Extracellular vesicles from regulatory macrophages

Abstract

The present invention relates to extracellular vesicles derived from immunoregulatory macrophage cells. These vesicles are useful in treating various diseases and conditions because they exhibit immunoregulatory, angiogenic, and tissue regenerative properties. In a further embodiment, the present invention relates to a pharmaceutical composition comprising the extracellular vesicles of the present invention. In yet another embodiment, the present invention relates to the pharmaceutical use of the extracellular vesicles or a pharmaceutical composition comprising such vesicles. In yet another embodiment, the present invention relates to the use of the extracellular vesicles or a pharmaceutical composition comprising such vesicles for the treatment or prevention of immunological and non-immunological diseases and conditions, including autoimmune diseases, inflammatory diseases, hypersensitivity reactions, graft rejection, and microvascular or macrovascular disorders of the lower extremities. The present invention also provides a method for preparing macrophage-derived extracellular vesicles from blood monocytes. Finally, the present invention provides a method for preparing immunoregulatory T cells using the extracellular vesicles.

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicles derived from immunoregulatory macrophage cells. These vesicles exhibit immunoregulatory, angiogenic, anti-inflammatory, and tissue regenerative properties, making them useful in the treatment of various diseases and conditions. In a further embodiment, the present invention relates to a pharmaceutical composition comprising the extracellular vesicles of the present invention. In yet another embodiment, the present invention relates to the pharmaceutical use of the extracellular vesicles or a pharmaceutical composition comprising such vesicles. In yet another embodiment, the present invention relates to the use of the extracellular vesicles or a pharmaceutical composition comprising such vesicles for the treatment or prevention of immunological and non-immunological diseases and conditions, including autoimmune diseases, inflammatory diseases, hypersensitivity reactions, graft rejection, and microvascular or macrovascular disorders. The present invention also provides a method for preparing macrophage-derived extracellular vesicles from blood monocytes. Finally, the present invention provides a method for preparing immunoregulatory T cells using the extracellular vesicles. [Background technology]

[0002] Transferring immunoregulatory cells from tolerant donors to intolerant recipients is a well-known technique in experimental immunology to establish tolerance in recipients, but its clinical application is only now receiving serious attention.[1] Currently, several immunoregulatory cell types, including regulatory T cells[2], tolerogenic dendritic cells[3], and regulatory macrophages[4], have reached the preclinical development stage and can be studied as immunosuppressants in early-phase clinical trials.

[0003] A wide range of immunological conditions, including T-cell and B-cell mediated autoimmune diseases, chronic inflammatory disorders, graft-versus-host diseases (GVHD), and graft rejection, may be candidates for treatment with cell-based immunoregulatory therapies. In these conditions, cell-based immunoregulatory therapies can reduce or even eliminate the need for conventional immunosuppressive or anti-inflammatory therapy, allowing patients to avoid its associated complications. Because the type of immune tolerance supported by regulatory cells is primary and self-sustaining, cell-based immunotherapy has the potential to offer a treatment option for diseases that would otherwise require long-term conventional immunosuppressive or anti-inflammatory therapy.

[0004] One particularly promising candidate cell type for use as an adjuvant immunosuppressant in transplantation is the immunoregulatory macrophage (referred to herein and in the literature as "Mreg"). Mreg cells reflect a unique state of macrophage differentiation, distinguished from other activated macrophages by their robust phenotype and potent T cell suppressor function [5]. Human Mregs potently suppress mitogen-stimulated T cell proliferation in vitro, which may be due to interferon (IFN)γ-induced indoleamine 2,3-dioxygenase activity and contact-dependent elimination of activated T cells. Furthermore, Mregs drive the development of activation-induced regulatory T cells, which then suppress the proliferation of effector T cells and inhibit dendritic cell maturation. Therefore, it is hypothesized that when Mregs are administered to a recipient, a feedforward loop of immune regulation is initiated, leading to long-term immune tolerance of the exogenous graft or prevention of immunopathological conditions. Mreg-containing cell preparations have been administered to kidney transplant recipients as a form of adjuvant immunosuppressive therapy in a series of case studies and two early-phase clinical trials.[5][9] These pilot studies have clearly demonstrated the feasibility of this approach in solid organ transplantation.

[0005] Despite significant advances in the field of immunoregulatory cells in recent years, concerns about the safety of cell therapy remain. In particular, the ability of inappropriate cell types, such as cells that differentiate into cancer cells, is a persistent concern for patient safety. Therefore, there is always a need for immunoregulatory cells or immunomodulators with reduced safety risks that can be used for therapeutic purposes, such as inducing immune tolerance of exogenous grafts in recipients, or for the treatment of various types of diseases. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2019 / 053091A1 [Non-patent literature]

[0007] [Non-Patent Document 1] Sambrook et al. (1989), Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. [Non-Patent Document 2] Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols Publishing, New York. [Overview of the Initiative] [Means for solving the problem]

[0008] Extracellular vehicles (EVs) represent heterogeneous groups of membrane-bound structures that are spontaneously released from most cells. EVs typically contain lipids, proteins, and RNA and are an efficient way to transfer functional cargo from cell to cell [10, 11]. Recent literature also supports the idea that EVs play a role in mediating complex and coordinated communication between different cell types [12, 13, 14].

[0009] This invention is based on the insight that Mreg cells prepared according to established protocols generate extracellular vesicles (EVs) that exhibit immunomodulatory, angiogenic, anti-inflammatory, and tissue-regenerative properties corresponding to the derived Mreg cells. Therefore, EVs can be isolated and used as active ingredients to treat or prevent a wide range of disorders and diseases. Since these vesicles are non-proliferative, they do not raise the same safety concerns as the derived cells. In particular, Mreg-derived vesicles are immunomodulatory and anti-inflammatory, meaning they can suppress undesirable T cell responses by inhibiting T cell proliferation. This can be tested as described below in Example 7. Furthermore, these vesicles are angiogenic, meaning they promote the formation of new blood vessels, for example, in hypoxic tissue. The ability to form new blood vessels can be tested in the vascular formation assay described below in Example 6. In addition, these vesicles are tissue-regenerative, meaning they can repair damaged tissue in wounds and ulcers, for example. The ability to regenerate damaged tissue can be tested by the curettage assay described below in Example 6.

[0010] Accordingly, in a first aspect, the present invention relates to macrophage-derived extracellular vesicles having a size of 0.1 to 10 μm and exhibiting immunomodulatory activity. The size of the vesicles is preferably analyzed using a Coulter counter, such as a MOXI counter. Preferably, the macrophage-derived vesicles of the present invention are 1 to 10 μm in size, meaning they belong to the so-called "large extracellular vesicle" or "L-EV" group. In practice, the vesicles of the present invention are typically provided as a population of vesicles of varying sizes. In these cases, it is preferable that at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the vesicles in the population are L-EVs, i.e., 1 to 10 μm.

[0011] Preferably, macrophage-derived extracellular vesicles do not contain a nucleus. Furthermore, the vesicles contain only trace amounts of double-stranded DNA. Preferably, the vesicles do not contain double-stranded DNA. The presence of double-stranded DNA can be measured by optical spectroscopy or gel electrophoresis. In a preferred embodiment, the presence of double-stranded DNA is measured by optical spectroscopy. In another preferred embodiment, the presence of double-stranded DNA is measured by gel electrophoresis. More preferably, the vesicles of the present invention do not contain any organelles selected from the group consisting of nuclei, mitochondria, endoplasmic reticulum, and Golgi complexes. The vesicles may contain fragments of such organelles, but preferably do not include complete functional organelles.

[0012] On the other hand, the vesicles may contain various proteins, lipids, and RNAs as functional cargos. In a preferred embodiment, macrophage-derived extracellular vesicles contain one or more proteins that induce angiogenesis, such as interleukin-8 and angiogenin. In one embodiment, the vesicles of the present invention contain one or more proteins selected from the group consisting of interleukin-8, platelet factor-4, serpin E1, serpin F1, TIMP-1, angiogenin, chemokine ligand 16 (CXCL16), dipeptidyl peptidase IV (DPPIV), endoglin (ENG), insulin-like growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), thrombospondin 1 (THBS1), and human plasminogen activator, urokinase (uPA). In yet another embodiment, the vesicles of the present invention contain one or more proteins selected from the group consisting of interleukin-8, platelet factor-4, serpin E1, serpin F1, TIMP-1, and angiogenin. Preferably, these proteins are human proteins.

[0013] In another embodiment, the vesicles of the present invention contain two, three, four, or five of these proteins. In the most preferred embodiment, the vesicles of the present invention contain all of these proteins. These proteins are also generated and secreted de novo when Mreg is cultured for 24 hours after recovery

[15] . Regarding angiogenesis, the protein factors do not show a uniform picture when viewed individually: interleukin-8 and angiogenin are strong stimulators of angiogenesis [16, 17], while platelet factor-4 is mainly known as an angiogenesis inhibitor

[18] . Serpin E1 (PAI-1) is thought to exert either an angiogenesis-promoting function or an anti-angiogenesis function

[19] , and the widely present multifunctional proteins serpin F1 and TIMP-1 similarly show opposing functions regarding angiogenesis [20, 21, 22]. However, rather than a single factor, it is possible that the combination of the factors within the vesicles and the composition of the membrane-bound CD molecules and receptors on or within the target cells (i.e., endothelial cells) are the factors responsible for the observed angiogenesis-promoting effect described in the following examples.

[0014] The extracellular vesicles of the present invention contain the surface markers described above in relation to Mreg cells. Preferably, the vesicles of the present invention contain on the surface one or more markers selected from the group consisting of CD11c, CD86, CD31, and CD45. At the same time, preferably, the above vesicles also contain on the surface one or more markers typically found in extracellular vesicles, such as LAMP-1, CD9, CD63, and CD81. Thus, in a preferred embodiment, the vesicles of the present invention contain on the surface one of the following marker patterns: (1) CD11c, CDB6, CD31; (2) CD11c, CD86, CD31, LAMP-1; (3) CD11c, CD86, CD31, LAMP-1, CD9; (4) CD11c, CD86, CD31, LAMP-1 CD9, CD63; (5) CD11c, CD86, CD31, LAMP-1 CD9, CD63, CD81; (6) CD11c, CD86, CD45; (7)CD11c, CD86, CD45, LAMP-1; (8)CD11c, CD86, CD45, LAMP-1, CD9; (9)CD11c, CD86, CD45, LAMP-1 CD9, CD63; (10)CD11c, CD86, CD45, LAMP-1 CD9, CD63, CD81; (11) CD11c, CD31, CD45; (12)CD11c, CD31, CD45, LAMP-1; (13)CD11c, CD31, CD45, LAMP-1, CD9; (14)CD11c, CD31, CD45, LAMP-1 CD9, CD63; (15)CD11c, CD31, CD45, LAMP-1 CD9, CD63, CD81; (16) CD86, CD31, CD45; (17)CD86, CD31, CD45, LAMP-1; (18)CD86, CD31, CD45, LAMP-1, CD9; (19) CD86, CD31, CD45, LAMP-1 CD9, CD63; or (20)CD86, CD31, CD45, LAMP-1 CD9, CD63, CD81.

