Manufacturing process of protein-associated extracellular vesicles

Abstract

The present invention relates to a process for producing protein-associated extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), comprising purifying EVs from a cell culture medium containing MSCs, the purification occurring by at least one filtration step of the medium, followed by concentrating the filtrate of the at least one filtration step by tangential flow filtration (TFF) in a TFF device, wherein the EVs are concentrated by TFF in a TFF device, during which the EVs are associated with one or more exogenous proteins within the TFF device or within a container fluidly connected to the TFF device to which the EVs are transferred. The present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of protein-associated EVs, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to a process for producing extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) and associated with proteins. The present invention also relates to compositions comprising therapeutically effective amounts of EVs associated with proteins, and uses thereof. [Background technology]

[0002] EVs are lipid bilayer-bound particles naturally released from cells and, unlike cells, are unable to replicate. EV diameters range from sizes approaching the smallest physically possible unilamellar liposomes (roughly 20–30 nanometers) to over 10 microns, although the vast majority of EVs are less than 200 nm. EVs transport cargo from parent cells, including proteins, nucleic acids, lipids, metabolites, and even organelles. Most cells studied to date, including some bacterial, fungal, and walled plant cells, are thought to release EVs. A wide variety of EV subtypes have been proposed and variously defined according to size, biogenesis pathway, cargo, cellular source, and function, leading to a historically heterogeneous nomenclature, including terms such as exosomes, microvesicles, and ectosomes.

[0003] EVs can be used for therapeutic purposes, such as delivering nucleic acids or other cargo to diseased tissues and cells. The growing interest in EVs has coincided with the establishment of companies and funding programs focused on developing EVs as biomarkers or treatments for disease, the founding of the International Society for Extracellular Vesicles (ISEV), and the launch of a scientific journal dedicated to this field, the Journal of Extracellular Vesicles.

[0004] With growing interest in EVs for therapeutic use, the need for clinical-grade EVs has also increased. To be clinically useful, EVs must be produced in a reproducible and controlled manner. Good manufacturing practice (GMP) for the production of EV-containing compositions is crucial to ensure uniformity and consistent quality of each batch produced. GMP is particularly stringent when dealing with cell-derived therapies. Several issues must be resolved before widespread clinical use of EVs is possible. Developing a platform for producing, storing, and handling clinical-grade EVs with reliable and reproducible quantification remains a challenge. MSC-derived EV-based medicines and subsequent clinical trials require the resolution of several technical, scientific, regulatory, and mechanistic challenges before MSC-derived EV-based therapies can be brought to the clinic.

[0005] Therefore, a protocol is needed that enables the stable and reproducible production of clinical-grade MSC-derived EVs under GMP conditions in accordance with the International Conference on Harmonization (ICH) 2020 GMP quality guidelines. Furthermore, EV-based products with good stability and commercially attractive shelf lives are needed. Additionally, there is a need to produce clinical-grade MSC-derived EVs with desired proteins bound to their membrane or surface. These proteins may improve the functionality and therapeutic value of EVs.

[0006] EVs have been engineered to express various therapeutic molecules (WO2019 / 198077). EVs have also been loaded with siRNA, oligonucleotides, or guide RNA (Reka Agnes Haraszti et al., 2018). Loading can be achieved by incubation of donor cells with the cargo molecules or by sonication of EVs, extrusion freeze-thaw cycling, and electroporation (DLuan et al., 2017). WO2021 / 084274A1 discloses compositions containing EVs with lipid-binding proteins bound to their outer surface.

[0007] Although EV purification methods have been reported, scalability remains an issue. Nordin Joel Z et al. (2019) disclose the use of tangential flow filtration (TFF) for EV purification as a method that can produce EVs on a large scale. The method employs labor-intensive bind / elute size-exclusion chromatography. Zheng Zhao et al. (2019) propose a method for producing surface antigen-engineered EVs using a microfluidic cell culture chip. The latter method is costly and not easily available.

[0008] There remains a need in the art for production methods that allow for the efficient production of protein-associated EVs that may be used therapeutically. Summary of the Invention

[0009] The present invention and its embodiments serve to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a process for producing protein-associated EVs derived from MSCs, as claimed in claim 1. More specifically, said process comprises: purifying EVs from a cell culture medium containing MSCs, said purification occurring by at least one filtration step of said medium; a step of concentrating the filtrate of said at least one filtration step, wherein said EVs are concentrated by tangential flow filtration (TFF) in a TFF device; During the TFF process, the EVs associate with one or more exogenous proteins within the TFF device or within a container fluidly connected to the TFF device into which the EVs are transferred from the TFF device.

[0010] In a second aspect, the present invention also relates to a composition comprising a therapeutically effective amount of EVs associated with a protein according to claim 11. In a last aspect, the present invention relates to the use of a composition according to claim 19. Embodiments of the process are given in claims 2 to 10, embodiments of the pharmaceutical composition are given in claims 12 to 17, and embodiments of the use of the pharmaceutical composition are given in claims 20 to 28. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows an embodiment of an MSC expansion unit and an EV processing unit that can be used to produce EVs of the present invention.

[0012] definition Unless otherwise defined, all terms used in the disclosed invention, including technical and scientific terms, have the meanings commonly understood by one skilled in the art to which this invention belongs. For further guidance, term definitions are included to better understand the teachings of the present invention.

[0013] As used herein, the following terms have the following meanings:

[0014] As used herein, "A," "an," and "the" refer to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0015] When used herein to refer to a measurable value such as a parameter, amount, duration in time, etc., "about" is intended to encompass a variation of + / -20% or less of and from the stated value, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less, to the extent that such variation is appropriate for practice in the disclosed invention. However, it will be understood that the value to which the modifier "about" refers is itself specifically disclosed.

[0016] As used herein, "comprise," "comprising," "comprises," and "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," or "contains," and are inclusive or open-ended terms that specify the presence of, for example, the following components, and do not preclude or exclude the presence of additional, unlisted components, properties, elements, members, steps that are known in the art or disclosed in the art.

[0017] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0018] The terms "wt %," "weight percent," "% wt," or "wt%," herein, and throughout the description unless otherwise defined, refer to the relative weight of each component based on the total weight of the formulation.

[0019] While the terms "one or more" or "at least one," such as one or more, or at least one member of a group of members, are clear in themselves, by way of further illustration, they specifically encompass reference to any one of said members, or any two or more of said members, such as, for example, any three or more, four or more, five or more, six or more, or seven or more of said members, up to and including all said members.

[0020] Unless otherwise defined, all terms used in the disclosed invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which the invention belongs. For further guidance, definitions of terms used in the description are included to better understand the teachings of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the present invention.

[0021] Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification may, but do not necessarily, all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, while some embodiments described herein include some features but not other features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the present invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0022] In the present invention, the term "extracellular vesicles" or "EVs" refers to micro- or nanometer-sized particles secreted by various types of cells in vivo and in vitro, containing proteins associated with said EVs. EVs contain proteins, growth factors, miRNA, and other molecules encapsulated in lipid spheres. EVs can be classified according to their size and intracellular origin. Exosomes are a subgroup of EVs, typically in the size range of 0.1 microns or less. Exosomes originate from multivesicular bodies, late endosomal compartments, and are secreted by fusion of multivesicular bodies with the plasma membrane. Another type of EV is shedding vesicles (also known as microvesicles), a heterogeneous population of membrane vesicles up to 1 micron in size that are released directly from the cell membrane by disruption of the cortical cytoskeleton. All types of vesicles secreted by cells are generally defined as EVs.

