treating acute lung injury of this embodiment can be administered orally in the form of, for example, tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, elixirs, etc., or parenterally in the form of injections, drip infusions, suppositories, inhalants, transmucosal absorbents, sprays, etc.

[0022] The composition for treating acute lung injury of this embodiment can be used without any particular limitation with pharmaceutically acceptable additives commonly used in the formulation of pharmaceutical compositions. More specifically, examples include excipients such as starch and crystalline cellulose; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; swelling agents such as alginic acid; injectable solvents such as water, ethanol, and glycerin; adhesives such as rubber-based adhesives and silicone-based adhesives; lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and benzyl alcohol; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc. The additives can be used alone or in combination of two or more. The additives can be used alone or in combination of two or more as a carrier for the therapeutic agent for acute lung injury.

[0023] The composition for treating acute lung injury of this embodiment may further contain recombinant ANXA1. ANXA1 is a membrane-localized protein that binds to phospholipids, inhibits phospholipase A2, and has anti-inflammatory activity. As an example, the amino acid sequence of the human ANXA1 protein used in the examples is shown in SEQ ID NO: 10. The recombinant ANXA1 contained in the composition for treating acute lung injury of this embodiment is not particularly limited as long as it significantly suppresses the expression of p-NF-κB, and includes mutants such as variants.

[0024] The composition for treating acute lung injury of this embodiment may further contain a formyl peptide receptor 2 (FPR2) agonist. FPR2 (FPRL-1, ALXA4) is a G protein-coupled receptor expressed in T cells and inflammatory cells. The FPR2 agonist is not particularly limited as long as it binds to the FPR2 receptor and has agonistic activity, and may be a peptide or a low-molecular-weight compound. Examples of FPR2 agonists include WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-CONH2) and MMK-1 (LESIFRSLLFRVM).

[0025] The composition for treating acute lung injury of this embodiment may further contain at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p. The miRNA contained in the composition for treating acute lung injury preferably contains at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, more preferably two types, more preferably three types, more preferably four types, more preferably five types, more preferably six types, even more preferably seven types, even more preferably eight types, and most preferably a combination of nine types: miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p.

[0026] Other Embodiments In one embodiment, the present invention provides airway epithelial cell-derived EVs for the treatment of acute lung injury.

[0027] In one embodiment, the present invention provides a method for treating acute lung injury, comprising administering an effective amount of EVs derived from airway epithelial cells to a patient in need of treatment.

[0028] In one embodiment, the present invention provides use of EVs derived from airway epithelial cells for producing a therapeutic agent for acute lung injury or a composition for treating acute lung injury.

[0029] [Quality Assessment Kit] In one embodiment, the present invention provides a quality assessment kit for the above-mentioned acute lung injury therapeutic drug, comprising an anti-ANXA1 antibody and / or a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or its amplification product. As described below in the Examples, nine highly expressed miRNAs encapsulated in HBEC-EVs significantly reduced IL-6 expression in cells treated with LPS or poly(I:C), and recombinant ANXA1 ligand significantly suppressed p-NF-κB expression in poly(I:C)-treated HBECs. These findings suggest that the expression levels of ANXA1 and / or miRNAs in airway epithelial cell-derived EVs are related to the efficacy of acute lung injury therapeutics. Therefore, evaluating the expression levels of ANXA1 and / or miRNAs in airway epithelial cell-derived EVs can be used to assess the efficacy of acute lung injury therapeutics.

[0030] Furthermore, in addition to the above antibody, the kit may include an ELISA kit for detecting the ANXA1 protein.

[0031] [Quality Assessment Method] In one embodiment, the present invention provides a method for assessing the quality of a therapeutic drug for acute lung injury, which involves confirming the expression of ANXA1 and / or at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in extracellular vesicles derived from airway epithelial cells. The quality assessment method of this embodiment may also use the quality assessment kit described above. For example, the quality of the therapeutic drug for acute lung injury can be controlled by assessing EVs recovered from the culture supernatant of airway epithelial cells using the quality assessment method of this embodiment.

[0032] [Prognosis Prediction Method] In one embodiment, the present invention provides a prognosis prediction method comprising measuring in vitro the expression level of at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in a sample from a patient with acute lung injury, and assessing the prognosis of the patient. For example, the expression level of the gene in the patient sample is compared with the control expression level of a control subject known to have a good prognosis, and if the expression level of the gene in the sample matches or is close to the control expression level of the control subject known to have a good prognosis, the patient's prognosis can be predicted to be good. Furthermore, for example, by comparing the expression level of the gene in a sample derived from a patient with the control expression level of a control subject known to have a poor prognosis, if the expression level of the gene in the sample matches or is close to the control expression level of the control subject known to have a poor prognosis, it can be predicted that the patient will have a poor prognosis.