[0015] Furthermore, in this specification, large extracellular vesicles (L-EVs) derived from regulatory macrophages are also referred to. Mreg It was also found that phosphatidylserine (PS) is present on the surface. PS is present on the cell membrane of macrophages, but Mreg and other macrophages are either PS-negative or have PS limited to a limited area on the cell surface. In contrast to Mreg, L-EV Mreg It shows a strong PS signal across the entire surface. Therefore, L-EV Mreg It can be distinguished from Mreg by detecting PS on the cell surface. In other words, L-EV Mreg The finding that PS is exposed on the cell surface suggests that L-EV in the sample Mregcan be used to specifically detect and / or visualize. Thus, PS is a surface marker of L-EV Mreg . Due to the surface exposure of PS, it becomes possible to identify L-EV in a heterogeneous cell sample Mreg , and it becomes possible to distinguish L-EV Mreg from Mreg. Also, the PS exposure on L-EV Mreg can be used for cell detection and cell sorting. In particular, L-EV Mreg can be separated from other vesicles or cells, particularly Mreg, based on the PS surface exposure.

[0016] The detection of the above surface marker can be achieved by various conventional methods. Preferably, the detection of the surface marker on the vesicles of the present invention includes flow cytometry. Flow cytometry is a method widely used for analyzing the expression of cell surface markers and intracellular molecules. Flow cytometry is commonly used for applications such as cell counting, cell sorting, and biomarker profiling. In particular, flow cytometry can be used to isolate various cell types in a heterogeneous cell population. Flow cytometry typically measures the fluorescence intensity generated by a fluorescently labeled antibody that detects markers on the surface of cells or particles. In a particularly preferred embodiment, the vesicles of the present invention exhibit one of the above marker patterns determined by flow cytometry. The present invention preferably relates to a composition such as a pharmaceutical composition containing a population of vesicles, and at least 50% of the above vesicles, preferably at least 60% of the above vesicles, at least 70% of the above vesicles, at least 80% of the above vesicles, at least 90% of the above vesicles, or at least 95% of the above vesicles exhibit one of the above marker patterns.

[0017] The present invention also relates to macrophage-derived extracellular vesicles that can be obtained by a method comprising, as a first step, differentiating CD14-positive blood monocytes into Mreg by culturing them in a culture medium in the presence of (i) M-CSF and / or GM-CSF, (ii) CD16 ligand and (iii) IFN-γ, and as a second step, obtaining the extracellular vesicles from the culture medium.

[0018] For therapeutic use, the Mreg-derived extracellular vesicles of the present invention are incorporated as a pharmaceutical composition. Accordingly, in a second aspect, the present invention relates to a pharmaceutical composition comprising macrophage-derived extracellular vesicles according to a first aspect of the present invention. The pharmaceutical composition comprises, as a first component, an effective amount of macrophage-derived extracellular vesicles, preferably Mreg-derived extracellular vesicles.

[0019] When used in this specification, an effective dose of vesicles administered to a patient is approximately 1 × 10⁶ for the patient being treated. 4 ~Approx. 1×10 9 It is within the range of / kg body weight, preferably about 1 × 10 4 ~Approx. 1×10 8 This is / kg body weight, and more preferably about 1 × 10⁻⁶ 5 ~Approx. 1×10 7 / kg body weight, and more preferably about 1 × 10 6 ~Approx. 1×10 7 This is / kg body weight, for example, approximately 1 × 10⁻⁶ 6 / kg, approximately 2 x 10 6 / kg, approximately 3 x 10 6 / kg, approximately 4 x 10 6 / kg, approximately 5 x 10 6 / kg, approximately 6 x 10 6 / kg, approximately 7 x 10 6 / kg, or approximately 8 x 10 6 Body weight is / kg.

[0020] In addition to the extracellular vesicles mentioned above, the pharmaceutical composition may contain further additives, such as buffers, pH regulators, and preservatives. The properties and amounts of additives included in the pharmaceutical composition depend on the intended route of administration.

[0021] Generally, various routes of administration can be used to provide macrophage-derived extracellular vesicles to patients in need of treatment. Preferably, the pharmaceutical compositions of the present invention are formulated for parenteral administration, for example, subcutaneous, intramuscular, intravenous, or intracutaneous administration. In one embodiment, the extracellular vesicles of the present invention, or a composition containing such vesicles, are administered to the patient by intravenous administration, for example, by injection or infusion. Pharmaceutical compositions suitable for intravenous administration by injection or infusion typically include a sterile aqueous solution or suspension, and a sterile powder for immediate preparation of the sterile solution or suspension. The incorporation of macrophage-derived extracellular vesicles into pharmaceutical compositions can be achieved by applying conventional methods known in the field of drug formulations. Suitable methods are, for example, described in standard textbooks.

[0022] Suitable carriers for administration by injection or infusion include physiological saline, bacteriostatic water, Cremophor EL® (BASF), or phosphate-buffered saline (PBS). The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. A sterile injection solution can be prepared by mixing the required amount of vesicles of the present invention with one or more of the above-mentioned raw materials in a suitable solvent, followed by sterile filtration. Generally, a suspension is prepared by mixing the active compound, i.e., macrophage-derived extracellular vesicles, with a sterile vehicle containing a basic dispersion medium and other necessary raw materials (from the above-mentioned raw materials). The pharmaceutical composition should be stable at the time of administration and preferably protected from contamination by microorganisms, such as bacteria and fungi, by including parabens, chlorobutanol, phenol, ascorbic acid, or thimerosal in the composition.

[0023] If the pharmaceutical composition is intended for injection, the total volume injected is 1 to 100 ml, preferably 10 to 50 ml, for example, 20 ml, 30 ml, or 40 ml. If the pharmaceutical composition is intended for infusion, the total volume injected is 50 to 500 ml, with a volume of 90 ml to 250 ml being particularly preferred, and a volume of 90 ml to 150 ml being even more preferred.

[0024] Macrophage-derived vesicles can be administered to patients requiring treatment with different dosing regimens. For example, when macrophage-derived vesicles are administered to a patient by intravenous infusion, the total amount of vesicles administered may be delivered in one or more infusions. The suspension containing macrophage-derived vesicles may be primed with 0.9% NaCl. The suspension may also be administered by a single infusion, more preferably by a short infusion of less than 60 minutes, for example, within 60 minutes, within 30 minutes, within 20 minutes, or within 15 minutes or less. Preferably, a central venous catheter is used to administer the macrophage-derived extracellular vesicle suspension.

[0025] Administration of macrophage-derived vesicles may be preceded, concurrently, or subsequently administered with other active agents. For example, when the vesicles are administered to patients suffering from microvascular or macrovascular complications, compounds that counteract blood viscosity, such as calcium dobesylate, or compounds that exert capillary stabilizing effects, such as naphthazone, may be administered before, concurrently, or after vesicle administration.

[0026] For safety reasons, it is recommended that the macrophage-derived vesicles of the present invention be administered within 24 hours of being recovered from the cell culture. Preferably, the vesicles are administered within 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours of being recovered from the culture.

[0027] In a third aspect, the present invention relates to a method for preparing macrophage-derived extracellular vesicles having immunomodulatory, angiogenic, anti-inflammatory, and tissue-regenerative properties. These vesicles have been found to be produced when monocytes are converted into immunomodulatory macrophages (Mregs). The preparation of Mregs is described in detail in the literature. Mregs are derived from human CD14+ blood monocytes. To induce the characteristic biological properties of Mregs, monocytes are treated with specific combinations of growth factors, cytokines, and receptor ligands. Cells obtained by this method are characterized by a distinctive phenotype that distinguishes them from blood monocytes, monocyte-derived dendritic cells, and other inhibitory myeloid cell products.

[0028] Therefore, the present invention relates to a method for preparing macrophage-derived extracellular vesicles having immunomodulatory activity, (a) A step of isolating CD14-positive monocytes from the target blood sample; (b) A step of culturing the above monocytes in a culture medium containing (i) M-CSF and / or GM-CSF and (ii) CD16 ligand; (c) The step of bringing the above cells into contact with IFN-γ; (d) A method comprising the step of obtaining macrophage-derived extracellular vesicles from the culture medium described above.

[0029] After performing steps (a) to (c), the culture medium contains both immunoregulatory Mregs and extracellular vesicles derived from immunoregulatory Mregs. Therefore, the separation of the vesicles from the culture medium in step (d) can be performed in two steps. In the first step (d1), the Mregs are separated from the medium by centrifugation at low gravity, for example, 300-400 × g. After centrifugation, the cells are present in the precipitate, while the vesicles remain in the supernatant. To recover the vesicles, the supernatant is then subjected to centrifugation at high gravity, for example, 3000-4000 × g, in the second step (d2). After centrifugation at high gravity, the vesicles are present in the precipitate and can be easily separated from the culture medium.

[0030] The above method for preparing macrophage-derived extracellular vesicles uses blood monocytes as a starting material. While the use of human blood monocytes is preferred, the method is not limited to the use of human-derived cells. In fact, it can also be applied to other types of non-human cells, particularly vertebrate cells, such as non-human primate or pig cells. However, the monocytes acting as the starting material are preferably obtained from the blood of a human donor, more preferably from the peripheral blood of a human donor. The donor may be a healthy individual or a patient suffering from one or more diseases. The monocyte donor may be the intended recipient of the vesicles obtained from differentiated Mreg cells (autologous method). Alternatively, the monocyte donor may be a different person from the intended recipient of the vesicles obtained from differentiated Mreg cells (allogeneic method). In the latter case, the donor and recipient may or may not be genetically related. The preferred relationship between the donor and recipient depends on the intended clinical application. The use of vesicles obtained from autologous Mreg cells may help avoid certain adverse reactions. Therefore, the use of vesicles obtained from autologous Mreg cells is preferred.

[0031] Various methods for concentrating monocytes from blood are known in the art, and each of these methods can be used in the context of the preparation method described above. For example, blood obtained by venous puncture can be treated with an anticoagulant and then separated using a separation medium, such as Ficoll-Paque Plus. In this case, the anticoagulant-treated blood sample is layered in a Ficoll-Paque Plus solution and centrifuged to form layers containing various cell types. The bottom layer contains erythrocytes that have agglutinated and settled with the Ficoll-Paque Plus reagent. The layer immediately above the erythrocyte layer mainly contains granulocytes that have migrated through the Ficoll-Paque Plus layer. Monocytes and lymphocytes are found at the interface between the plasma and Ficoll-Paque Plus due to their lower density. Concentration of the mononuclear cell fraction can be achieved by washing and centrifugation after the isolation of the layers.

