Method for manufacturing protein-bound extracellular vesicles

Abstract

The present invention relates to a method of manufacturing extracellular vesicles (EVs) bound to proteins derived from mesenchymal stromal cells (MSCs), said method comprising the steps of: - purifying EVs from a cell culture medium comprising MSCs, wherein said purification is performed by subjecting said medium to at least one filtration step; next - a step of concentrating the filtrate of said at least one filtration step, wherein said EVs are concentrated by tangential flow filtration in a TFF device; and - wherein during said TFF step, EVs bind to one or more exogenous proteins inside said TFF device or in a vessel that is in fluid connection with said TFF device and to which said EVs are transferred from said TFF device. The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of EVs bound to proteins, and uses thereof.

Description

Technical Field

[0001] This invention relates to a method for manufacturing extracellular vesicles (EVs) that bind to proteins derived from mesenchymal stromal cells (MSCs). The invention also relates to a composition comprising a therapeutically effective amount of protein-bound EVs, and its use. Background Technology

[0002] EVs are lipid bilayer-separated particles naturally released by cells, but unlike cells, they are not replicable. EV diameters range from near the physically smallest possible monolayer liposome (approximately 20-30 nanometers) to over 10 micrometers, but the vast majority are smaller than 200 nm. They transport large quantities of proteins, nucleic acids, lipids, metabolites, and even organelles derived from parent cells. It is believed that most cells studied to date can release EVs, including some bacteria, fungi, and plant cells surrounded by cell walls. Various EV subtypes have been proposed and defined differently based on size, biogenetic pathway, transporter, cellular origin, and function, leading to different historical nomenclature, including terms such as exosome, microvesicle, and extranuclear granule.

[0003] EVs can be used for therapeutic purposes, such as delivering nucleic acids or other transporters to diseased tissues and cells. Parallel to this growing interest are the formation of companies and funding programs focused on developing EVs as biomarkers or disease therapies, the establishment of the International Society for Extracellular Vesicles (ISEV), and the founding of the Journal of Extracellular Vesicles.

[0004] With the growing interest in using EVs for therapeutic purposes, the demand for clinical-grade EVs has also increased. For clinical application, EVs need to be manufactured in a reproducible and controlled manner. Good Manufacturing Practices (GMP) for manufacturing EV-containing compositions are crucial to ensure batch-to-batch consistency and quality. GMP is particularly challenging when cell-derived therapies are involved. Several issues need to be overcome before EVs can be widely used clinically. Developing platforms for the reliable and reproducible production, storage, and handling of clinical-grade EVs remains a challenge. Before introducing MSC-derived EV therapies into the clinic, MSC-derived EV drugs and subsequent clinical trials require addressing a variety of technical, scientific, regulatory, and mechanistic issues.

[0005] Therefore, there is a need for protocols that can stably and reproducibly produce clinical-grade MSC-derived EVs under GMP conditions conforming to the International Council for Harmonisation of Technical Requirements for Quality (ICH) 2020. Furthermore, EV products with good stability and commercially attractive shelf lives are also required. Additionally, there is a need to produce clinical-grade MSC-derived EVs containing the desired proteins bound to their membranes or surfaces. These proteins can enhance the function and therapeutic value of EVs.

[0006] EVs have been modified to express various therapeutic molecules (WO 2019 / 198 077) or loaded with siRNA, oligonucleotides, or guide RNA (Reka Agnes Haraszti et al., 2018). Loading can be performed by incubating donor cells with the transport molecules or by sonication, extrusion, freeze-thaw cycles, and electroporation of the EVs (DLuan et al., 2017). WO2021 / 084274A1 discloses a composition comprising EVs having lipid-binding proteins bound to their outer surface.

[0007] Purification methods for EVs have been reported, but scalability remains an issue. Nordin Joel Z et al., in 2019, disclosed a proposed method for purifying EVs using tangential flow filtration (TFF) as a prospective method for large-scale EV production. This method employs a labor-intensive approach combined with elution size exclusion chromatography. Zheng Zhao et al., in 2019, proposed a method for producing surface antigen-engineered EVs using microfluidic cell culture chips. The latter is expensive and not readily available.