[0033] In this embodiment, examples of the specimen include biopsy samples, blood, urine, saliva, sweat, tissue exudates, etc. Examples of blood include serum and plasma. Methods for extracting nucleic acids (e.g., total RNA) from the specimen include methods using an RNA extraction reagent containing acidic phenol, and are performed according to standard procedures.

[0034] Methods for detecting miRNA in patient samples include amplifying specific miRNA fragments by PCR using primers and analyzing the amplified products, or analyzing specific miRNAs by hybridization using probes complementary to the specific miRNAs. From the perspective of quantitative analysis, it is preferable to amplify specific miRNA fragments by PCR and analyze the amplified products. Specific quantitative methods include next-generation sequencing (NGS) and real-time PCR (RT-PCR).

[0035] [Prognosis Prediction Kit] In one embodiment, the present invention provides a prognosis prediction kit for patients with acute lung injury, comprising a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or its amplification product. The kit of this embodiment is suitable for use in the prognosis prediction method described above.

[0036] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0037] Characterization of HBEC-EVs: HBEC-EVs were isolated from conditioned medium obtained from primary cultures of HBECs by ultracentrifugation and extensively characterized. Cryo-transmission electron microscopy confirmed the presence of typical bilayer vesicles exhibiting size heterogeneity (see Figure 1(A)). NanoSight tracking analysis confirmed that the average diameter of HBEC-EVs was approximately 100 nm (see Figure 1(B)).

[0038] We assessed the RNA content of HBEC-EVs and confirmed that the RNA profile of HBEC-EVs contained minimal ribosomal RNA but significant amounts of small RNAs (Fig. 2(A)(B)). To comprehensively profile the surface markers of HBEC-EVs, we performed multiplex bead-based cytometry to simultaneously detect and semiquantitate 37 distinct EV surface epitopes.

[0039] In total, 24 of 37 markers (64.9%) were detected on HBEC-EVs. These surface markers were identified to encompass various categories, including tetraspanins (CD9, CD63, CD81), antigen-presenting proteins (MHC-1, MHC-2), immune cell markers (CD2, CD3, CD11c, CD14, CD20, CD24, CD25, CD41b, CD45, CD49e, CD56, CD86), endothelial cell markers (CD31, CD105, CD142), MSC markers (SSEA-4, CD29, CD44), and epithelial cell markers (EpCAM) (see Figure 3). The data indicated the presence of several immune-related markers, mostly at low to moderate intensity levels. Notably, MHC-1 showed the highest intensity, suggesting a potential immunomodulatory role for HBEC-EVs.

[0040] Furthermore, Western blot analysis confirmed the presence of EV marker proteins such as CD9, CD63, CD81, and MHC-2, but the actin cytoskeleton was not abundant in EVs (see Figure 4).

[0041] Immunomodulatory Potential of HBEC-EVs in a Cellular Model of ALI Several immune cells, including alveolar macrophages, play important roles in initiating the immune cascade that contributes to the development of ALI. To mimic immune activation in ALI, PMA-induced THP-1 macrophages were treated with LPS and HBEC-EVs. Exposure to LPS in THP-1 cells increased the expression levels of proinflammatory cytokines, such as IL-6, IL-8, and TNF-α, whereas treatment with HBEC-EVs attenuated these elevated levels (Figure 5). The immunosuppressive potential of HBEC-EVs in THP-1 cells was comparable to that of treatment with 1 μM dexamethasone (DEX). In contrast, lung epithelial cells respond to similar injuries and infections by regulating leukocyte influx through the production of proinflammatory cytokines and chemokines. To simulate proinflammatory epithelia, we evaluated HBEC-EVs and HBECs after the addition of poly(I:C). In particular, transfection of HBECs with poly(I:C) increased the expression levels of proinflammatory cytokines, including IL-6, IL-8, and TNF-α. However, this response was attenuated by HBEC-EV treatment (see Figure 6). The immunosuppressive potential of HBEC-EVs was consistent with that of 10 μM DEX. These findings highlight the immunomodulatory role of HBEC-EVs during and after the onset of pulmonary inflammation.

[0042] [Effect of HBEC-EV miRNAs on Immune Regulation] To elucidate the underlying mechanisms, we investigated the composition of the top 10 miRNAs encapsulated in HBEC-EVs (see Figure 7(A)). To understand the biological functions of these top 10 miRNAs in HBEC-EVs, we performed GO analysis using DIANA-mirPath and found that 12 of the top 50 pathways were related to the Toll-like receptor (TLR) signaling pathway (see Figure 7(B)). Further GO analysis revealed that miRNAs related to the regulation of immune responses and various inflammatory pathways were enriched.