[0032] Another method commonly used to separate mononuclear leukocytes from blood samples is leukocyte apheresis. Leukocyte apheresis is a special type of apheresis in which leukocytes are obtained from peripheral blood in a sequential process by relative density. In this procedure, the blood in question is passed through a special centrifuge that collects a fraction of selected leukocytes and returns the remaining blood cells and plasma to the donor. Leukocyte apheresis is currently a common clinical method for obtaining leukocytes or stem cells from peripheral blood. Various devices are available from several manufacturers and can be used to perform leukocyte apheresis in the context of this invention, for example, the Spectra Optia® Apheresis System from Terumo BCT. When leukocyte apheresis is performed using the COBE® Spectra Apheresis System, it is preferable to use the manual protocol provided by the manufacturer, as it has been found that higher quality monocytes are obtained with this protocol compared to the AutoPBSC protocol.

[0033] By using a separation medium such as Ficoll-Paque Plus and a leukocyte apheresis apparatus, a cell fraction containing lymphocytes in addition to monocytes can be obtained. According to the present invention, monocytes can be concentrated and separated from lymphocytes by known methods, for example, magnetic bead separation, elutriation, filtration, or plastic adhesion, before the cells are used for the preparation of Mregs and vesicles. However, it is not essential to use a homogeneous monocyte fraction for Mreg preparation. In fact, the presence of 0.1–20%, preferably 10–20%, of lymphocytes in the monocyte fraction can have a positive effect on the differentiation of monocytes into Mregs.

[0034] To obtain a mononuclear cell preparation enriched with monocytes, peripheral blood mononuclear cells may be contacted, for example, with CD14 microbeads to which CD14-positive monocytes bind. The monocytes from step (a) of the above method can be isolated by leukocyte apheresis and then subjected to a separation step using a CD14 affinity molecule, preferably a CD14 antibody. Such a purification step significantly reduces contamination of the starting material with non-monocytes. Isolation of CD14 monocytes can be achieved by an automated system. For example, the CD14 monocytes used in the above method can be isolated using CliniMACS® Technology (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0035] Monocyte fractions isolated by leukocyte apheresis and / or other methods can be used directly for cell differentiation by incubation with M-CSF and / or GM-CSF and CD16 ligand, or they can be stored overnight at 4°C in autologous plasma supplemented with the anticoagulant dextrose citrate solution (ACD-A) or any other suitable buffer until further use. If the isolated monocyte fraction must be transported to another location where differentiation processing will be carried out, care should be taken to ensure that differentiation of the cells by incubation with M-CSF / GM-CSF is initiated within 24 hours of cell isolation, preferably within 18 hours, 12 hours, 6 hours, 4 hours, or 2 hours of monocyte isolation. For long-term storage, the monocyte fraction can be resuspended in a suitable cryopreservation solution and stored for extended periods at temperatures below 20°C, preferably below 80°C.

[0036] After isolation, monocytes are incubated in the presence of macrophage colony-stimulating factor (M-CSF, also known as CSF1), granulocyte-macrophage colony-stimulating factor (GM-CSF), or both, along with a CD16 ligand. M-CSF is also known in the art as a hematopoietic growth factor that influences the proliferation, differentiation, and survival of monocytes, macrophages, and myeloid progenitor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF, also known as CSF2) is a monomeric glycoprotein that functions as a cytokine and is secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts. M-CSF and GM-CSF proteins from different species have been described and can be purchased from various manufacturers. The choice of M-CSF and / or GM-CSF depends on the origin of the monocytes to be differentiated into Mreg cells. For example, when differentiating human monocytes into Mreg cells using the above process, the culture medium used contains human M-CSF and / or human GM-CSF, preferably recombinant human M-CSF and / or recombinant human GM-CSF. Similarly, when using porcine monocytes in the differentiation method, the M-CSF and / or GM-CSF added to the culture medium are of porcine origin.

[0037] For example, the cells can be suspended in a medium containing M-CSF and / or GM-CSF and CD16 ligand. Alternatively, M-CSF / GM-CSF and CD16 ligand can be added some time after the start of cell culture. The culture medium used in step (b) of the above method may be any medium described in the literature as suitable for use in the culture of monocytes and / or macrophages. Suitable culture media include, for example, PromoCell Macrophage Generation Medium (PromoCell, Heidelberg, Germany), Dulbecco's Modified Eagle Medium (DMEM), DMEM:F12 Blend, Medium 199, or RPMI-1640 Medium. The culture medium is preferably a chemically defined medium. In addition to M-CSF / GM-CSF, the culture medium may contain other factors that promote Mreg survival and differentiation, including: growth factors and cytokines, e.g., epidermal growth factor (EGF) or IL-4; fatty acids, cholesterol and other lipids; vitamins, transferrin and trace elements; insulin, glucocorticoids, cholecalciferol or ergocalciferol and other hormones; nonspecific immunoglobulins and other plasma proteins. In preferred embodiments, the culture medium is RPMI-1640 or a medium derived therefrom.

[0038] Typically, the concentration of M-CSF in the culture medium in step (b) of the above method is in the range of 1 to 100 ng of protein per ml of medium. Time-course experiments measuring the amount of M-CSF in the culture medium revealed that M-CSF is consumed or degraded over time; for example, a culture with an initial dose of 5 ng / ml M-CSF would have a content below physiological levels by the second day of culture, while a culture with an initial dose of 25 ng / ml M-CSF would maintain a concentration above 10 ng / ml throughout the 7-day culture period. Therefore, the concentration of M-CSF in the culture medium is usually in the range of 20 to 75 ng / ml, for example, 20 to 25 ng / ml. When GM-CSF is used in place of M-CSF, the same concentrations outlined above for M-CSF can be used in the medium. Since GM-CSF appears to be more potent than M-CSF, a GM-CSF concentration of 0.1 to 100 ng of protein per ml of medium is recommended. When both M-CSF and GM-CSF are used in the culture medium, the total concentration of these two growth factors falls within the aforementioned range, namely, 20–75 ng / ml.

[0039] In addition to M-CSF and / or GM-CSF, the culture medium used in step (b) of the above method also contains a CD16 ligand. Stimulation of the CD16 cell surface receptor on monocytes is necessary to induce differentiation of monocytes into Mreg cells. Stimulation of the CD16 cell surface receptor can be achieved by adding human or non-human immunoglobulin, more preferably human immunoglobulin or a fragment thereof. The immunoglobulin fragment may be, for example, an Fc fragment of immunoglobulin. It is hypothesized that the immunoglobulin acts via FcγRIII(CD16) to induce the Mreg phenotype. A simple way to achieve CD16 ligand stimulation is to add human serum to the culture medium. Therefore, the medium used to create Mreg cells may contain 1-20% human AB serum.

[0040] If the Mreg-derived vesicles are intended for use in therapeutic applications where induction of angiogenesis is desirable, the culture medium used to culture monocytes in step (b) of the above method may contain, in addition to M-CSF / GM-CSF and CD16 ligand, Toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), or high-mobility groupbox protein 1 (HMGB1), to enhance the production of angiogenic factors such as VEGF-A. The TLR ligand can be added to the culture medium at a concentration range of 1000 ng / ml to 1 μg / ml. The TLR ligand can be added at any stage of the production method. The TLR ligand may be present in the initial medium used to culture monocytes, i.e., on day 0 of culture, or it may be added at a later stage, for example, on day 5, 6, or 7 of culture. Preferably, the TLR ligand is added at the same time as the IFN-γ is added.

[0041] In step (c) of the above method, the cells are brought into contact with the cytokine interferon-γ (IFN-γ). The selection of IFN-γ used in the method of the present invention depends on the origin of the monocytes subjected to differentiation. When differentiating human monocytes into Mreg, the IFN-γ added is usually recombinant human IFN-γ. The amount of IFN-γ added to the monocyte culture is in the range of 5 to 100 ng / ml. An amount of 25 ng IFN-γ per 1 ml of culture medium is particularly preferred.

[0042] IFN-γ can be added to the culture medium simultaneously with M-CSF / GM-CSF and CD16 ligand, meaning that this cytokine can be added, for example, when initiating monocyte culture. In this method, the monocytes to be differentiated are cultured in the presence of M-CSF / GM-CSF, CD16 ligand, and IFN-γ for the entire culture period. However, the culture period in the presence of IFN-γ is usually considerably shorter than the culture period in the presence of M-CSF / GM-CSF, meaning that IFN-γ is typically added after the cells have been cultured in the presence of M-CSF / GM-CSF for, for example, 3 days, and then the culture is continued for a further 18-72 hours in the presence of IFN-γ.

[0043] In step (d) of the above method, macrophage-derived extracellular vesicles are obtained from the culture medium. Particularly good results have been achieved when the above cells are cultured for 6 days in the presence of M-CSF / GM-CSF and CD16 ligand, followed by pulsed IFN-γ for 18-24 hours. Subsequently, macrophage-derived extracellular vesicles, and optionally differentiated Mreg cells, can be collected on day 7. Various methods such as centrifugation or filtration can be used to collect the above vesicles. For example, the culture medium obtained at the end of step (c) of the above method contains both Mreg cells derived from these cells and the above vesicles, and can be subjected to centrifugation at a low speed, for example, 400×g or less, 300×g or less, 200×g or less, or 100×g or less for a time in the range of 10-60 minutes.

[0044] Preferably, step (d) includes centrifugation at 500 × g for 10 to 20 minutes. The pellet obtained from this centrifugation consists essentially of complete Mreg cells. These cells can be further processed or stored for further use. The supernatant obtained from this centrifugation consists essentially of L-EV-Mreg cells and extracellular vesicles containing smaller extracellular vesicles and soluble components. This supernatant can be subjected to faster centrifugation, for example, at 3000 × g or more, 4000 × g or more, or 5000 × g or more for a time ranging from 30 to 180 minutes. Preferably, step (d) includes centrifugation at 4000 × g for 20 to 60 minutes. The pellet obtained from this centrifugation consists essentially of L-EV-Mreg cells, while smaller vesicles and soluble factors may be found in the supernatant.

[0045] The isolated L-EV-Mreg can be washed with a buffer generally suitable for use with macrophages, such as phosphate-buffered saline (PBS) supplemented with 5% human serum albumin. The vesicles can then be transferred to a blood transfusion bag, glass infusion device, or another closed-system container that allows for transport and stored.

[0046] The extracellular vesicles of the present invention are derived from immunoregulatory macrophages that can differentiate from monocytes. It is particularly preferable that the vesicles of the present invention are derived from immunoregulatory macrophage cells expressing CD258, DHRS9, and IDO markers. The combination of these three markers provides a reliable description of immunoregulatory Mreg cells, distinguishing them from other macrophages.