[0023] The term "associated with an EV", in relation to a substance, means that the substance is either a) attached to or bound to the surface of an EV (by any type of bond, such as covalent or non-covalent), preferably non-covalently; and / or b) attached to or bound within the surface of said EV; and / or c) incorporated within said EV.

[0024] The substances associated with EVs can be any type of substance, including, but not limited to, amino acids, proteins, peptides, nucleic acids such as RNA and DNA (e.g., non-coding RNA, miRNA, mRNA), sugars, carbohydrates, fats, vitamins, growth factors, pro-angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, antioxidant molecules, pro-neurogenic molecules, and antiviral molecules; and ions such as metal ions or calcium ions.

[0025] The terms "cell medium" or "cell culture medium" or "medium" refer to an aqueous solution of nutrients and other components of a defined composition, which can be used for the maintenance or growth of cells.

[0026] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to a subject and does not suppress the biological activity and properties of the administered composition. For example, a pharmaceutically acceptable carrier may function as a stabilizer and / or adjuvant. Examples of carriers include, but are not limited to, propylene glycol, saline, emulsions, and mixtures of water and organic solvents.

[0027] The term "sufficient amount" means an amount sufficient to produce a desired measurable effect, eg, an amount sufficient to alter a protein expression profile.

[0028] The term "therapeutically effective amount" refers to an amount effective to ameliorate symptoms of a disease. Since prevention of disease onset can be considered treatment, a therapeutically effective amount can also be a "prophylactically effective amount."

[0029] The term "treatment" refers to both therapeutic and prophylactic or preventative treatment, which aims to prevent or slow (alleviate) the target pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0030] The term "composition" refers to a composition at any stage in the manufacturing process, including a pharmaceutically acceptable end product and any intermediates in its manufacture.

[0031] The term "treating" refers to reversing, preventing, alleviating, or suppressing the progression of a disease, disorder, or disorder, or one or more symptoms of a disease, disorder, or disorder. As used herein, "treating" may refer to reducing the likelihood or frequency of occurrence of a disease, disorder, or disorder in a mammal compared to an untreated control population, or compared to the same mammal before treatment. For example, as used herein, "treating" may refer to preventing a disease, disorder, or disorder, and may include delaying or preventing the onset of a disease, disorder, or disorder, or delaying or preventing the symptoms of a disease, disorder, or disorder. As used herein, "treating" may refer to reducing the severity of a disease, disorder, or disorder, or the symptoms of such a disease, disorder, or disorder, before the onset of the disease, disorder, or disorder. Such prevention or reduction of the severity of pre-painful diseases, disorders, or disorders refers to the administration of the compositions of the present technology as described herein to a subject who is not suffering from the disease, disorder, or disorder at the time of administration. As used herein, "treating" may also refer to preventing the recurrence of a disease, disorder, or disorder, or the recurrence of one or more symptoms of such a disease, disorder, or disorder. As used herein, the terms "treatment," "treatment," and "therapeutically" refer to the therapeutic action as defined above.

[0032] The term "fibrosis," as used herein, refers to the formation of excess fibrous connective tissue in an organ or tissue undergoing a repair or reactive process.

[0033] In the present invention, the term "mesenchymal stromal cells" or "MSCs" should be understood as adherent stromal cells that can differentiate into various cell types. Sources of MSCs are bone marrow, umbilical cord cells, adipose tissue, amniotic fluid, mammary gland, and blood. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention relates to a process for producing EVs associated with proteins, compositions comprising EVs associated with proteins, and uses thereof.

[0035] In a first aspect, the present invention provides a process for producing protein-associated EVs derived from MSCs, comprising: Purifying EVs from a cell culture medium containing MSCs, said purification occurring by at least one filtration step of said medium, followed by a step of concentrating the filtrate of said at least one filtration step, wherein said EVs are concentrated by tangential flow filtration (TFF) in a TFF device; During the TFF process, the EVs bind or associate with one or more exogenous proteins within the TFF device or within a container fluidly connected to the TFF device into which the EVs are transferred from the TFF device.

[0036] "Binding" and "binding to" in the context of the present invention may refer to any type of bond or attachment, either covalent or non-covalent, or any other type of bond or attachment known in the art.

[0037] The MSCs of the present invention are grown and expanded in a concentrated cell culture medium. When the MSCs reach a desired concentration, the diluted cell culture medium containing EVs, called the cell supernatant, from which the EVs are harvested, is collected for further processing. In one embodiment, the supernatant contains at least 40 x 10 MSCs. 6 They are harvested when a minimum concentration of cells / L is reached. Cell concentration and viability may be defined by cell counting, for example, with a hemocytometer such as a Burker Counting Chamber and trypan blue staining.

[0038] To purify EVs from the supernatant, the cell culture medium in which MSCs are maintained (also called cell supernatant) is filtered to remove contaminants present in said cell culture medium, which contributes to the purity and stability of the final product.

[0039] In this context, "stability" of a product such as an EV refers to the ability of a particular formulation or product to remain within predefined ranges of values ​​for parameters of physical, chemical, microbiological, toxicological, and functional specifications or characteristics in a particular environment, such as a container or closed system, over a period of time. Non-limiting examples of such parameters include particle number, particle size, leakage of internal components, and activity.

[0040] This purification occurs by at least one filtration step of the medium, hi one embodiment, the at least one filtration step is a dead-end filtration.

[0041] In a preferred embodiment, the filtration comprises at least two steps of filtration. Again, in one embodiment, at least one filtration step is dead-end filtration. In a further embodiment, both filtration steps are performed by dead-end filtration. Preferably, at least one of the filtration steps is also used as a product sterilization means to comply with the GMP regulations defined above. It has been found that a series of dead-end filtrations is sometimes necessary to sufficiently remove impurities from the supernatant. In some cases, it has been found that a single filtration often clogs the filter used, reducing the purity and quantity of the final product.

[0042] In the first filtration step, the cell supernatant is filtered through a dead-end filter. In a further preferred embodiment, the filtration is performed by passing the supernatant through a filter with a mesh size of 1 to 5 microns, more preferably 1 to 3 microns. In one embodiment, the first filtration step is performed by dead-end filtration. In a further embodiment, this is preferably performed in a closed system with a peristaltic pump providing a constant flow through the filter, even more preferably about 100 ml / min.

[0043] In one embodiment, the filtrate from the first filtration is sent to a second filter, this time with a smaller pore size than that used in the first filtration step. Preferably, this second filtration step is a sterilization step to make the final product compliant with GMP regulations as defined above. In a more preferred embodiment, the filter has a mesh size of less than 1 micron, more preferably 0.05-1 micron, even more preferably 0.1-0.5 micron, and most preferably 0.1-0.22 micron. In one embodiment, the second filtration step is carried out by dead-end filtration. In a further embodiment, this is preferably carried out in a closed system interconnected with the first filtration step. In another further embodiment, this is carried out with a peristaltic pump providing a constant flow through the filter, even more preferably at about 100 ml / min.