[0008] The field still needs efficient methods for producing protein-bound EVs that can be used for therapeutic purposes. Summary of the Invention

[0009] This invention and its embodiments are intended to provide solutions to one or more of the aforementioned disadvantages. Therefore, according to claim 1, the present invention relates to a method for manufacturing protein-bound EVs derived from MSCs. More specifically, the method includes the following steps:

[0010] - Purify EVs from a cell culture medium containing MSCs, wherein the purification is performed by at least one filtration step on the culture medium;

[0011] - A step of concentrating the filtrate from the at least one filtration step, wherein the EV is concentrated by tangential flow filtration in a TFF device; and

[0012] -During the TFF step, the EV binds to one or more exogenous proteins inside the TFF device or in a container fluidly connected to the TFF device and into which the EV is transferred from the TFF device.

[0013] In a second aspect, the invention also relates to a composition comprising a therapeutically effective amount of an EV that binds to the protein of claim 11. In a final aspect, the invention relates to the use of the composition of claim 19. Embodiments of the invention are shown in the methods of claims 2 to 10, the pharmaceutical compositions of claims 12 to 17, and the uses of the pharmaceutical compositions of claims 20 to 28. Attached Figure Description

[0014] Figure 1 An embodiment of an MSC amplification unit and an EV processing unit that can be used to produce the EVs of the present invention is shown.

[0015] definition

[0016] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes definitions of terms to better understand the teachings of this invention.

[0017] As used herein, the following terms have the following meanings:

[0018] Unless the context clearly specifies otherwise, the terms “a,” “an,” and “the” used herein refer to the singular and plural pronouns, respectively. For example, “a compartment” means one or more compartments.

[0019] As used herein, “approximately” refers to a measured value such as a parameter, quantity, duration, etc., and is intended to include variations of less than + / -20%, preferably less than + / -10%, more preferably less than + / -5%, even more preferably less than + / -1%, and still more preferably less than + / -0.1%, such variations apply to the disclosed invention to date. However, it should be understood that the value referred to by the modifier “approximately” is itself specifically disclosed.

[0020] As used herein, “comprising” is synonymous with “including” or “containing” and is an open-ended term that specifies the presence of subsequent items such as components, and does not exclude or exclude the presence of additional, unlisted ingredients, features, elements, components, or steps known in the art or disclosed herein.

[0021] The range of values ​​listed by endpoints includes all numbers and fractions contained within that range, as well as the listed endpoints.

[0022] Unless otherwise defined, the expressions “% by weight,” “percentage by weight,” “%wt” or “wt%” herein and throughout the specification mean the relative weight of the various components based on the total weight of the formulation.

[0023] While the terms “one or more” or “at least one”, such as one or more or at least one of a group of components, are self-explanatory, by further example, the term specifically refers to any one of the said components, or any two or more of the said components, such as ≥3, ≥4, ≥5, ≥6 or ≥7, or at most all of the said components.

[0024] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes definitions of terms to better understand the teachings of this invention. The terms or definitions provided herein are merely illustrative of the invention.

[0025] Throughout this specification, "one embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrase "in one embodiment" throughout different places in this specification does not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, particular features, structures, or characteristics may be combined with each other in any suitable manner that is obvious to those skilled in the art. Moreover, while some embodiments described herein include some features included in other embodiments but not others, combinations of features from different embodiments are still within the scope of the invention and form different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiments may be used in any combination.

[0026] For the purposes of this invention, the term "extracellular vesicle" or "EV" should be understood as micron- or nanon-sized particles secreted in vivo and in vitro by different types of cells, including proteins bound to said EV. EVs contain proteins, growth factors, miRNAs, and other molecules encapsulated in lipid globules. EVs can be classified according to size and intracellular origin. Exosomes are a subtype of EV, typically smaller than 0.1 micrometers. Exosomes originate from multivesicular bodies, a type of secondary endosomal compartment that is secreted through fusion of the multivesicular body with the plasma membrane. Another subtype of EV is exfoliated vesicles (also known as microvesicles), a heterogeneous group of membrane vesicles released directly from the cell membrane by disrupting the cortical cytoskeleton, reaching up to 1 micrometer in size. All types of vesicles secreted by cells are generally referred to as EVs.