[0047] The CD258 marker, also known as LIGHT or TNFSF14 in the literature, is a secreted protein of the TNF superfamily. The human sequence of CD258 can be found at NCBI gene_ID 8740. IDO refers to indoleamine 2,3-dioxygenase. The sequence of the human gene encoding this marker can be found at NCBI gene_ID 3620. Synonyms for IDO are IDO1 or INDO. DHRS9 is a retinol dehydrogenase of the SDR family of retinol dehydrogenases. The sequence of the human gene encoding this marker can be found at NCBI gene_ID 10170.

[0048] In a preferred embodiment of the present invention, the immunoregulatory macrophages that generate the extracellular vesicles of the present invention further express at least one marker selected from the group consisting of TGFβ1 and PAEP. TGFβ1 refers to transforming growth factor β, a multifunctional cytokine belonging to the transforming growth factor superfamily. The sequence of the human gene encoding this marker can be found at NCBI gene_ID 7040. PAEP refers to progestagen-associated endometrial protein. The sequence of the human gene encoding this marker can be found at NCBI gene_ID 5047.

[0049] Furthermore, the immunoregulatory macrophages that generate the extracellular vesicles of the present invention may express common macrophage markers. Therefore, the Mreg immunoregulatory macrophages from which the above extracellular vesicles originate may further express at least one marker selected from the group consisting of CD33, CD11b, and HLA-DR.

[0050] In a particularly preferred embodiment, the immunoregulatory macrophages that generate the extracellular vesicles of the present invention express one of the following marker patterns: (1) CD258, DHRS9, IDO; (2)CD258, DHRS9, IDO, TGFβ1; (3) CD258, DHRS9, IDO PAEP; (4)CD258, DHRS9, IDO, TGFβ1, PAEP; (5)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33; (6)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33, CD11b; (7)CD258, DHRS9, IDO, TGFβ1, PAEP, CD33, CD11b, HLA-DR; (8)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR; (9)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR, TGFβ1; (10)CD258, DHRS9, IDO, CD33, CD11b, HLA-DR, PAEP.

[0051] Vesicles derived from immunoregulatory macrophages expressing CD258, DHRS9, IDO, TGFβ1, and PAEP are particularly preferred in this specification.

[0052] The expression of markers by macrophages that produce vesicles according to the present invention can be determined at the mRNA or protein level. In a particularly preferred embodiment, detection of the marker is performed at the transcription level. Suitable methods for monitoring gene expression at the transcription level include those that enable quantitative or semi-quantitative detection at the mRNA level, such as quantitative RT-PCR (e.g., TaqMan® RT-PCR), real-time RT-PCR, Northern blot analysis, or other methods commonly known in the art.

[0053] The detection of transcription levels typically requires, as a first step, the isolation of mRNA from the macrophages to be analyzed. Methods for isolating RNA, such as mRNA, are well known in the art and have been discussed in detail in the literature (see, e.g., Sambrook et al. (1989), Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, and Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols Publishing, New York). Such methods usually involve cell lysis. Cell lysis can be carried out by using a detergent that can disrupt the cell's plasma membrane. For example, a buffer containing guanidine thiocyanate and / or SDS can be used for cell lysis. The above method may include a step of enzymatic digestion of the cell's DNA to obtain pure RNA that does not contain even trace amounts of DNA (which may interfere with further downstream uses, e.g., monitoring of expression levels). Inhibitors of enzymes that lead to RNA degradation may also be added to the lysis buffer. Kits for preparing highly purified RNA are available from several manufacturers, such as Qiagen, Ambion, Stratagene, Clontech, Invitrogen, and Promega.

[0054] RNA isolated from cell or tissue samples using commercially available kits typically contains different types of RNA. Preferably, the RNA obtained from tissue samples is total RNA, including mRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). In the method of the present invention, it is desirable to enrich the mRNA fraction relative to the fractions of other cellular RNAs. Preferably, mRNA is separated from other RNA molecules. Methods for enriching or purifying mRNA are known in the art. Since mRNA contains a poly(A) tail at its 3' end, affinity chromatography can be performed using oligo(dT) or poly(U) linked to a solid matrix such as cellulose or Sephadex® matrix (see, for example, Ausubel et al., (1994), Current Protocols in Molecular Biology, Current Protocols Publishing, New York).

[0055] Commonly used methods for detecting marker expression at the transcriptional level in immunoregulatory macrophages include, for example, RT-PCR, TaqMan RT-PCR, and microarray analysis, as described in more detail in WO2019 / 053091A1. Primers or probes used in PCR or RT-PCR detection methods are designed to enable specific hybridization to target sequences within each marker gene, such as CD258, DHRS9, or IDO genes, and subsequent amplification of said sequences. Those skilled in the art can easily design oligonucleotide primers and / or probes that can be used to detect the expression of each marker gene based on the disclosure in WO2019 / 053091A1.

[0056] If high levels of marker mRNA, such as CD258, DHRS9, and IDO, are detected, it can be inferred that the cell is Mreg. IDO and DHRS9 are intracellular markers and can be detected, for example, by RT-PCR. Non-Mreg cells, such as resting macrophages, may also express one or more of the markers mentioned above to some extent, but such expression is thought to be relatively low. Therefore, according to the present invention, it is preferable to use the expression of markers by non-Mreg cells, particularly resting macrophages, as a negative control. If the measured levels of marker expression at the RNA level by quantitative RT-PCR are at least approximately 50%, at least approximately 75%, at least approximately 150%, at least approximately 200%, at least approximately 250%, at least approximately 300%, at least approximately 350%, at least approximately 400%, at least approximately 450%, at least approximately 500%, at least approximately 550%, at least approximately 600%, at least approximately 750%, or at least approximately 1000% higher than the respective levels in the negative control, i.e., resting macrophages, then the cells tested are clearly indicated to be Mreg cells.

[0057] In yet another particularly preferred embodiment, the detection of Mreg markers, e.g., CD258, DHRS9, and IDO, involves the detection of marker gene expression at the translational level, i.e., at the protein level. This means determining the amount of each marker protein, e.g., CD258, DHRS9, and IDO. The amount of marker protein can be determined by any preferred method capable of specifically detecting the protein in a biological sample. The detection of the protein may be based on a molecule that specifically binds to the protein, or on the separation of the protein from other proteins present in the sample. Molecules that specifically bind to marker proteins include antibodies and antibody fragments having binding activity for each marker. Numerous antibodies targeting marker proteins such as CD258, DHRS9, and IDO are commercially available. Such antibodies or fragments can be used for the detection of marker proteins in immunohistochemical methods including Western blotting, quantitative Western blotting, enzyme-linked immunosorbent assay (ELISA), polarization analysis, (quantitative) surface plasmon resonance (SPR), or quantitative electron microscopy. In a particularly preferred embodiment of the present invention, the detection of the markers includes ELISA. Other methods for detecting specific binding include, for example, fluorescence resonance energy transfer (FRET). Methods that enable quantitative detection of marker proteins by separating them from other components in a biological sample include quantitative mass spectrometry, electrophoresis, such as two-dimensional gel electrophoresis, and chromatography, such as size exclusion chromatography or ion exchange chromatography.

[0058] As mentioned above, when markers are detected at the protein level, such detection is particularly preferable to include flow cytometry. Flow cytometry may involve measuring the fluorescence intensity produced by a fluorescently labeled antibody that detects the marker on the cell surface. Flow cytometry can also be used to detect intracellular markers, but such detection is less desirable because the antibody must permeate the cells, thereby killing them.

[0059] As mentioned above, non-Mreg cells may also produce small amounts of the aforementioned Mreg markers, such as CD258, DHRS9, and IDO. Therefore, protein levels in non-Mreg cells, such as unregulated macrophages, should also be measured to obtain a negative control. If the protein levels of the tested cells clearly show that marker expression is higher than that of the negative control, it can be concluded that the tested cells are Mreg. Specifically, if the measured levels of marker expression at the protein level are at least approximately 50%, at least approximately 75%, at least approximately 150%, at least approximately 200%, at least approximately 250%, at least approximately 300%, at least approximately 350%, at least approximately 400%, at least approximately 450%, at least approximately 500%, at least approximately 550%, at least approximately 600%, at least approximately 750%, or at least approximately 1000% higher than the respective levels in the negative control, i.e., non-Mreg cells, such as unregulated macrophages, it is clearly indicated that the tested cells are Mreg cells. Measurement of marker expression at the protein level is preferably performed by quantitative ELISA.

[0060] In a fourth aspect, the present invention relates to the pharmaceutical use, i.e., the use for therapeutic purposes, of vesicles according to the first aspect of the present invention or pharmaceutical compositions according to the second aspect of the present invention. Hereinafter, it is demonstrated that macrophage-derived extracellular vesicles exhibit essentially the same characteristics as the derived Mreg cells. Accordingly, the vesicles exhibit many therapeutically useful properties, such as immunosuppressive, immunomodulatory, angiogenic, and anti-inflammatory properties, which make them highly suitable for use in immunosuppressive, anti-inflammatory, or tissue repair therapies. For example, the extracellular vesicles of the present invention, like the derived macrophages, are thought to be T-cell suppressive and mediate the active removal of activated T cells. Therefore, the vesicles are highly suitable for use as adjuvant immunosuppressive therapy in various immunologically mediated diseases and in the context of organ or tissue transplantation.

[0061] Accordingly, in one embodiment of the present invention, extracellular vesicles according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention are used in a method for suppressing graft rejection and / or extending the graft survival period in a recipient. Accordingly, the present invention also relates to a method for suppressing graft rejection and / or extending the graft survival period in a recipient, comprising the steps of (i) administering an effective amount of macrophage-derived extracellular vesicles according to the first aspect of the present invention, or (ii) administering a pharmaceutical composition according to the second aspect of the present invention. Preferably, the transplantation is an organ, tissue, or cell transplantation. The type of organ to be transplanted is not limited by the present invention, but is preferably a kidney, liver, heart, lung, or pancreas. The organ to be transplanted to the recipient is particularly preferably a human organ.

[0062] When the transplant is a tissue transplant rather than an organ transplant, the tissue transplanted to the recipient is not particularly limited. Rejection of tissue derived from an allogeneic donor in the recipient can be prevented or mitigated by the macrophage-derived extracellular vesicles of the present invention. The transplanted tissue is preferably human tissue, such as the intestine, cornea, skin, composite tissue, bone marrow, or pancreatic islet tissue.

[0063] The macrophage-derived vesicles of the present invention can also support cell transplantation to a recipient by suppressing the immune response in the recipient. When transplantation is cell transplantation, the nature of the transplanted cells is not generally limited, but it is preferable that the transplanted cells be selected from the group consisting of adult stem cell grafts, isolated hepatocyte grafts, or leukocyte grafts. The macrophage-derived vesicles of the present invention may also include soluble factors that promote the homing and engraftment of adult stem cells, such as cathelicidin. In preferred embodiments of the present invention, the vesicles are used to promote the engraftment of hematopoietic stem cells (HSCs) after bone marrow or HSC transplantation.