[0044] The filtrate of said one or more filtration steps will contain the EVs according to the present invention.

[0045] In the final step, the EVs are washed and concentrated. Washing and concentration can be performed by conventional means known in the art, such as membrane filtration, either microfiltration or ultrafiltration. Concentration is a process that involves removing fluid from a solution while retaining solute molecules. Membrane filtration is a separation technique widely used in life science laboratories. Depending on the membrane porosity, membrane filtration can be classified as a microfiltration or ultrafiltration process. Microfiltration membranes, typically with pore sizes of 0.1 μm to 10 μm, are commonly used for clarification, sterilization, and particulate removal, or for cell collection. Ultrafiltration membranes, with smaller pore sizes of 0.001 to 0.1 μm, are used for concentration and desalting of solute molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), buffer exchange, and gross fractionation. Ultrafiltration membranes are typically classified by their molecular weight cutoff (MWCO) or nominal molecular weight cutoff (NMWCO), rather than by pore size.

[0046] There are two main membrane filtration styles that can use either microfiltration or ultrafiltration membranes: 1) direct flow filtration (DFF), also known as "dead-end" filtration, brings the feed stream perpendicular to the membrane surface and attempts to force 100% of the fluid through the membrane, and 2) tangential flow filtration (TFF), also known as cross-flow filtration, moves the feed stream parallel to the membrane surface, so that some passes through the membrane (permeate) while the remainder (retentate) is recycled back to the feed reservoir.

[0047] Said washing and concentration will preferably be carried out by a tangential flow filtration step (TFF). Tangential flow filtration (TFF) or cross-flow filtration is a process in which the feed stream travels parallel to the membrane surface. Under pressure, part of the stream is forced through the membrane (permeate), while the remainder (retentate) is recycled back to the feed reservoir.

[0048] In one embodiment, the filtrate of at least one filtration step, comprising said EVs derived from MSCs, will be used in a TFF concentration step in a TFF device.

[0049] In a further preferred embodiment, the TFF will have a molecular weight cutoff range (MWCO) of 100 kDa. MWCO is defined as the minimum molecular weight of a solute that is 90% retained by the membrane. Therefore, a TFF with a 100 kDa cutoff range will remove most, if not all, of the particles and components in the TFF permeate that have a molecular weight less than 100 kDa. As a result, the final composition remaining in the retentate does not contain any free (i.e., not associated with EVs) constituents, components, or substances that have a molecular weight less than 100 kDa. The constituents, components, or substances can be any particles, such as proteins or peptides, that are normally present in or are part of the cell culture medium.

[0050] In one embodiment, during the TFF process, EVs bind or associate with one or more exogenous proteins within the TFF device or within the container fluidically connected to the TFF device into which the EVs are transferred. In one embodiment, the proteins are replenished into the TFF device or the connected container during the concentration of the EVs. In a further embodiment, the replenishment of proteins into the TFF device or the connected container can be performed, by way of non-limiting example, by infusion or injection into the TFF device, which is preferably a closed system.

[0051] The protein will bind or associate with EVs in the TFF or in a container connected to the TFF. It is important that the container should be fluidically connected to the TFF in a closed system. The container can be any container known in the art that can be connected to a TFF device and can combine the TFF retentate containing EVs with the required protein. In a non-limiting example, the container can be a bag or pouch. The retentate and the product bound to the protein will then be returned to the TFF.

[0052] Unbound or unassociated protein will be washed away during one or more washing steps preferably carried out in TFF.Washing step can be carried out using one or more washing buffers, such as buffered saline.Washing buffer should be compatible with EV and protein, so that the buffer does not contain any harmful substances.

[0053] In one embodiment, the protein is selected from the group consisting of annexin, thioredoxin, or lactadherin. In a preferred embodiment, the protein is annexin V, Trx, or Mfge8, preferably recombinant annexin V, Trx, or Mfge8.

[0054] Annexins are calcium-dependent phospholipid-binding proteins, and therefore, their binding to EVs enhances the anti-inflammatory properties of EVs. Some annexins, such as annexin V, are associated with pro-inflammatory activity. However, the association of annexin V with EVs has been shown to enhance the anti-inflammatory activity of EVs. Annexin V has a molecular weight of approximately 37 kDa.

[0055] Thioredoxins are small redox proteins known to exist in all living organisms. Thioredoxin 1 (Trx) has strong antioxidant activity and exhibits anti-inflammatory and anti-fibrotic properties. In addition, binding to EVs enhances the anti-inflammatory properties of EVs.

[0056] Lactadherin, also known as milk fat globule-EGF factor 8 protein (Mfge8), is a secreted protein found in vertebrates, including mammals and birds. Lactadherin contains a phosphatidylserine-binding domain and an Arg-Gly-Asp-binding sequence. Mfge8 binds in a calcium-independent manner.

[0057] In a further embodiment, calcium is replenished to the TFF device or the fluidically connected container during the EV concentration. Preferably, calcium is replenished at a concentration of about 1 to about 10 mM. In one example, the calcium concentration is 1 to 9 mM, in another example, 1 to 8 mM, in another example, 1 to 7 mM, in another example, 1 to 6 mM, in another example, 1 to 5 mM, in another example, 1 to 4 mM, or in another example, 1 to 3 mM. Preferably, the calcium concentration is 1.5 to 3 mM, preferably 1.5 to 2.5 mM. Most preferably, about 2 mM calcium is replenished to the TFF device during the EV concentration.

[0058] Calcium in human blood is generally maintained within a fairly narrow range of about 2.2 to about 2.7 mM. When the EVs are administered to a (human) patient (see below), the calcium range is preferably maintained below 10 mM in the final administered product.

[0059] Injecting calcium into the TFF device or the fluidically connected vessel aids in the binding of calcium-dependent proteins, such as annexins and thioredoxins, to EVs. For calcium-independent proteins, such as Mfge8, binding or association with EVs, calcium injection is not required.

[0060] In a further embodiment, the calcium and the protein are mixed in the TFF or fluidically connected vessel and incubated with the EVs present in the TFF device or vessel.

[0061] In a further embodiment, the protein-bound EVs / protein-associated EVs are washed, re-concentrated and subsequently collected outside the TFF device.

[0062] As already mentioned, proteins that are not bound to or associated with EVs will be removed during one or more washing steps, which are preferably also performed in TFF. The retentate will contain EVs. One or more washing steps can be performed using a washing buffer, such as, but not limited to, buffered saline. The washing buffer should be compatible with EVs and EV-associated proteins, in the sense that EVs and EV-associated proteins remain viable and retain their properties.

[0063] Once unbound or non-associated proteins have been thoroughly washed away, the protein-bound EVs / EVs associated with the protein are concentrated, preferably by circulation through the TFF device. The concentrated end product containing EVs is then collected outside the device, preferably in a collection container such as, but not limited to, a collection bag or cryotube.

[0064] The final product may be stored below 10°C, preferably below 4°C, or more preferably by cryopreservation, wherein the composition is frozen at temperatures between -20°C and -196°C, preferably between -40°C and -196°C, more preferably between -80°C and -196°C. The freezing procedure is preferably a flash freezing or vitrification procedure, which ensures that the product remains viable during the freezing process and after thawing. Another freezing procedure may be controlled-rate freezing, which preferably compensates for the exothermic reaction at the crystallization point to improve the stability of the product. For the latter procedure, a controlled-rate freezer may be used.