[0043] Next, we examined IL-6 mRNA expression in PMA-induced THP-1 macrophages after LPS exposure and pre-miRNA transfection. qRT-PCR revealed that pre-miR-7-5p, -miR-125b-5p, let-7a-5p, -miR-125a-5p, -let-7b-5p, -let-7f-5p, -let-7i-5p, -miR-26a-5p, and -let-7c-5p (except -miR-16-5p) significantly reduced IL-6 expression in LPS-treated THP-1 cells (Figure 8). Next, we evaluated IL-6 mRNA expression in HBECs after poly(I:C) exposure and pre-miRNA transfection. Indeed, we confirmed that overexpression of these nine miRNAs significantly reduced IL-6 expression in HBECs treated with poly(I:C) (see Figure 9).

[0044] Proteomic Analysis of HBEC-EVs and Immune Modulation Through EV Protein Cargo Next, we performed proteomic analysis of HBEC-EVs using liquid chromatography-mass spectrometry (LC-MS / MS). To elucidate the biological roles of proteins in HBEC-EVs, we utilized DAVID 2021 for gene ontology (GO) enrichment analysis, focusing on 751 overlapping proteins found in two different HBEC-EVs. Consistent with the characteristics of EVs, GO analysis of cellular components demonstrated that the majority of identified proteins were exosome-associated and membrane-related (Figure 10). Furthermore, biological process analysis showed that HBEC-EV cargo was significantly enriched for proteins involved in regulating cell-cell adhesion, translation initiation, and mRNA stability. Notably, among the top 10 biological processes identified by GO analysis, enrichment of proteins related to the WNT signaling and NF-κB signaling pathways was observed (Figure 11). To elucidate novel molecular mechanisms underlying HBEC-EV-mediated immunomodulation, we investigated changes in the NF-κB signaling pathway in THP-1 cells and HBECs in response to EV treatment. THP-1 treatment resulted in a clear activation of NF-κB signaling, as indicated by p-NF-κB expression. Furthermore, HBEC-EV treatment significantly reduced p-NF-κB and IL-6 protein levels in poly(I:C)-treated HBECs (see Figures 12(A) and 12(B)) and LPS-treated THP-1 cells (see Figures 13(A) and 13(B)). Based on these data, we investigated the top 10 miRNAs present in HBEC-EVs involved in Toll-like receptor (TLR) signaling, as all TLR signaling pathways ultimately lead to activation of the NF-κB pathway. These top 10 miRNAs have been shown to directly target various NF-κB pathway-related genes in humans and mice (see Figure 14). These findings suggest that the TLR-NF-κB signaling pathway is involved in the mechanism responsible for HBEC-EVs-mediated immune regulation via protein and miRNA cargo.

[0045] Regulation of the NF-κB Pathway via ANXA1 / FPR Signaling in HBEC-EVs: To better understand how HBEC-EVs mediate immune regulation through NF-κB signaling, we focused on the interaction between EV ligands and receptors on recipient cells. Using the FANTOM5 tool, we identified eight common ligands from a pool of 751 EV surface proteins and 2558 ligands: transferrin (TF), guanine nucleotide-binding protein alpha inhibitory activity polypeptide 2 (GNAI2), laminin alpha 3 (LAMA3), annexin A1 (ANXA1), phospholipid transfer protein (PLTP), laminin alpha 5 (LAMA5), apolipoprotein E (APOE), and fibrillin 1 (FBN1), listed in descending order of expression. Among the eight overlapping ligands, ANXA1, a protein recognized for its potential to suppress NF-κB signaling, was selected for further analysis. Furthermore, Western blot analysis confirmed the presence of ANXA1 in HBEC-EVs (see Figure 15). Using recombinant ANXA1 ligand (rANXA1), we assessed the role of ANXA1 in HBEC-EV-mediated immunomodulation. We confirmed that rANXA1 treatment significantly suppressed p-NF-κB expression in poly(I:C)-treated HBECS (see Figure 16). rANXA1 treatment did not significantly suppress p-NF-κB expression in LPS-treated THP-1 cells. Using the FANTOM5 tool, ANXA1 interacts with receptors such as dysferlin (DYSF), epidermal growth factor receptor (EGFR), formyl peptide receptor (FPR)1, FPR2, and FPR3. Because FPRs, especially FPR2, are the primary ligands for ANXA1 in humans and are known for their important roles in regulating host defense and inflammatory responses, we focused on FPRs as potential receptors for EV ANXA1. ANXA1 primarily binds to FPR2, triggering an anti-inflammatory response and suppressing both innate and adaptive immune processes via the NF-κB and MAPK pathways.Furthermore, FPR2 has been reported to be primarily expressed in leukocytes, endothelial cells, and epithelial cells. After treatment with poly(I:C), we observed increased gene expression of FPR2 and FPR3 in HBECs (see Figure 17). In humans, FPR2 shares 72% amino acid sequence identity with FPR3. Consequently, we focused on investigating the role of EV ANXA1-FPR2 signaling in lung injury and inflammation. Importantly, blockade of FPR2 using the specific antagonist WRW4 reversed the HBEC-EV-mediated immunomodulatory effect on NF-κB signaling (see Figure 18). To further validate this experiment, we used type 2 alveolar epithelial cells (ATII cells) as recipient cells for HBEC-EV treatment. Primary ATII cells were isolated from human lung samples, cultured in a 2D environment, and immunofluorescence staining confirmed the expression of HT2-280 cells. + / SFTPC + Notably, blockade of FPR2 using WRW4 reversed the HBEC-EV-mediated immunomodulatory effect on NF-κB signaling in ATII cells (see Figure 19). ANXA1 was identified as playing a central role in the immunomodulation of HBEC-EVs via FPR2-NF-κB signaling in inflamed bronchial and alveolar epithelial cells.