[0064] To suppress graft rejection in recipients and to induce acceptance of allogeneic organ, tissue, or cell transplantation, the vesicles of the present invention or pharmaceutical compositions containing such vesicles can be administered intravenously by injection or infusion, as described above. The injection or infusion can be performed before or after surgery. When administered before surgery, the vesicles are administered to the recipient at least once, preferably twice, and more preferably three times, prior to the surgery. Preferably, the vesicles are administered to the recipient starting one week before surgery, for example, six, five, four, three, two, or one day before surgery. When administered after surgery, the first administration is preferably within 24 hours after surgery, more preferably within 36, 48, 60, or 72 hours after surgery. In stably immunosuppressed transplant recipients, vesicle therapy can be administered at any time after transplantation. Alternatively, the vesicles can be administered to transplant recipients experiencing acute or chronic graft rejection. Subsequently, these vesicles suppress the recipient's immune system's T-cell response to the graft and remain in the recipient's body for a sufficiently long period, thereby conferring long-term graft tolerance to the recipient.

[0065] When the vesicles of the present invention are used to suppress graft rejection or extend graft survival in a recipient, the graft is typically an allogeneic graft, i.e., a graft produced from a donor that is genetically different but belongs to the same species as the recipient. In this case, the vesicles are derived from macrophages prepared from blood monocytes obtained from the donor. Monocytes can be obtained from living or deceased donors. In the case of a deceased donor, i.e., cadaver donation, the donor's body is usually perfused with a medium by opening the main arteries for the purpose of organ preservation. Venous blood can be taken from the body, collected, and used to prepare the macrophage-derived extracellular vesicles described herein. When extracellular vesicles prepared from monocytes obtained from a deceased donor are used, prevention of organ rejection can be achieved by administering immunosuppressants, which are commonly used for this purpose during organ transplantation.

[0066] In another embodiment, macrophage-derived extracellular vesicles according to the first aspect of the present invention or pharmaceutical compositions according to the second aspect of the present invention are used in a method for promoting or sustaining the engraftment or effect of a cell-based pharmaceutical for regulatory T cells. Accordingly, the present invention also relates to a method for promoting or sustaining the engraftment or effect of a cell-based pharmaceutical for regulatory T cells in a subject, comprising the steps of (i) administering an effective amount of macrophage-derived extracellular vesicles according to the first aspect of the present invention, or (ii) administering a pharmaceutical composition according to the second aspect of the present invention.

[0067] In addition to their immunomodulatory and immunosuppressive properties, the macrophage-derived vesicles of the present invention possess anti-inflammatory properties and are therefore highly useful in the remission of inflammatory processes. Accordingly, the vesicles provided herein are also useful in treating diseases or disorders characterized by dysregulated immune states or excessive inflammatory responses. Such diseases or disorders include, for example, autoimmune diseases, inflammatory diseases, and hypersensitivity reactions.

[0068] Accordingly, in yet another embodiment of the present invention, vesicles according to the first aspect of the present invention or pharmaceutical compositions according to the second aspect of the present invention are used in methods for treating or preventing autoimmune diseases, inflammatory diseases, or hypersensitivity reactions.

[0069] When macrophage-derived extracellular vesicles are used to treat autoimmune diseases, the diseases may be (a) primarily T cell-mediated, (b) primarily antibody-mediated, or (c) primarily mediated by other cellular components of the immune system. The diseases may be focal or systemic autoimmune conditions. The types of autoimmune conditions treated with macrophage-derived vesicles are not limited to systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritis; ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous microvasculitis, granulomatosis with polyangiitis Examples include eosinophilic granulomatosis with polyangiitis, Behçet's disease, thromboangiitis obliterans, Kawasaki disease, and other large, medium, or small vasculitis of autoimmune etiologies; multiple sclerosis (MS) and neuroimmunological disorders; type 1 diabetes mellitus, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction, and other autoimmune endocrine disorders; hemolytic anemia, thrombocytopenic purpura, and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, pemphigus, and other autoimmune skin conditions.

[0070] Macrophage-derived vesicles are also effective in treating acute or chronic inflammatory diseases and diseases involving pathophysiologically important inflammatory components. The inflammatory diseases treated may be localized or systemic. The types of inflammatory diseases treated with the vesicles of the present invention are not limited to, but include, arterial occlusive diseases, e.g., peripheral artery occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or stenosis; microvascular angina, also known as cardiac syndrome X; inflammation systemically associated with metabolic disorders, including type II diabetes and obesity-related metabolic syndrome; and skin diseases, including eczema. Preferably, the inflammatory diseases treated are characterized by chronic inflammation of the intima of the arterial wall, e.g., myocardial infarction, stroke, severe limb ischemia, and pAOD.

[0071] Treatment of pAOD is particularly desirable. In patients unsuitable for revascularization due to the degree or location of arterial occlusion or significant comorbidities, pAOD is a severely debilitating and prevalent condition for which amputation is known to be the only treatment option. Amputation remains a last resort treatment, with a relatively high mortality rate, and only a small number of patients fully recover motor function after amputation. Mreg cells have been previously found to be angiogenic. These cells greatly promote angiogenesis, i.e., the formation of new blood vessels, through the basal and stimulated expression of angiogenic growth factors, such as VEGF, FIGF (VEGF-D), PDGFB, and MDK. In particular, Mreg cells produce high levels of vascular endothelial growth factor (VEGF) when stimulated with TLR4 ligands. It has been described that Mreg cells induce the expression of vascular endothelial growth factor C (VEGF-C), an angiogenic factor that effectively stimulates angiogenesis in vivo

[27] . This invention demonstrates that extracellular vesicles derived from Mreg cells share the angiogenic properties of the Mreg cells from which they originate. Therefore, these vesicles also promote angiogenesis.

[0072] In one embodiment, macrophage-derived extracellular vesicles are injected intramuscularly or subcutaneously into an ischemic limb. In ischemic tissue, the vesicles are inevitably exposed to microbial and necrotic tissue components (e.g., HMGB1) that act as TLR4 agonists. Thus, the vesicles can be used to promote tissue regeneration through local secretion of pro-angiogenic growth factor. In another embodiment, macrophage-derived extracellular vesicles can be stimulated ex vivo with a TLR ligand during the manufacturing process to ensure high levels of VEGF production. Examples of TLR ligands include, but are not limited to, lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). The pAOD treated with the vesicles of the present invention may be of any grade or category. For example, the pAOD may be a Grade I pAOD, Category 1-4, or Grade II-IV pAOD.

[0073] The vesicles of the present invention are also useful for treating or preventing hypersensitivity reactions. Preferably, the hypersensitivity reactions treated or prevented by the macrophage-derived extracellular vesicles of the present invention are selected from the group consisting of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity, and mast cell disease.

[0074] Because the extracellular vesicles of the present invention are angiogenic, their use in other diseases or conditions requiring angiogenesis is also intended herein. Accordingly, the present invention also relates to vesicles according to a first aspect of the present invention, or pharmaceutical compositions according to a second aspect of the present invention, used in methods for inducing angiogenesis or vascularization in hypoxic tissue, promoting tissue repair processes by participating in tissue remodeling and tissue regeneration, preventing or reducing fibrosis, reducing ischemic pain, or avoiding limb amputation. Accordingly, the present invention also relates to methods for inducing angiogenesis or vascularization in hypoxic tissue, promoting tissue repair processes by participating in tissue remodeling and tissue regeneration, preventing or reducing fibrosis, reducing ischemic pain, or avoiding limb amputation, comprising the steps of (i) administering an effective amount of macrophage-derived extracellular vesicles according to a first aspect of the present invention, or (ii) administering a pharmaceutical composition according to a second aspect of the present invention.

[0075] The extracellular vesicles of the present invention are also useful for treating microvascular or macrovascular complications in subjects. Accordingly, the present invention also relates to vesicles according to a first aspect of the present invention or pharmaceutical compositions according to a second aspect of the present invention for use in a method for treating microvascular or macrovascular complications of the lower extremities of a subject. The present invention also relates to a method for treating microvascular or macrovascular complications of the lower extremities of a subject, comprising the steps of (i) administering an effective amount of macrophage-derived extracellular vesicles according to a first aspect of the present invention, or (ii) administering a pharmaceutical composition according to a second aspect of the present invention.

[0076] As used herein, microvascular complications refer to vascular diseases affecting small blood vessels and capillaries in the body, such as small cerebral blood vessels, small coronary arteries, or small blood vessels in the lower leg. During microvascular complications, the capillary basement membrane thickens and hardens, causing occlusion or rupture of capillaries or small arteries, resulting in tissue necrosis and loss of function. Macrovascular complications, on the other hand, refer to vascular diseases affecting large blood vessels, such as arteries. Aortic occlusion leads to a high incidence of heart attacks, strokes, and peripheral vascular diseases in diabetic patients. Arterial occlusion in the lower leg often leads to slow-healing ulcers on the foot and lower leg.

[0077] One of the causes of microvascular and macrovascular complications is long-term diabetes mellitus. In diabetic patients, high blood glucose levels cause vascular endothelial cells to absorb more glucose than normal. Subsequently, these cells form more glycoproteins on their surface than usual, and the basement membrane of the blood vessel wall becomes abnormally thick and weak. As a result, the blood vessel wall leaks, reducing blood flow throughout the body, and some tissues are damaged due to insufficient oxygen supply.

[0078] According to the present invention, the microvascular disorders to be treated are preferably selected from the group of diseases consisting of vasculitis, arteritis, angioplasia, white atrophy, sclerosis, Determann syndrome, diabetic vascular disorders, occlusive endarteritis, erythromelalgia, fibromuscular dysplasia, perforation, Mönkberg sclerosis, Aussler's disease, compartment syndrome, Paget-von Schröder syndrome, Raynaud's syndrome, and leg ulcers. In a particularly preferred embodiment, the microvascular disorder treated by the present invention is a leg ulcer. As used herein, leg ulcers include diabetic leg ulcers and venous leg ulcers.

[0079] According to the present invention, the macrovascular disorders treated with the extracellular vesicles of the present invention are preferably selected from the group consisting of aneurysms, dissections, atherosclerosis, atherosclerosis, peripheral artery occlusive disease (pAOD), intermittent claudication, necrosis and gangrene, vascular malformations, Lulish syndrome, or compression syndromes.