[0065] In one embodiment, the medium in which MSCs are cultured and expanded prior to the start of dilution and EV collection is a cell culture medium containing purified human serum albumin.

[0066] Albumin in the final product aids in the stability and functionality of the EVs. As previously defined, the "stability" of a product, such as an EV, in this context refers to the ability of a particular formulation or product to remain within a predefined range of values ​​for parameters of physical, chemical, microbiological, toxicological, and functional specifications or characteristics in a particular environment, such as a container or closed system, over a period of time. Non-limiting examples of such parameters include particle number, particle size, leakage of internal components, and activity.

[0067] Albumin should preferably be present in the cell culture medium for culture and expansion, as it aids in EV stability and functionality. In one embodiment, the medium therefore contains human albumin, either recombinant albumin or purified albumin, e.g., purified from human plasma. In a further embodiment, the albumin is present in the medium at a concentration of 1 g / L to 5 g / L. The latter concentration has been shown to be particularly useful in obtaining a viable, stable end product.

[0068] In a second aspect, the present invention also relates to a composition comprising therapeutic protein-bound EVs / protein-associated EVs, and optionally calcium. The composition is obtainable by the process described above. In a preferred embodiment, the size of the EVs is less than 1 μm.

[0069] In a preferred embodiment, the size of the EVs in the composition is less than about 750 nm, preferably less than 500 nm, preferably less than 400 nm, preferably less than 300 nm. In another or further preferred embodiment, the size of the EVs in the composition is at least 5 nm, more preferably at least 10 nm, more preferably at least 25 nm, more preferably at least 50 nm. In another or further preferred embodiment, the size of the EVs in the composition is 25 to 500 nm, preferably 25 to 400 nm, more preferably about 50 to about 300 nm.

[0070] Optical techniques are routinely used to measure and count extracellular vesicles (EVs). In one embodiment of the present invention, the particle size of the EVs is measured by nanoparticle tracking analysis (NTA), a method suitable for quantifying and sizing nanoparticles suspended in a buffer solution. In another or further embodiment, particle size is measured using tunable resistive pulse sensing (TRPS), which is used as a reference method for NTA. In another or further embodiment, particle size is measured using high-resolution flow cytometry.

[0071] An effective amount is the amount of agent that alone stimulates the desired result.The absolute amount will depend on various factors, including the substance selected for administration, whether it is administered once or multiple times, and individual patient parameters, including age, physical condition, size, weight, and stage of disease.These factors are well known to those skilled in the art and can be addressed with just routine experimentation.Dosage may also vary depending on the subject to which the composition is administered.

[0072] In one embodiment of the composition, the composition may further contain calcium, and preferably, the calcium concentration of the composition is about 1 to about 10 mM. In one embodiment, the calcium concentration is 1 to 9 mM, or 1 to 8 mM, or 1 to 7 mM, or 1 to 6 mM, or 1 to 5 mM, or 1 to 4 mM, or 1 to 3 mM. Preferably, the calcium concentration is 1.5 to 3 mM, preferably 1.5 to 2.5 mM. Most preferably, the calcium concentration of the composition is about 2 mM.

[0073] In one embodiment, said calcium (at least in part) is bound to or associated with EVs.

[0074] Calcium levels below 1 mM will result in decreased binding of calcium-dependent binding proteins. Because calcium in human blood is generally maintained within a fairly narrow range of about 2.2 to about 2.7 mM, administration of compositions to human patients is avoided if the composition contains more than 10 mM calcium.

[0075] In one embodiment, the composition of the present invention will further comprise human albumin at a concentration of 10-30 g / l, more preferably at a concentration of 10-20 g / l, more preferably at a concentration of 15-20 g / l.

[0076] The source of the albumin is the cell culture medium for MSCs. Therefore, since albumin is not produced by MSCs, it may theoretically be considered a contaminant in the production process. However, surprisingly, it has been found that the presence of albumin is actually necessary to ensure the stability and activity of the final product, and thus the composition of the present invention. Albumin at the required concentration functions as a drug stabilizer and activity enhancer. Forced removal of albumin has been found to reduce the activity of the product (Hyungtaek Jeon et al., 2020).

[0077] The albumin concentration may be measured by a colorimetric method using an anti-albumin antibody or by ELISA. In one embodiment, the colorimetric measurement is a bromocresol green assay.

[0078] In one embodiment, at least 90% of the albumin present in the composition is associated with the EVs in the composition.

[0079] Human albumin has a MW of approximately 66 kDa. Albumin that is not associated with EVs is therefore removed by the TFF process, and the albumin remaining in the composition is associated with EVs. In a further preferred embodiment, at least 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, and more preferably 99% of the albumin in the composition is associated with EVs.

[0080] In one embodiment, the composition of the present invention comprises human albumin and calcium, and preferably the composition comprises about 25-750 mg of albumin per mg of calcium, more preferably about 25-500 mg of albumin per mg of calcium, more preferably about 50-250 mg of albumin per mg of calcium, more preferably about 50-500 mg of albumin per mg of calcium, more preferably about 75-400 mg of albumin per mg of calcium, more preferably about 100-350 mg of albumin per mg of calcium, more preferably about 125-300 mg of albumin per mg of calcium, more preferably about 150-275 mg of albumin per mg of calcium, and most preferably about 175-250 mg of albumin per mg of calcium.

[0081] In one embodiment of the composition, the protein associated with EVs is selected from annexin, thioredoxin, and lactadherin, preferably the protein is a recombinant protein. In a further embodiment of the composition, the protein is annexin V, Trx, or Mfge8, preferably recombinant annexin V, Trx, or Mfge8.

[0082] As previously shown, annexins are calcium-dependent phospholipid-binding proteins, and therefore, their binding to EVs enhances their anti-inflammatory properties. Some annexins, such as annexin V, are associated with pro-inflammatory activity. However, annexin V association with EVs has been shown to enhance their anti-inflammatory activity. Thioredoxins are small redox proteins known to be present in all living organisms. Thioredoxin 1 (Trx) has strong antioxidant activity and anti-inflammatory and anti-fibrotic effects. In addition, binding to EVs enhances their anti-inflammatory properties. Lactadherin, also known as milk fat globule-EGF factor 8 protein (Mfge8), is a secreted protein found in vertebrates, including mammals and birds. Lactadherin contains a phosphatidylserine-binding domain and an Arg-Gly-Asp-binding sequence. Mfge8 binds in a calcium-independent manner.

[0083] In one embodiment, the composition comprises EVs associated with annexin V, preferably recombinant annexin V, and further comprises calcium. In one embodiment, the ratio of EV-associated annexin V to albumin is preferably 1:90,000 to 1:3,000,000.

[0084] In one embodiment of the present invention, the composition may further comprise one or more secondary therapeutic agents. As used herein, a therapeutic agent refers to any agent that can be used to prevent, treat, and / or manage a disease, such as those described herein. Such agents may be intravesicular (i.e., contained within EVs) or associated with EVs. Suitable therapeutic agents are known to those skilled in the art and include non-coding RNA, miRNA, mRNA, growth factors, pro-angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, antioxidant molecules, pro-neurogenic molecules, antiviral molecules, and the like. In some embodiments, isolated EVs are used in conjunction with a secondary agent. In some embodiments, the secondary agent is a steroid, an antioxidant, or inhaled nitric oxide. In some embodiments, the steroid is a corticosteroid. In some embodiments, the corticosteroid is methylprednisolone or dexamethasone. In some embodiments, the antioxidant is superoxide dismutase.