[0027] The term “bound to EV” in relation to a substance means that the substance a) is attached to or bound to the surface of the EV (by any means, such as covalent or non-covalent bonding), preferably in the form of non-covalent bonds; b) is attached to or bound to the surface of the EV; or c) is internalized into the EV.

[0028] The substance that binds to EV can be any type of substance, including, but not limited to, the following molecules: amino acids, proteins, peptides, nucleic acids such as DNA and RNA (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.

[0029] The term "cell culture medium" or "culture medium" refers to an aqueous solution containing nutrients and other defined components that can be used for cell maintenance or growth.

[0030] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the administered composition. For example, pharmaceutically acceptable carriers can serve as stabilizers and / or excipients. Examples of carriers are, but are not limited to, propylene glycol, saline, emulsions, and mixtures of organic solvents with water.

[0031] The term "sufficient amount" refers to an amount sufficient to produce the desired measurable effect, such as an amount sufficient to alter protein expression properties.

[0032] The term "therapeutic effective dose" refers to the amount that can effectively improve the symptoms of a disease. A therapeutic effective dose can also be a "preventive effective dose," since prevention can be considered treatment.

[0033] The term "treatment" refers to therapeutic and preventative measures aimed at preventing or alleviating a target pathological condition or disease. Those who require treatment include those who already have the disease, those who are susceptible to the disease, and those who want to prevent the disease.

[0034] The term "composition" refers to a composition at any stage of the manufacturing process, including the final pharmaceutically acceptable product and any intermediates in the process.

[0035] The term "treatment" refers to reversing, preventing, improving, or inhibiting the progression of a disease, symptom, or condition, or one or more symptoms of a disease, symptom, or condition. As used herein, "treatment" can also refer to reducing the likelihood or incidence of a disease, symptom, or condition in a mammal compared to an untreated control group or to the same mammal before treatment. For example, as used herein, "treatment" can refer to preventing a disease, symptom, or condition and may include delaying or preventing the onset of a disease, symptom, or condition, or delaying or preventing the symptoms of a disease, symptom, or condition. As used herein, "treatment" can also refer to reducing the severity of a disease, symptom, or condition or alleviating the symptoms of such a disease, symptom, or condition before the onset of the disease, symptom, or condition. Such prevention or reduction of the severity of a disease, symptom, or condition before the onset of the disease involves administering the compositions of the present technology described herein to a subject who does not have a disease, symptom, or condition at the time of administration. As used herein, "treatment" can also refer to preventing the recurrence of a disease, symptom, or condition or one or more symptoms of such a disease, symptom, or condition. As used in this article, the terms “therapeutic,” “treatment,” and “in therapy” refer to the therapeutic acts defined above.

[0036] As used in this article, the term "fibrosis" refers to the formation of an excessive amount of fibrous connective tissue in an organ or tissue during a repair or reaction process.

[0037] For the purposes of this invention, the term "mesenchymal stromal cells" or "MSCs" should be understood as matrix adherent cells capable of differentiating into various cell types. MSCs are derived from bone marrow, umbilical cord cells, adipose tissue, amniotic fluid, mammary glands, and blood. Detailed Implementation

[0038] This invention relates to a method for manufacturing protein-bound EVs, a composition comprising protein-bound EVs, and their uses.

[0039] In a first aspect, the present invention relates to a method for manufacturing protein-bound EVs derived from MSCs, the method comprising the following steps:

[0040] - Purify EVs from a cell culture medium containing MSCs, wherein the purification is performed by at least one filtration step in the culture medium; Next

[0041] - A step of concentrating the filtrate from the at least one filtration step, wherein the EV is concentrated by tangential flow filtration in a TFF device;

[0042] -During the TFF step, the EV binds to or associates with one or more exogenous proteins inside the TFF device or in a container fluidly connected to the TFF device and transferred therefrom.

[0043] In the context of this invention, “bonding” and “bonding to” can refer to any type of bonding or attachment, covalent or non-covalent, or any other type of bonding or attachment known in the art.