[0080] In another aspect, the extracellular vesicles of the present invention are for use in methods of promoting surgical wound healing, traumatic wound healing, or other wound healing in a subject. Vesicle therapy can be optionally used in combination with conventional management (i.e., washing, occlusion, and dressing) to promote the healing of acute or chronic wounds. The wounds to be treated may be open or closed wounds. Open wounds may include incisions, lacerations, abrasions, avulsions, penetrating injuries, or punctures. Closed wounds may include crush injuries or hematomas. Incisions may be traumatic or iatrogenic (i.e., surgical incisions). The extracellular vesicles of the present invention can be used to promote the engraftment of autologous or allogeneic skin grafts. The extracellular vesicles of the present invention can be used, or not used, in combination with conventional management for the healing of burns caused by skin exposure to heat, cryogenics, chemicals, friction, radiation, or electric currents. The references made herein with respect to the treatment of ulcers are similarly applicable to the treatment of wounds or burns.

[0081] In yet another aspect of the present invention, the macrophage-derived extracellular vesicles of the present invention can be used to generate immunoregulatory T cells. It has been previously described that T cells co-cultured with Mregs develop a regulatory T cell phenotype that suppresses the proliferation of effector T cells and inhibits the maturation of dendritic cells. Therefore, the macrophage-derived extracellular vesicles of the present invention can also be used to generate immunoregulatory T cells instead of Mregs.

[0082] Therefore, according to a fifth aspect, the present invention is a method for preparing immunoregulatory T cells, (a) The process of obtaining the target T cells; (b) The step of co-culturing T cells with macrophage-derived extracellular vesicles as described herein; (c) A method comprising the step of obtaining the immunoregulatory T cells from the culture medium described above.

[0083] The immunoregulatory T cells obtained by the above method can be used alone or in combination with Mreg or macrophage-derived extracellular vesicles of the present invention to treat any of the diseases or disorders discussed elsewhere in this specification. In the first step of the above method, T cells are obtained from a blood sample of interest. These cells can be obtained, for example, from a blood sample or apheresis solution, or from a tissue of interest such as bone marrow or spleen. These cells can be obtained, for example, from blood obtained by venipuncture, by conventional methods. The T cells used in the above method are, for example, CD3+ T cells or a subset thereof. Before co-culturing with macrophage-derived extracellular vesicles, CD3+ T cells can be purified or concentrated by conventional methods, for example, by magnetic microbead separation or flow cytometry sorting. The T cells can also be activated before the co-culturing step. For example, T cells can be treated with CD3 / CD28 activating beads, i.e., superparamagnetic beads that are similar in size to antigen-presenting cells for this purpose and are covalently linked to anti-CD3 and anti-CD28 antibodies.

[0084] Next, the T cells are brought into contact with the macrophage-derived vesicles of the present invention. The cells can be brought into contact with different vesicle:Treg ratios. For example, the cell fractions can be brought into contact with vesicle:Treg ratios of 1:1 to 50:1, preferably 5:1 to 10:1. More preferably, the vesicle:Treg ratio is about 15:1. Various culture media can be used in the co-culture method. The media may be those described above relating to the method for preparing Mreg cells. In a preferred embodiment, the medium is Gibco RPMI1640 supplemented with 10% human serum. The medium may contain further additives, such as M-CSF and / or GM-CSF, preferably human recombinant M-CSF and / or GM-CSF. The amount of M-CSF and / or GM-CSF is in the range described elsewhere herein, for example, 5 to 100 ng / ml, preferably 20 to 25 ng / ml. The culture medium may also contain other additives, such as Glutamax, in an amount of 1 to 5 mM, preferably 2 mM.

[0085] T cells are co-cultured with macrophage-derived vesicles for 1 to 8 days, preferably at least 3, 4, or 5 days, more preferably at least 3 days. After the predetermined culture period, immunoregulatory T cells are obtained from the culture medium. For example, T cells can be isolated by conventional methods, for example, by enriching CD4+ CD25+ TIGIT+ FoxP3+ Treg. The activation state of the T cells can be determined, for example, by FACS. If necessary, the above cells can be further formulated into pharmaceutical products. [Brief explanation of the drawing]

[0086] [Figure 1] This is a schematic diagram of the in vitro differentiation of human monocytes (to Mreg and L-EV-Mreg) (left) and the differential centrifugation process leading to an L-EV-Mreg enriched pellet (right). SN: Supernatant; EV: Extracellular vesicle. [Figure 2] This figure shows the results of the analysis of the vesicles and cells described above. A. Representative analysis of samples containing Mreg and L-EV-Mreg using a MOXI counter. B. Representative analysis of samples containing Mreg and L-EV-Mreg using a nucleo counter. Vesicles lacking a nucleus are not detected using the nucleo counter method. C. Quantitative results from the analysis of vesicles and cells. [Figure 3] This figure shows the morphology of L-EV-Mreg cells observed by microscopy. A. Bright-field microscopy after harvesting on day 7. B. Transmission electron microscopy after harvesting on day 7. L-EVMreg cells share several morphological features of Mreg cells, such as numerous vesicles and / or vacuoles and pseudopod-like extensions. N, nucleus; V, vesicle / vacuole; P, pseudopod. [Figure 4]This figure shows the results of characterizing cell surface markers in L-EV-Mreg cells. A. Representative scatter plots (FCS vs. SSC) showing L-EV-Mreg and Mreg populations on day 7. Flow cytometry analysis of surface marker characteristics in BL-EV-Mreg and Mreg cells. FSC, forward scattering; SSC, side scattering; *, P<0.05; **, P<0.01. [Figure 5] This figure shows the results of characterizing angiogenic proteins in L-EV-Mreg. A. Representative proteome profiling array membrane incubated with sonicated L-EV-Mreg, showing the relative abundance of 55 proteins involved in angiogenesis. Overlapping spots on each membrane correspond to individual proteins. 3D heatmap analysis of BA. C. Arrangement of protein spots on the membrane. Proteins with the highest signal intensity are shown in bold. [Figure 6] This figure shows the effects of L-EV-Mreg on in vitro wound healing and angiogenesis. A. Micrographs of representative wound healing assays using human endothelial cells (HUVEC) with and without the addition of 106 L-EV-Mreg / ml. Note that the treatment group contains a large number of L-EVs (arrows). The scale bar represents 250 μm. B. Statistical analysis of the effect of adding L-EV-Mreg on relative wound closure (control=1) after 8h. The bars show the mean ± SD of three independent experiments;*, P<0.05 (1-sample Student's t-test, relative to 1). Representative images of tubulation analysis using HUVEC cell cultures with and without the addition of L-EV-Mreg. The scale bar represents 500 μm. D. Effects of adding L-EV-Mreg on various tubulation parameters. The horizontal bars show the mean ± SD of three independent experiments. *, P<0.05; **, P<0.01 (one-sample Student's t-test, relative to 0). Edge color, branch-related parameters; yellow, segment-related parameters; cyan, mesh-related parameters; blue, node-related parameters; red, junction-related parameters. [Figure 7]This figure shows the results of a T cell expansion inhibition experiment. T cells were activated with CD3 / CD28 beads and then incubated with Mreg cells or L-EV-Mreg cells at various concentrations. The amount of granzyme B-positive cells was significantly reduced by the addition of Mreg or L-EV-Mreg cells. [Figure 8] Figures 8A to 8D show the results of phosphatidylserine analysis on the cell membrane of Mreg and L-EVMreg. (A) Cells and vesicles were stained with Hoechst 33342 (violet) and PSVue 480 (border color) respectively to label DNA and PS. Signals were merged onto a bright-field background to distinguish morphological features. Arrows indicate L-EVMreg. Scale bar indicates 50 μm. (B) Left: Magnified view of two ranges selected from (A). Right: Same range as left, showing only nuclear staining. Arrows indicate L-EVMreg. It has been shown that L-EVMreg lacks a nucleus and is positive for PS, while Mreg contains a nucleus and is negative for PS, or PS is limited to a limited area on the cell surface. Scale bar indicates 20 μm. (C) Typical PS staining of one Mreg cell and one L-EVMreg. Arrows indicate L-EVMreg. The scale bar represents 15 μm. D Left: Flow cytometry and gating of L-EVMreg (red frame). Right: Gated L-EVMreg is positive for PS (annexin V staining) and negative for nucleic acids (propidium iodide staining). [Modes for carrying out the invention] [Examples]

[0087] All experiments were approved by the local ethics committee of University Medical Center Schleswig-Holstein, Kiel, Germany (protocol identification numbers: D519 / 18 and D518 / 13). Mreg was manufactured in accordance with current GMP principles for the production of sterile pharmaceuticals. Care was taken at all processing steps to ensure that the product, raw materials, and equipment were protected from contamination and impurities.

[0088] All experiments were conducted using L-EVMreg derived from 3–9 Mreg preparations from different donors. Groups were compared using statistical software GraphPad Prism 5.01 for Windows (GraphPad Software, San Diego, USA). All data were tested for normality using the Kolmogorov-Smirnov test. If normality was not obtained, the data were transformed (arcsine of the square root of x) and analyzed using one-way ANOVA and Tukey's test. A p-value < 0.05 was considered statistical significance. All values ​​are expressed as mean ± SD (standard deviation) or ± SEM (standard error mean) as specified for each case.

[0089] (Example 1) Mreg differentiation and isolation of large extracellular vesicles Peripheral blood mononuclear cells (PBMCs) were obtained from a leukocyte reduction system (LRS) chamber provided by the Department of Transfusion Medicine (University Hospital of Schleswig-Holstein, Kiel, Germany). As shown in Figure 1 (left panel), monocytes were isolated and differentiated into Mreg cells. Briefly, PBMCs were purified using a Ficoll-Paque PLUS gradient (GE Healthcare, Chicago, USA), and monocytes were collected using a CD14-positive magnetic bead cell sorting system (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Isolated CD14+ monocytes were cultured in RPMI 1640 medium containing GlutaMax (GIBCO, Billings, MT, USA) supplemented with 10% human AB serum (Access Biological, Vista, CA, USA) and 4200 IU / ml human M-CSF (R&D Systems, Wiesbaden, Germany), in bags (Miltenyi) at a rate of 0.83 × 10⁶ 6Cells were cultured at a concentration of cells / ml.

[0090] The culture bags were placed in an incubator under standard culture conditions (humidified atmosphere of 5% carbon dioxide / 95% air, 37°C). After 6 days of culture, 500 IU / ml of human interferon (IFN)γ (R&D Systems, McKinley Place MN, USA) was added to the culture, and the cells were incubated for a further 24 hours. On day 0 (after cell seeding) and day 6 (after IFNγ was added), the cell culture bags were inverted. On day 7, Mreg cells were harvested and separated from the culture medium by centrifugation at 300×g for 10 min at room temperature (Figure 1, right panel).

[0091] The pellets containing Mreg were resuspended in PBS and subjected to further analysis. The remaining culture supernatant containing EV was further centrifuged at 4000×g for 1 hour at 4°C, and the L-EV-Mreg-containing pellets were resuspended in PBS and subjected to further analysis. Alternatively, L-EV-Mreg was resuspended in a 1:1 ratio in Cryostore 5 (Stemcell Technologies, Cologne, Germany) and stored at -80°C until further use.