[0085] Specific second-line therapeutic agents used in the treatment or management of certain pulmonary diseases, including, but not limited to, pulmonary hypertension, include oxygen, anticoagulants such as warfarin (Coumadin); diuretics such as furosemide (Lasix®) or spironalactone (Aldactone®); calcium channel blockers; potassium such as K-dur®; inotropes such as digoxin; vasodilators such as nifedipine (Procadia®) or diltiazem (Cardizem®); endothelin receptor blockers such as bosentan (Tracleer®) and ambrisentan (Retailis®); prostacyclin analogs such as epoprostenol (Floran®), treprostinil sodium (Remodulin®, Tyvaso®), and iloprost (Bentavis®); and PDE-5 inhibitors such as sildenafil (Revatio®) and tadalafil (Adcirca®).

[0086] The composition is preferably formulated as a liquid.Storage can be carried out by vials, IV bags, ampoules, cartridges, inhalers such as liquid inhalers, nebulizers or powder inhalers, and pre-filled syringes.The liquid formulation can further contain various compounds other than the active ingredient or EV as formulation to ensure stable and active drug therapy after storage.These compounds include solubilizers, stabilizers, buffers, osmotic pressure adjusters, bulking agents, viscosity enhancers / reducers, surfactants, chelating agents, and adjuvants.

[0087] In another embodiment, the composition may be lyophilized or freeze-dried. The lyophilized formulation may be stored in a vial, cartridge, inhaler such as a liquid inhaler, nebulizer, or powder inhaler, dual-chamber syringe, and pre-filled mixing system. Prior to administration, the lyophilized composition is reconstituted as a liquid. Reconstitution may be performed by combining a diluent with the freeze-dried powder, mixing, and then injecting. Reconstitution usually requires a reconstitution and delivery system to ensure that the drug is mixed and administered correctly.

[0088] In a preferred embodiment, the composition is aqueous. In one embodiment of the present invention, the EVs will be formulated as a composition further comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier or diluent is selected so that the EVs of the present invention remain viable and maintain their properties. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a prophylactically or therapeutically active agent. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Examples of substances that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as magnesium hydroxide, magnesium stearate, and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations.

[0089] The composition may be stored at 4°C, or more preferably by cryopreservation, wherein the composition is frozen at temperatures between -20°C and -196°C, preferably between -40°C and -196°C, more preferably between -80°C and -196°C. The freezing procedure is preferably a flash freezing or vitrification procedure, which ensures that the product remains viable during the freezing process and after thawing. Another freezing procedure may be controlled-rate freezing, which preferably compensates for the exothermic reaction at the crystallization point to improve the stability of the product. For the latter procedure, a controlled-rate freezer may be used.

[0090] In one embodiment, the composition will be suitable and / or formulated for administration to a patient by injection, intravenous administration, subcutaneous administration, intramuscular administration, cutaneous or transdermal application, inhalation, intratracheal instillation, oral instillation, systemic instillation, or intranasal instillation. The composition may be formulated for use externally, alone or in combination with a hydrogel, polymer, or polymeric medical device for sustained release of EVs.

[0091] In some embodiments, the composition is suitable for intravenous administration. In some embodiments, the composition is suitable for administration to the lungs or trachea of ​​a subject. In some embodiments, the composition is formulated for administration by inhalation. In some embodiments, the composition is formulated for administration as an aerosol. In some embodiments, the EVs are administered using a nebulizer. In some embodiments, the isolated EVs are administered using an endotracheal tube.

[0092] In some embodiments, EVs are administered or formulated with a surfactant, preferably a pulmonary surfactant. The surfactant is preferably selected so as not to affect the stability of the composition. In some embodiments, the pulmonary surfactant is an isolated, naturally occurring surfactant. In some embodiments, the pulmonary surfactant is derived from bovine or porcine lungs. In some embodiments, the pulmonary surfactant is a synthetic surfactant. A pulmonary surfactant is a lipoprotein mixture useful for keeping lung airways open (e.g., by preventing alveolar walls from adhering to each other). Pulmonary surfactants may be composed of phospholipids such as dipalmitoylphosphatidylcholine (DPPC), -phosphotidylcholine (PC), phosphatidylglycerol (PG); cholesterol; and proteins such as SP-A, B, C, and D. Pulmonary surfactants may be derived from natural sources, such as bovine or porcine lung tissue. Examples include Alveofact™ (from cow lung lavage), Curosurf™ (from minced pig lung), Infasurf™ (from calf lung lavage), and Survanta™ (from minced cow lung with additional components including DPPC, palmitic acid, and tripalmitin). Pulmonary surfactants may be synthetic. Examples include Exosurf™ (composed of DPPC with hexadecanol and tyloxapol), Pumactant™ or artificial lung expansion compound (ALEC) (composed of DPPC and PG), KL-4 (composed of DPPC, palmitoyloleylphosphatidylglycerol, palmitic acid, and a synthetic peptide that mimics SP-B), and Venticute™ (composed of DPPC, PG, palmitic acid, and recombinant SP-C). Pulmonary surfactants may be obtained from commercial suppliers.

[0093] The present invention also encompasses packaged and labeled pharmaceutical products. The article of manufacture or kit includes a suitable unit dosage form in a suitable container or container, such as a glass vial or plastic ampoule, or other hermetically sealed container. The unit dosage form should be suitable for pulmonary delivery, for example, by aerosol. Preferably, the article of manufacture or kit further includes instructions on how to use, including how to administer the pharmaceutical product. The instructions may further include informational material advising a physician, technician, or subject on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instructions indicating or suggesting a dosing regimen for use, including, but not limited to, actual dosages, monitoring procedures, and other monitoring information.

[0094] As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transport and may contain desiccants to ensure stability.

[0095] The kit may contain EVs in a sterile aqueous suspension, which may be used as is, or diluted with saline for intravenous injection or nebulizer use, or diluted with surfactant or combined with surfactant for intratracheal administration. Therefore, the kit may also include a diluent or a drug such as saline or surfactant. The kit may also include a pulmonary delivery device such as a nebulizer, or disposable components therefor, such as a mouthpiece, nosepiece, or mask.

[0096] In a final aspect, the present invention equally relates to the use of the composition as described above. More specifically, the composition is suitable for therapeutic or prophylactic use. The present invention contemplates preventing and treating certain diseases. Preventing a disease means reducing the likelihood of the disease appearing and / or delaying the onset of the disease. Treating a disease means reducing or eliminating the symptoms of the disease. Therefore, the present invention also contemplates providing a method for treating and / or preventing a disease in a subject.

[0097] The subject is preferably a human subject, although certain aspects of the invention may be practiced on any subject that may benefit therefrom, including human subjects, agricultural livestock (e.g., cows, pigs, etc.), prized animals (e.g., horses), companion animals (e.g., dogs, cats, etc.), etc. In preferred embodiments, the subject is a human, preferably a human patient, and the patient may be an adult, infant, or neonate.