[0046] HBEC-EVs attenuate ALI in vivo through the FPR-NF-κB signaling pathway. In this study, we used a mouse model of ALI induced by both LPS and poly(I:C) to investigate the physiological immunomodulatory effects of intratracheal HBEC-EV administration on lung inflammation during and after the onset of the disease (see Figure 20). Mice treated with HBEC-EVs showed significantly reduced BAL hemorrhage compared with the PBS control group, indicating a lower degree of lung injury after HBEC-EV treatment (see Figure 21). Furthermore, HBEC-EVs significantly reduced the total number of BAL cells and neutrophils compared with the PBS control group (see Figure 22). HE staining of lung tissue demonstrated that both LPS- and poly(I:C)-induced lung injury were alleviated by HBEC-EVs (see Figure 23). HBEC-EV treatment also significantly reduced peribronchial and perivascular inflammation (see Figure 24). Proinflammatory cytokine levels were measured using ELISA, and IL-6 and TNF-α levels were significantly reduced in the BALF of HBEC-EV-treated mice compared with the PBS control group (Figure 25). Furthermore, we evaluated the effect of HBEC-EVs on FPR-NF-κB signaling in the lungs of mice with ALI. In an LPS- and poly(I:C)-induced lung injury model, the presence of FPR2-positive alveolar epithelial cells and macrophage clusters was confirmed (Figure 26). Consistently, HBEC-EV treatment significantly suppressed p-NF-κB expression in the lungs of mice compared with the PBS control group (Figure 27). Collectively, this study provides evidence that intratracheal administration of HBEC-EVs can attenuate acute lung inflammation through cargo transport-mediated FPR-NF-κB signaling. HBEC-EVs may be useful as a potential therapeutic strategy for ALI through immunomodulation (Figure 28).

[0047] The present invention contributes to the treatment of acute lung injury.

Claims

1. A drug for treating acute lung injury that contains extracellular vesicles derived from airway epithelial cells as the active ingredient.

2. The therapeutic agent for acute lung injury described in claim 1, wherein the extracellular vesicles derived from airway epithelial cells express ANXA1 (annexin A1).

3. The therapeutic agent for acute lung injury described in claim 1, wherein the extracellular vesicles derived from airway epithelial cells contain at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p.

4. The therapeutic agent for acute lung injury according to claim 1, which is administered via the airway.

5. A composition for treating acute lung injury, comprising the therapeutic agent for acute lung injury according to any one of claims 1 to 4 and a pharmaceutically acceptable additive.

6. A composition for treating acute lung injury according to claim 5, further comprising recombinant ANXA1 protein.

7. The composition for treating acute lung injury according to claim 5, further comprising a formyl peptide receptor agonist.

8. The composition for treating acute lung injury according to claim 5, further comprising at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p.

9. A quality evaluation kit for a therapeutic drug for acute lung injury according to any one of claims 1 to 4, comprising an anti-ANXA1 antibody and / or a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or its amplification product.

10. A method for evaluating the quality of a therapeutic drug for acute lung injury according to any one of claims 1 to 4, comprising confirming the expression of ANXA1 and / or at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in extracellular vesicles derived from airway epithelial cells.

11. A method for predicting prognosis, comprising measuring in vitro the expression level of at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p in a sample from a patient with acute lung injury, and evaluating the prognosis for the patient.

12. A prognosis prediction kit for patients with acute lung injury, comprising a primer set for amplifying at least one miRNA selected from the group consisting of miR-7-5p, miR-125b-5p, let-7a-5p, miR-125a-5p, let-7b-5p, let-7f-5p, let-7i-5p, miR-26a-5p, and let-7c-5p, and / or a probe that binds to the miRNA or its amplification product.

Images