[0092] Results: L-EVs were found to be detectable in Mreg cultures on day 7, the end of the differentiation period. The mean L-EV yield was 1.97 ± 0.64 L-EVs per Mreg cell (L-EVMreg / Mreg). L-EVMreg / Mreg was negatively correlated with the Mreg collection rate (the ratio of the number of Mregs collected on day 7 to the number of CD14+ monocytes seeded on day 0), while the lactate concentration in the culture medium was positively correlated with the pH of the culture medium and not with the glucose concentration in the culture medium on day 7 (data not shown).

[0093] (Example 2) Analysis of cell counters and nucleo counters In automated cell and vesicle analysis, basic parameters, such as the number of particles (cells or vesicles), were evaluated using: (i) the MOXI cell counter (Orflo, Ketchum, ID, USA), which analyzes membrane-bound vesicles and cells with a size of 3–20 μm based on the Coulter principle; and (ii) the nucleo counter (NC-200 Chemometec, Allerod, Denmark), which stains cell nuclei using two different dyes that allow for the distinction between living and dead cells.

[0094] Results: Mregs were found to have an average size of 13.46 ± 1.17 μm and an average volume of 1.31 ± 0.34 pl, while the corresponding L-EVMreg population were confinable vesicles with an average size of 7.34 ± 0.71 μm and an average volume of 0.21 ± 0.06 pl (Figure 2). Furthermore, L-EV-Mregs were clearly detectable by a method using the Coulter counter principle (MOXI analysis; Figure 2A), but could not be visualized by nucleic acid stain-based analysis (nucleo counter analysis; Figure 2B), suggesting that Mreg-derived L-EVs lack double-stranded nucleic acid (i.e., a nucleus).

[0095] (Example 3) Microscopic analysis Mreg cells and vesicles were analyzed by bright-field and transmission electron microscopy. For transmission electron microscopy, Mreg and L-EV-Mreg pellets were fixed in 3% glutaraldehyde in PBS for 30 minutes. After fixation with 2% osmium oxide for 2 × 15 minutes and dehydration in a series of gradually increasing ethanol doses, Mreg and L-EV-Mreg were embedded overnight in araldite. The araldite blocks were trimmed for ultrathin sectioning, and ultrathin sections (40–50 nm) were excised using an Ultramicrotome Leica UC7 with a diamond knife (Diatom, Hatfield, PA, USA). Sections were contrast-enhanced with uranyl acetate for 15 minutes and lead citrate for 7 minutes. Analysis was performed using a transmission electron microscope (Jeol JEM1400plus) connected to a digital imaging system (Firma TVIPS TemCam-F416).

[0096] Results: L-EVMregs can be easily observed by bright-field microscopy in Mreg cultures immediately after cell harvesting (Figure 3A). Using high-magnification TEM, Mregs exhibit the typical appearance of nucleated pseudopod-containing macrophages with numerous intracellular vesicles / vacuoles. L-EVMregs are smaller in size, lack a nucleus, and reveal a clear and homogeneous intravesicular structure consisting of numerous vesicles / vacuoles ranging in size from approximately 1 μm (Figure 3B). Note that L-EVs are not present in cell culture media containing 10% FCS (data not shown) and are created de novo during Mreg differentiation.

[0097] (Example 4) FACS analysis Fluorescence-activated cell sorting (FACS) was performed using a MACS Q10® cytometer (Miltenyi). Specific antibodies and their corresponding isotypes (all from BD Biosciences) were conjugated directly with fluorescein isothiocyanate (FITC): CD31, CD16, CD45, anti-mouse IgG1κ. Conjugated with phycoerythrin (PE): CD86, CD38, CD11c, anti-mouse IgG1κ. Conjugated with allophycocyanin (APC): CD206, CD103, anti-mouse IgG1κ, CD14, anti-mouse IgG2a. The gating strategy consists of: (i) identification based on the size and granularity of major Mreg and L-EV populations (FSC / SSC profile), (ii) exclusion of non-viable cells (by 7-AAD exclusion, BD Biosciences), (iii) identification of Mregs using each identity marker, and (iv) analysis of the same markers on L-EVs.

[0098] Results: Flow cytometry experiments were performed using antibodies against nine different specific differentiation antigen (CD) molecules, which had also been previously used to characterize Mreg

[15] . The presence of these specific markers was analyzed in L-EV-Mreg and Mreg, and both populations showed similar CD expression patterns on the surface for some of the molecules examined (CD11c was 90.33±2.74% in L-EV-Mreg and 99.14±0.29% in Mreg; CD86 was 81.74±5.11% in L-EV-Mreg and 98% in Mreg). The percentages were 54±0.80%; CD14 was 5.34±4.13% in L-EV-Mreg and 24.06±9.22% in Mreg; CD16 was 0.70±0.20% in L-EV-Mreg and 17.88±5.88% in Mreg; and CD38 was 26.93±1.57% in L-EV-Mreg and 44.56±11.80% in Mreg). However, compared to Mreg, the number of L-EV-Mreg positive cells was significantly lower for CD31 (L-EV-Mreg: 60.62±7.01%; Mreg: 95.50±3.29%; P<0.01), CD206 (L-EV-Mreg: 18.26±7.69%; Mreg: 59.66±10.78%; P<0.05), CD103 (L-EV-Mreg: 10.35±4.27%; Mreg: 68.92±6.92%; P<0.01), and CD45 (L-EV-Mreg: 94.84±0.86%; Mreg: 99.02±0.46%; P<0.05) (Figure 4).

[0099] Flow cytometry analysis demonstrated the presence of typical extracellular vesicle membrane markers (LAMP-1, CD9, CD63, and CD81) on L-EVMreg, based on the guidelines of Minimal Information for Studies of Extracellular Vesicles ((MISEV 2018)[24, 25, 26]).

[0100] (Example 5) Human angiogenesis proteome profiling array L-EV-Mreg was semi-quantitatively evaluated for the presence of 55 angiogenesis-related proteins using the Human Proteome Profiler Array Kit (R&D Systems, Minneapolis, MN, USA) as described in the manufacturer's protocol. Briefly, isolated L-EV-Mreg samples resuspended in PBS were sonicated on ice for 20 minutes to release intravesicular proteins and applied to array membranes containing antibodies against each pro-angiogenic protein. After incubation, a cocktail of biotinylated antibodies and HRP-streptavidin was added to the membranes, and the signals were visualized by chemiluminescence detection as per the provided manual. Membrane images were taken using Fusion FX Vilber (Vilber Lourmat, Eberhardzell, Germany), and signal intensity was analyzed using ImageJ 1.41 software (NIH) and Vilber Smart imaging (Vilber Lourmat).

[0101] Results: Proteomic profiling was performed to assess whether L-EV-Mreg contained intravesicular angiogenesis-promoting proteins. Semi-quantitative analysis revealed that L-EV-Mreg was rich in mediators that may promote angiogenesis. The results are shown in Figure 5. Of the 55 angiogenesis-related proteins evaluated, 23 were found to be at detectable levels (signal intensity > 10% of the reference spot). The most abundant proteins detected in L-EV-Mreg were interleukin-8 (IL-8; signal intensity 104.9% compared to the reference spot), platelet factor 4 (PF4; 98.5%), serpin E1 (97.2%), serpin F1 (96.5%), tissue metalloproteinase inhibitor 1 (TIMP-1; 96.2%), and angiogenin (95.8%).

[0102] (Example 6) In vitro wound healing and angiogenesis assay To investigate whether L-EV-Mreg may have a beneficial effect on wound healing and angiogenesis, a curettage and tubulation assay was performed using human endothelial cells (HUVECs). For this purpose, human umbilical vein endothelial cells (HUVECs) were isolated from the umbilical cord as previously described

[28] . These cells were cultured in endothelial cell growth medium ECGM (PromoCell, Heidelberg, Germany) supplemented with 4 μL / mL endothelial cell growth supplement, 0.1 ng / mL epidermal growth factor, 1 ng / mL basic fibroblast growth factor, 90 μg / mL heparin, 1 μg / mL hydrocortisone (all from PromoCell), and 10% fetal bovine serum (Thermo Fisher, Dreieich, Germany). HUVECs were maintained in a humid atmosphere (5% carbon dioxide / 95% air) at 37°C.

[0103] In the in vitro wound healing assay ("scratching assay"), 15,000 HUVEC cells / cm² were observed. 2 The cells were seeded and grown to confluence. The cell monolayer was scraped to create an in vitro wound, and 1 × 10⁶ cells were collected. 6 The culture was further maintained in or without the presence of L-EV-Mreg / ml. After 8 hours (T8h), the size of the remaining cell-free gap was evaluated using ImageJ 1.41 software (NIH) and compared to the gap size at T0h. In the in vitro tube formation assay, HUVEC cells were seeded in a special cell culture dish (Ibidi, Munich, Germany) according to the protocol provided by the manufacturer. Briefly, 10,000 HUVEC cells were seeded in culture medium into Matrigel® precoated wells. After 1 hour, the cells were 1 × 10⁶ 6 HUVECs were stimulated in or without the presence of L-EV-Mreg / ml. Micrographs were taken 8 hours after culture, and tubulation and angiogenesis-related parameters were analyzed using the angiogenesis analysis tool in ImageJ software 1.41 (NIH)

[25] .

[0104] Results: The curettage assay revealed that the presence of L-EV-Mreg resulted in a slightly higher percentage of endothelial cell-covered area compared to the control, indicating that L-EV-Mreg has a positive effect on wound healing (L-EV-Mreg: 86.64±11.67%; control without L-EV-Mreg: 78.65±8.58%; L-EV / control ratio: 1.10±0.08%; P<0.05; Figures 6A and 6B). The tube formation assay showed that the presence of L-EV-Mreg during the culture period significantly affected 6 of the 18 angiogenesis-related parameters examined (total segment length: +20.70±2.98%; P<0.05; number of isolated segments: -6.10±2.78%; P<0.05; number of segments: +23.00±6.65%; P<0.05; total master segment length: +17.50±1.84%; P<0.01; number of master segments: +33.10±7.00%; P<0.05; number of meshes: +41.30±7.33%; P<0.05; Figures 6C and 6D).