[0098] In one embodiment, the composition according to any of the above embodiments is used in the prevention or treatment of a lung disorder. In one embodiment, the lung disorder may be an inflammatory lung disease, a pulmonary vascular disease, or an acute lung injury. More preferably, the inflammatory lung disease is pulmonary hypertension, also known as pulmonary arterial hypertension (PAH), asthma, bronchopulmonary dysplasia (BPD), allergy, idiopathic pulmonary fibrosis, or pneumonia. The inflammatory lung disease may be caused by an infection, such as a viral infection. In a preferred embodiment, the viral infection is influenza, SARS-CoV-1, MERS, or SARS-CoV-2. In another embodiment, the acute lung injury is associated with sepsis or is acute respiratory distress syndrome (ARDS).

[0099] These diseases also include pulmonary vascular diseases that do not have an inflammatory component. Still other pulmonary conditions that can be treated according to the present invention include acute lung injury associated with sepsis or mechanical ventilation. Examples of this latter condition include acute respiratory distress syndrome.

[0100] Pulmonary hypertension is a lung disease characterized by blood pressure in the pulmonary arteries that is much higher than normal. Symptoms include shortness of breath, chest pain especially during physical activity, weakness, fatigue, fainting, lightheadedness especially during exercise, dizziness, abnormal heart sounds and murmurs, distended jugular veins, fluid retention in the abdomen, legs, and ankles, and bluish nail beds.

[0101] Bronchopulmonary dysplasia (BPD) is a condition that affects newborns receiving oxygen or mechanical ventilation, or those born with low birth weight, especially those born very prematurely (e.g., before 32 weeks of gestation). Bronchopulmonary dysplasia is also known as neonatal chronic lung disease. Causes of BPD include mechanical injury, e.g., as a result of mechanical ventilation, oxygen toxicity, e.g., as a result of oxygen therapy, and infection. The disease may progress from non-inflammatory to inflammatory over time. Symptoms include pale skin, chronic cough, rapid breathing, and shortness of breath. Subjects with BPD are more susceptible to infections, such as respiratory syncytial virus infection. Subjects with BPD may develop pulmonary hypertension.

[0102] Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome, is a condition resulting from lung injury or acute illness. Lung injury can be the result of mechanical ventilation, trauma, burns, and / or aspiration. The acute illness can be infectious pneumonia or sepsis. This is considered a severe form of acute lung injury and is often fatal. It is characterized by pulmonary inflammation, impaired gas exchange, and release of inflammatory mediators, hypoxemia, and multiple organ failure. ARDS can also be defined as bilateral infiltrates on chest radiography and a ratio of arterial oxygen tension (PaO2) to fraction of inspired oxygen (FiO2) of less than 200 mmHg. Bilateral infiltrates and a PaO2 / FiO2 ratio of less than 300 mmHg indicate acute lung injury, which is often a precursor to ARDS. Symptoms of ARDS include shortness of breath, tachypnea, and confusion due to low oxygen levels.

[0103] Idiopathic pulmonary fibrosis is characterized by scarring or thickening of the lungs for unknown reasons. Idiopathic pulmonary fibrosis most often occurs in people between the ages of 50 and 70. Symptoms include shortness of breath, a persistent cough (typically dry), chest pain, and decreased activity levels.

[0104] For prophylaxis and / or treatment, EV may be used alone or in combination with one or more secondary agents or active ingredients, as the case may be. The subject may also receive mechanical intervention, such as mechanical ventilation, with or without exogenous oxygen administration.

[0105] The subject may be suffering from a pulmonary disease (or lung abnormality) suitable for treatment using the EVs of the present invention, or may be at risk of developing such a disease (or abnormality). Such subjects include newborns, particularly newborns born at short gestational ages. As used herein, a human newborn refers to a human from birth to approximately four weeks. As used herein, a human infant refers to a human from approximately four weeks to approximately three years of age. As used herein, short gestational age refers to birth (or delivery) occurring before the normal gestational age for a given species. In humans, a full gestational age is approximately 40 weeks and can range from 37 weeks to over 40 weeks. Similar to preterm birth, short gestational age in humans is defined as birth occurring before 37 weeks of gestation. As such, the present invention contemplates the prevention and / or treatment of subjects born before 37 weeks of gestation, including subjects born at shorter gestational ages (e.g., before 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 weeks of gestation). Typically, such premature infants would be treated as newborns, but the present invention contemplates treatment beyond the newborn stage, into infancy and / or adulthood. Certain subjects may have a genetic predisposition to particular types of lung disease, such as, for example, pulmonary hypertension, and may also be treated according to the present invention.

[0106] For neonates, particularly those of short gestational age, the present invention contemplates administration of EVs within 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, or 1 hour of delivery. In some important instances, MSC exosomes are administered within 1 hour of delivery.

[0107] The present disclosure further contemplates administration of EVs even in the absence of symptoms indicative of a pulmonary disease, such as, but not limited to, BPD.

[0108] In one embodiment, the composition containing the EVs of the present invention may be used in the treatment of COVID-19, more specifically, COVID-19-induced or acute pneumonia. COVID-19, a novel infectious disease caused by severe acute respiratory syndrome, attacks the human respiratory system and lung epithelial tissue. It has been reported that a subpopulation of patients is more likely to suffer from more severe symptoms of COVID-19 disease. Major complications include pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, septic shock, and death.

[0109] COVID-19, also known as SARS-CoV-2, has been shown to be associated with various mechanisms of pulmonary infection that can progress to acute respiratory distress syndrome (ARDS), likely caused by cytokine storm, multiorgan failure, septic shock, and blood clots. Compared to bacterial pneumonia, bacterial pneumonia is a common pulmonary infection in which the entire lung or portions of the alveoli become inflamed and filled with fluid, pus, and cellular debris. It is primarily caused by viruses, fungi, or bacteria and is often treated with antibiotics. In other cases, cardiovascular complications occur, liver enzymes are elevated reflecting liver damage, and neurological signs appear. In children, as the infection progresses, they may suffer from pediatric multisystem inflammatory syndrome, which has symptoms similar to Kawasaki disease and can result in death. Based on current data, children account for a small proportion of reported cases, with approximately 1% of cases occurring in those under 10 years of age and 4% in those aged 10–19 years.

[0110] The compositions containing EVs provide a multi-target therapeutic effect with a primary mode of action of suppressing the inflammatory process.

[0111] EVs target multiple mechanisms of lung injury, including hyper-inflammation and cytokine storm, fibrosis, oxidative stress resulting from (mechanical) ventilation, and epithelial cell apoptosis due to viral activity and inflammatory responses.

[0112] In another embodiment, the composition comprising EVs is used as a supplemental treatment for COVID-19, more particularly COVID-19 induced or acute pneumonia.

[0113] In another embodiment, the composition according to any of the above embodiments is used to prevent or treat inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis. IBD is a group of diseases that result in inflammation of the gastrointestinal (GI) tract, with ulcerative colitis (UC) and Crohn's disease (CD) being the two most common forms of IBD. UC is a disease that causes long-term inflammation and pain in the innermost layers of the colon and rectum. CD can occur anywhere in the digestive tract and can penetrate deeper into the affected tissues. Symptoms of CD include the development of fistulas around the anus. However, these diseases are similar in that both can cause abdominal pain, severe diarrhea, fatigue, and weight loss.

[0114] Compositions according to the invention comprising a therapeutically effective amount of EVs associated with annexin V, preferably recombinant annexin V, have been shown to be particularly effective for use in the prevention or treatment of inflammatory bowel diseases, in particular Crohn's disease and ulcerative colitis, preferably Crohn's disease.

[0115] For administration of EVs in connection with pulmonary diseases, the preferred method of administration is intratracheal instillation or inhalation. For neurological diseases, this is preferably systemic instillation, intranasal instillation, or inhalation. For Crohn's disease fistulas, ulcers, or cartilage repair, this is preferably by local injection. For wound healing, burn, and / or ulcer treatment, EVs will preferably be administered externally, preferably in combination with a hydrogel or polymer medical device for sustained release of EVs.

[0116] The EV of the present invention is administered in an effective amount. An effective amount is the amount of agent that alone stimulates the desired result. The absolute amount will depend on various factors, including the substance selected for administration, whether the administration is single or multiple, and individual patient parameters, including age, physical condition, size, weight, and stage of disease. These factors are well known to those skilled in the art and can be addressed with just routine experimentation. The dosage may also vary depending on the subject to whom the composition is administered.

[0117] In one embodiment of the invention, the EV is administered to the patient at a dose of 10 9 EV / kg~10 12 EV / kg or 10 10 EV / kg~10 12 In a further embodiment, in children (0 months to 12 years), the dose is 10 9 EV / kg~10 11 In teenagers and adults, the dose ranges from 10 EV / kg to 10 EV / kg. 9 EV / kg~10 12 It may be in the range of EV / kg.

[0118] In one embodiment of the invention, the EVs are administered at a dose of about 10 per patient for the treatment of perianal fistulas in CD. 10 ~about 10 12 EV doses.

[0119] The present disclosure also contemplates repeated administration of EVs, including 2, 3, 4, 5, or more administrations. In some cases, the EVs may be administered continuously. Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 days), or several weeks (e.g., 1-2, 1-3, or 1-4 weeks), depending on the severity of the condition being treated. When administration is repeated but not continuous, the interval between administrations may be several hours (e.g., 4, 6, or 12 hours), several days (e.g., 1, 2, 3, 4, 5, or 6 days), or several weeks (e.g., 1, 2, 3, or 4 weeks). The intervals between administrations may be the same or different. As an example, if disease symptoms appear to be worsening, EVs may be administered more frequently, and once symptoms have stabilized or abated, EVs may be administered less frequently.

[0120] In some cases, repeated intravenous administration of small amounts of EV may be performed. Thus, the present disclosure contemplates repeated administration of low dosage forms of EV and single administration of high dosage forms of EV. Low dosage forms include, but are not limited to, 10 per kilogram or per local injection. 10 ~10 11 High dosage forms may range from, but are not limited to, 10 EV per kilogram or per local injection. 11 ~10 12 It will be appreciated that the severity of the disease will influence the health status of the subject and the route of administration, particularly single or repeated administration of low or high doses of EV.

[0121] EVs may be administered by any route that results in delivery to the lungs or gastrointestinal tract. Systemic administration routes, such as bolus injection or continuous infusion, are preferred. More direct routes, such as intranasal administration, intratracheal administration (e.g., via intubation), and inhalation (e.g., via aerosol through the mouth or nose), are also contemplated by the present invention and may be more appropriate in some cases, especially when a rapid effect is required. As used herein, an aerosol refers to a suspension of small particles dispersed in a gas, including a mist or spray containing such particles. As used herein, aerosolization refers to the process of producing an aerosol by converting a suspension into small particles or droplets. This may be achieved using an aerosol delivery system such as a pressurized pack or a nebulizer. Nebulizers include air-jet (i.e., pneumatic), ultrasonic, and vibrating mesh nebulizers using a suitable propellant, such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In addition to nebulizers, other devices for pulmonary delivery include, but are not limited to, metered dose inhalers (MDIs) and dry powder inhalers (DPIs). Capsules and cartridges, for example, of gelatin, for use in inhalers or insufflators may be formulated containing lyophilized exosomes and a suitable powder base, such as lactose or starch.

[0122] When systemic delivery is desired, the EVs may be formulated for parenteral administration by injection, including, for example, by bolus injection or continuous infusion. Formulations for injection may be provided in unit dosage form, for example, in ampoules or in multi-dose containers, with or without added preservatives.

[0123] The compositions may take the form of aqueous suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulations such as suspending agents, stabilizers, and / or dispersing agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may contain suitable stabilizers or agents that increase solubility. Alternatively, the exosomes may be lyophilized or in other powder or solid form for constitution with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0124] The EVs and compositions as described herein will preferably be obtained by a process as described above.

[0125] The invention is explained in more detail below with reference to non-limiting examples.

[0126] Description of the Examples and / or Figures Example 1 - Isolation of EVs from MSCs Umbilical cord (UC)-derived MSCs, obtained from umbilical cord tissue or Wharton's gelatin, were expanded in an expansion unit comprising a stirred bioreactor (2) in fluid connection with a cell culture medium supply vessel (1). Referring to Figure 1, the following numbers refer to the following: 1. Cell Culture Medium Container 2. Stirred Bioreactor 3.Cell supernatant container 4. Refrigerator 5a, 5b, 5c. Peristaltic pump 6. First filter unit 7. Second filter unit 8.TFF Cassette 9. Final product

[0127] Cells are grown in the presence of the three-dimensional microcarriers present in the bioreactor (2) and in a xeno-free and serum-free medium containing purified human albumin at a concentration of 3 g / L, and further containing recombinant purified transferrin at a concentration of 60 mg / L.

[0128] At least 40 × 10 cells in the bioreactor (2) 6 MSCs were expanded and grown until a minimum concentration of 100 cells / L was reached and MSC confluency reached 80%. Cell concentration was determined by cell counting and fluorescent labeling of MSCs attached to microbeads. At this stage, the cells were ready to begin EV secretion. To this end, fresh serum-free and xeno-free medium was diluted 1:10 with basal medium (DMEM / high glucose / phenol red-free / glutamax, Thermo Scientific) and added to the cell culture medium. After 24 hours, the system was ready to begin EV collection. To this end, the conditioned cell medium or cell supernatant from the bioreactor (2) was pumped into the cell supernatant container (3) located in a refrigerator (4) set at 4 °C. This supernatant contained EVs produced by the MSCs. The supernatant collected in the container (3) was then pumped by a peristaltic pump (5c) into the first filter unit (6) containing a 2-micron filter. The supernatant was filtered by dead-end filtration in a closed system with a peristaltic pump (5c) delivering a constant flow through the filter (6) at 100 mL / min. The flow-through fraction of the first filtration step was subsequently filtered through a second filter unit (7) fluidly connected to the first filter unit (6) with a 0.2 micron filter. Filtration was performed by dead-end filtration in a closed system with a peristaltic pump (5c) delivering a constant flow through the filter (7) at 100 mL / min. The flow-through fraction containing the EVs was collected in a 1 L bag and subsequently stored at 4 °C.