[0105] (Example 7) Inhibition of T cell activation and expansion To investigate T cell activation and expansion by Mreg and L-EV-Mreg cells, lymphocytes purified by Ficol gradient centrifugation were activated with CD3 / CD28 beads and then incubated with Mreg cells or L-EV-Mreg cells at varying concentrations. The ThermoFisher activation system (Dynabeads® human T-Activator CD3 / CD28; #11131D) consists of uniform beads with a diameter of 4.5 μm. These beads are inert and superparamagnetic. The beads are similar in size to antigen-presenting cells and are covalently linked to anti-CD3 and anti-CD28 antibodies. These two antibodies provide primary and costimulatory signals optimized for effective T cell activation and expansion. The ability of Mreg and L-EV-Mreg cells to control T cell activation and expansion was determined by the percentage of granzyme B-positive lymphocytes. The corresponding analysis was performed by flow cytometry using a MACS Q10® cytometer (Miltenyi) and the corresponding antibody (#12-8899-41) from Invitrogen.

[0106] Results: The results are shown in Figure 7. Activation of the lymphocyte population by administration of CD3 / CD28 beads resulted in the generation of a granzyme B-positive cell population (control = 100%). Adding Mreg to activated lymphocytes (Mreg / lymphocyte ratio = 1.34) significantly reduced the amount of granzyme B-positive cells (20.78 ± 4.61). A similar effect was also obtained by the application of L-EV-Mreg, and the inhibition of T cell activation (granzyme B-positive cells) was dose-dependent, achieving an effect comparable to that induced by Mreg (25.85 ± 7.04) with the administration of 3.2 million L-EV-Mreg / ml (L-EV Mreg / lymphocyte ratio = 9.14).

[0107] (Example 8) Mreg and L-EV Mreg Phosphatidylserine exposure on the membrane To evaluate PS exposure on the extracellular membrane by flow cytometry, the Annexin V-FITC kit (Miltenyi Biotec, Germany) was used according to the manufacturer's instructions. Briefly, immediately after isolation, cells were incubated in Annexin V buffer for 20 minutes, washed twice with the same buffer, and the final pellet was divided into four groups. Cells were incubated (i) in the absence of the fluorescent dye, (ii) in the presence of Annexin V, (iii) in the presence of propidium iodide (PI), or (iv) in the presence of both Annexin V and PI. Annexin V was added for an additional 20 minutes of incubation, while PI was added immediately before performing the flow cytometry scan. Co-staining of cells with PI and Annexin V was considered to indicate dead cells.

[0108] To visualize the PS of the outer membrane of cells / vesicles, L-EV Mreg The contained Mreg pellet was resuspended in a buffer containing 10 mM HEPES and 145 mM NaCl. The resuspended Mreg L-EV Mreg 10 μM PSVue 480 (Molecular Targeting Technologies, West Chester, PA, USA) and 0.2 ng / ml Hoechst 33342 for nucleus staining were added. The mixture was then held in the dark at 37°C for 15 min, followed by centrifugation at 500 × g for 5 min. The resulting pellet was then resuspended in HEPES buffer. A small amount of resuspended Mreg and L-EV was added. Mreg The samples were placed on a glass slide, covered with a glass coverslip, and examined immediately. The analysis was performed using a Leica DM2000 LED fluorescence microscope equipped with DAPI, L5, and rhodamine filter cubes; an HC PL FLUOTAR × 40 / 0.80 objective lens; and a Leica DFC7000 T fluorescence camera, with Image Overlay software.

[0109] All experiments involved cells derived from at least five healthy donors and L-EVs. MregThe study was conducted using the following statistical software: GraphPad Prism 5.01 for Windows (GraphPad Software, San Diego, USA). Group comparisons were performed using the Kolmogorov-Smirnov test. If normality was not obtained, the data were transformed (arcsine of the square root of x) and analyzed using one-way ANOVA and Tukey's test. A p-value < 0.05 was considered statistical significance. All values ​​are expressed as mean ± SEM (standard error mean).

[0110] Results: Nuclear staining and PS staining were performed on viable cells of Mreg and L-EVMreg. The results showed that Mreg was either negative for PS or PS was limited to a limited area of ​​the cell surface. In contrast to Mreg, L-EVMreg lacked a nucleus and showed a strong PS signal across its entire surface (Figures 8A-8C). Further analysis of the vesicle fraction by cytometry confirmed the fluorescence microscopy findings that define L-EVMreg as a PS-positive vesicle structure lacking a nucleus.

[0111] (References) TIFF2026517332000002.tif227158TIFF2026517332000003.tif228159TIFF2026517332000004.tif61161

Claims

1. Macrophage-derived extracellular vesicles having a size of 0.1–10 μm that exhibit immunomodulatory, angiogenic, anti-inflammatory, and / or tissue regenerative activities.

2. The macrophage-derived extracellular vesicle according to claim 1, comprising one or more markers selected from the group consisting of CD11c, CD86, CD31, and CD45 on its surface.

3. The macrophage-derived extracellular vesicle according to claim 1 or 2, comprising one or more markers selected from the group consisting of LAMP-1, CD9, CD63, and CD81 on its surface.

4. Macrophage-derived extracellular vesicles according to any one of claims 1 to 3, which are derived from immunoregulatory macrophages (Mreg), preferably immunoregulatory macrophages expressing CD258, DHRS9, and IDO markers.

5. Macrophage-derived extracellular vesicles according to any one of claims 1 to 4, comprising one or more proteins selected from the group consisting of interleukin-8, platelet factor-4, serpin E1, serpin F1, TIMP-1, and angiogenin.

6. The macrophage-derived extracellular vesicle according to claim 5, comprising interleukin-8, platelet factor-4, serpin E1, serpin F1, TIMP-1, and angiogenin.

7. A macrophage-derived extracellular vesicle according to any one of claims 1 to 6, which does not contain a nucleus.

8. An extracellular vesicle derived from a macrophage according to any one of claims 1 to 7, which does not include any organelle selected from the group consisting of the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex.

9. An extracellular vesicle derived from a macrophage according to any one of claims 1 to 8, comprising phosphatidylserine on its surface.

10. Macrophage-derived extracellular vesicles according to any one of claims 1 to 9, which can be obtained by a method comprising the steps of differentiating CD14-positive blood monocytes into immunoregulatory macrophages by culturing the monocytes in a culture medium in the presence of (i) M-CSF and / or GM-CSF, (ii) CD16 ligand, and (iii) IFN-γ, and then obtaining the extracellular vesicles from the culture medium.

11. A pharmaceutical composition comprising macrophage-derived extracellular vesicles according to any one of claims 1 to 10.

12. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in pharmaceuticals.

13. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in a method for suppressing graft rejection and / or extending the graft survival period in a subject receiving transplantation.

14. A macrophage-derived extracellular vesicle or pharmaceutical composition for use in the method according to claim 13, wherein the graft is an allogeneic graft.

15. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in methods for promoting or sustaining the engraftment or effect of cell-based pharmaceuticals of regulatory T cells.

16. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in methods of treating or preventing autoimmune diseases, inflammatory diseases, or hypersensitivity reactions.

17. The aforementioned autoimmune diseases include systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritis; ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous microvasculitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behçet's disease, thromboangiitis obliterans, and Kawasaki disease. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method of claim 16, selected from the group consisting of large, medium, or small vasculitis of other autoimmune etiologies; multiple sclerosis (MS) and neuroimmunological disorders; type 1 diabetes mellitus, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction, and other autoimmune endocrine disorders; hemolytic anemia, thrombocytopenic purpura, and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, pemphigus, and other autoimmune skin conditions.

18. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method of claim 16, wherein the inflammatory disease is selected from the group consisting of arterial occlusive diseases, such as peripheral artery occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or stenosis; microvascular angina pectoris, also known as cardiac syndrome X; inflammation systemically associated with metabolic disorders, including type II diabetes mellitus and obesity-related metabolic syndrome; and skin diseases, including eczema.

19. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method according to claim 16, wherein the hypersensitivity reaction is selected from the group consisting of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity, and mast cell disease.

20. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in methods for inducing wound healing or promoting the tissue repair process, or preventing / limiting fibrosis, by being involved in tissue remodeling, tissue regeneration, angiogenesis, and vascular formation.

21. Macrophage-derived extracellular vesicles according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in a method for treating microvascular or macrovascular disorders of the target lower limb.

22. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method according to claim 21, wherein the macrovascular disorder is selected from the group consisting of aneurysm, dissection, atherosclerosis, atherosclerosis, peripheral artery occlusive disease (PAD), intermittent claudication, necrosis and gangrene, vascular malformation, Lulish syndrome, or compression syndrome.

23. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method according to claim 21, wherein the microvascular disorder is selected from the group consisting of vasculitis, arteritis, angioplasia, white atrophy, sclerosis of the skin, Determann syndrome, diabetic vascular disorder, endarteritis obliterans, erythromelalgia, fibromuscular dysplasia, perforation, Mönkeberg sclerosis, Aussler's disease, compartment syndrome, Paget-von Schröder syndrome, Raynaud's syndrome, and lower leg ulcers.

24. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method according to claim 23, wherein the lower leg ulcer is diabetic or venous lower leg ulcer.

25. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method of claim 24, wherein the method comprises the step of directly administering vesicles to an ulcer by subcutaneous or intramuscular injection.

26. Macrophage-derived extracellular vesicles or pharmaceutical compositions for use in the method according to any one of claims 21 to 25, wherein the microvascular or macrovascular disorder is diabetic microvascular or macrovascular disorder.

27. A method for preparing macrophage-derived extracellular vesicles having immunomodulatory activity, (a) A step of isolating CD14-positive monocytes from the target blood sample; (b) A step of culturing the monocytes in a culture medium containing (i) M-CSF and / or GM-CSF and (ii) CD16 ligand; (c) The step of bringing the cells into contact with IFN-γ; (d) A step of obtaining macrophage-derived extracellular vesicles from the culture medium. A method that includes this.

28. The method according to claim 27, wherein the culture medium in step (b) comprises human blood serum, for example, human AB serum.

29. The method according to claim 27 or 28, wherein the concentration of M-CSF and / or GM-CSF in step (b) is in the range of 5 to 100 ng / ml, preferably 20 to 25 ng / ml.

30. The method according to any one of claims 27 to 29, wherein the monocytes of step (b) are cultured for at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to IFN-γ stimulation.

31. The method according to any one of claims 27 to 30, wherein the culture in step (b) is carried out in a breathable bag, which is preferably made of plastic.

32. The method according to any one of claims 27 to 31, wherein the concentration of IFN-γ in step (c) is in the range of 5 to 100 ng / ml, preferably 20 to 25 ng / ml.

33. The method according to any one of claims 27 to 32, wherein the macrophage-derived extracellular vesicles are obtained by centrifugation from the culture medium of step (d).

34. The method according to claim 33, wherein the macrophage-derived extracellular vesicles of step (d) are separated from macrophage cells by centrifugation.

35. A method for preparing immunoregulatory T cells, (a) The process of obtaining the target T cells; (b) A step of co-culturing T cells with macrophage-derived extracellular vesicles according to any one of claims 1 to 10; (c) A step of obtaining the immunoregulatory T cells from the culture medium. A method that includes this.

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