[0129] In the final step, the flow-through fraction was delivered to a TFF cassette (8) with a 100 kDa cutoff, fluidly connected to a dead-end filter unit (6, 7). The retentate from the TFF cassette (8) containing the EVs was washed with buffered saline and concentrated to a final volume of 10 mL by circulation through the TFF cassette (8) using a peristaltic pump (5c) with a rotor speed of 300 rpm. The final product (9) was collected from the TFF (8) and frozen at -80 °C or below in cryotubes, bags, or other suitable containers or maintained at 4 °C.

[0130] The final product was analyzed to ensure its quality. Quality controls included measuring the concentration of albumin associated with EVs and particle analysis by techniques such as colorimetry, ELISA, nanoparticle tracking analyzer (NTA), tunable resistive pulse sensing (TRPS), electron microscopy, and / or Raman microscopy to ensure product purity and stability. Product samples were tested for the presence of endotoxin and / or mycoplasma. Other quality controls included qualitative chromatography, mass spectrometry, ELISA, sequencing, qRT-PCR, and activity tests such as in vitro T cell, B cell, and macrophage polarization.

[0131] Under the culture conditions described above, the MSCs reach a cell density of at least 0.25 x 10 per ml of cell culture medium within 18 to 24 hours of culturing the MSCs. 9 Experiments were performed with MSCs derived from UC, but were also repeated with MSCs derived from other tissues or sources, including but not limited to, mammary gland, bone marrow, Wharton's gel, (umbilical cord) blood, peripheral blood, amniotic membrane, adipose tissue, dental pulp, fallopian tube, liver, and lung tissue.

[0132] Example 2 - Protein binding to EVs EVs were produced as described in Example 1 until collection after the second filtration step. Subsequently, the flow-through fraction collected in the filtration step was transferred to a TFF cassette with a 100 kDa cutoff for annexin V labeling / binding of EVs, washing, and concentration of vesicles. All steps were performed in a closed system.

[0133] The flow-through fraction of the filtration step was recirculated in the TFF cassette using a peristaltic pump with a rotor speed of 300 rpm until a volume of 15 ml was obtained. EVs remained in the retentate, while the permeate was disposed of as waste. The calcium concentration in the retentate was then determined, and calcium was replenished until a Ca concentration of 2 mM was obtained.

[0134] Next, 0.5 mg of Annexin V was added to every 1 ml of retentate in the TFF cassette, followed by recirculation of the product at room temperature for 15 minutes. A small sample was taken and the concentration of Annexin V was confirmed using an ELISA test.

[0135] The retentate (approximately 15 ml) was then washed with saline wash buffer (approximately 10 ml) to remove unbound annexin and unwanted contaminants less than 100 kDa. The retentate was then recirculated in the TFF at 200 rpm and reconcentrated to approximately 15 ml. This wash step was repeated two more times, and the waste (permeate) was checked each time (by ELISA) to determine the amount of unbound annexin V before disposal. The third wash permeate contained only a small amount of unbound annexin V, indicating that most of the unbound annexin had been washed away. The peristaltic pump was then stopped, and the concentrated final product was then collected in a collection syringe. The product could be aliquoted prior to storage or stored as is, frozen at -80°C or below in cryotubes, bags, or other suitable containers, or maintained at 4°C.

[0136] The experiments described for annexin V binding to EVs were repeated to obtain thioredoxin-bound EVs and lactadherin-bound EVs.

[0137] Example 3: EV activity using an in vivo model of Crohn's disease The experiment demonstrates the activity of annexin V-associated EVs, produced in a manner similar to Example 2, in an in vivo model of Crohn's disease. Briefly, the experiment is performed in female mice. Colitis is induced by administration of 3% sodium dodecyl sulfate (DSS) in the drinking water, optionally for more than 5 days. This is an established and widely used animal model, referenced hundreds of times in PubMed (for a review, see Kawada et al., 2007). All animals are treated in accordance with appropriate ethical regulations. Animals are subdivided into three experimental groups: Group 1 (normal controls): Daily intraperitoneal (ip) injections of PBS (vehicle only), 0.2 ml on days 1-5. - Group 2 (colitis induction): Similar to Group 1, 3% DSS was added to the drinking water. - Group 3 (colitis induction and treatment with EVs): similar to group 2, with the addition of MSC-EVs suspended in 0.2 ml of PBS via the IP route.

[0138] On day 6, the animals are sacrificed using CO. The colon is excised and one portion of the tissue is fixed in formalin for subsequent histological analysis, while another portion is immediately frozen in liquid nitrogen and then stored at -80°C for subsequent RNA extraction.

[0139] EV dose and administration route EVs are isolated and administered according to previously described procedures. Route of administration: EVs suspended in 0.2 ml of PBS are administered intraperitoneally every day from day 0 to day 5.

[0140] Assessment of intestinal injury Animal weight Disease activity index (fecal assessment; see Tanaka F (2008)) - Histopathological signs of flogosis - Titration of inflammatory mediator expression in colonic tissue extracts: TNFalfa, IL6, IL-1beta, and Cox2 by RT-PCR.

[0141] Statistical analysis of results Data are expressed as mean ± SD. Group differences were analyzed by t-test, with p<0.05 being significant.

[0142] result In conclusion, the results show that EVs according to the invention associated with annexin V are particularly suitable for reducing the inflammatory response in experimental colitis, resulting in a reduction in disease activity index and an improvement in general condition. EVs associated with annexin V improve the clinical picture and phlogistic response in animal models of intestinal inflammatory disease.

[0143] The present invention is in no way limited to the embodiments described in the examples and / or shown in the drawings, on the contrary, the method according to the invention may be realized in many different ways without departing from the scope of the invention.

Claims

1. A process for producing extracellular vesicles (EVs) derived from MSCs and associated with one or more exogenous proteins, comprising: Purifying EVs from a cell culture medium containing MSCs, said purification occurring by at least one filtration step of said cell culture medium, followed by a step of concentrating the filtrate of said at least one filtration step, wherein said EV is concentrated by tangential flow filtration (TFF) in a TFF device; During the TFF step, the EVs associate with the exogenous protein within the TFF device or within a container fluidly connected to the TFF device to which the EVs are transferred from the TFF device.

2. 10. The process of claim 1, wherein the exogenous protein is replenished to the TFF device or a connected vessel during the concentration of the EVs.

3. 10. The process of claim 1, wherein the TFF device or a connected container is supplemented with calcium during the concentration of the EVs.

4. 4. The process of claim 3, wherein the calcium and the exogenous protein are mixed in the TFF device or a connected vessel and incubated with the EVs present in the TFF device or a connected vessel.

5. The process described in claim 4, wherein the EVs associated with the exogenous protein are washed, reconcentrated, and collected outside the TFF device.

6. The process of any one of claims 1 to 5, wherein the exogenous protein is selected from annexin, thioredoxin, and lactadherin.

7. The process of any one of claims 1 to 6, wherein the exogenous protein is Annexin V, Trx, or Mfge8.

8. The process of claim 1, wherein the exogenous protein is recombinant annexin V, Trx, or Mfge8.

9. The process of any one of claims 1 to 8, wherein the TFF device has a cutoff range of 100 kDa.

10. The process according to any one of claims 1 to 9, wherein the MSCs are cultured and expanded in a cell culture medium containing purified human serum albumin.

11. 11. The process of claim 10, wherein the albumin concentration in the cell culture medium is between 1 g / l and 5 g / l.

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