[0044] The MSCs of this invention can be grown and expanded in concentrated cell culture medium. Once the MSCs reach the desired concentration, the diluted cell culture medium containing EVs (referred to as cell supernatant, from which EVs are extracted) is collected for further processing. In one embodiment, when the MSCs reach at least 40 x 10⁻⁶ cells / mL... 6 The supernatant was collected at a minimum concentration of 100 cells / L. Cell concentration and cell viability can be determined by cell counting, for example, using a hematology analyzer such as a Bürker counting chamber and trypan blue staining.

[0045] To purify EVs from the supernatant, the cell culture medium used to maintain MSCs (also known as cell supernatant) is filtered out, thereby removing contaminants present in the medium. The latter contributes to the purity and stability of the final product.

[0046] In the context of this invention, the "stability" of a product such as an EV refers to the fact that a particular formulation or product, under specific conditions (e.g., a container or a closed system), remains within predetermined ranges of physical, chemical, microbiological, toxicological, and functional specifications or characteristics over a given period of time. Non-limiting examples of such parameters include particle number, particle size, and leakage and activity of internal components.

[0047] This purification is performed by subjecting the culture medium to at least one filtration step. In one embodiment, the at least one filtration step is dead-end filtration.

[0048] In a preferred embodiment, the filtration includes at least two filtration steps. Furthermore, in one embodiment, at least one filtration step is dead-end filtration. In a further embodiment, both filtration steps are performed via dead-end filtration. Preferably, at least one filtration step is used as a method of product sterilization to comply with GMP specifications as defined above. In some cases, continuous dead-end filtration is necessary to adequately remove impurities from the supernatant. In some cases, it has been found that single filtration often leads to clogging of the filter used and reduces the purity and quantity of the final product.

[0049] In the first filtration step, the cell supernatant is filtered by dead-end filtration. In a further preferred embodiment, the filtration is performed by placing the supernatant on a filter with a sieve size of 1 to 5 micrometers, more preferably a filter with a sieve size of 1 to 3 micrometers. In one embodiment, the filtration is performed by dead-end filtration. In a further preferred embodiment, it is preferably performed in a closed system with a peristaltic pump that provides a constant flow rate through the filter, even more preferably at about 100 ml / min.

[0050] In one embodiment, the filtrate from the first filtration is passed through a secondary filter, the pore size of which is smaller than that of the filter used in the first filtration step. Preferably, this second filtration step is a sterilization step to ensure the final product conforms to the GMP specifications defined above. In a more preferred embodiment, the pore size of the filter is less than 1 micrometer, more preferably between 0.05 micrometers and 1 micrometer, even more preferably between 0.1 micrometers and 0.5 micrometers, and most preferably between 0.1 micrometers and 0.22 micrometers. In one embodiment, the second filtration step is performed by dead-end filtration. In a further embodiment, it is preferably performed in a closed system interconnected with the first filtration step. In another further embodiment, it is preferably performed by a peristaltic pump providing a constant flow rate through the filter, even more preferably at about 100 ml / min.

[0051] The filtrate from one or more of the filtration steps will contain the EV of the present invention.

[0052] In the final step, the EV will be washed and concentrated. Washing and concentration can be performed using methods conventional in the art, such as membrane filtration, microfiltration, or ultrafiltration. Concentration is the process of removing liquid from a solution while retaining solute molecules. Membrane filtration is a commonly used separation technique in life science laboratories. Based on the membrane porosity, membrane filtration can be classified as microfiltration or ultrafiltration. Microfiltration membranes typically have pore sizes between 0.1 μm and 10 μm and are commonly used for clarification, sterilization, and removal of particulates or for cell collection. Ultrafiltration membranes have much smaller pore sizes, from 0.001 μm to 0.1 μm, and are used to concentrate and desalinate dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchange buffers, and crude fractions. Ultrafiltration membranes are typically classified according to molecular weight cutoff (MWCO) or nominal molecular weight cutoff (NMWCO), rather than according to pore size.

[0053] There are two main types of membrane filtration, which can be done using microfiltration or ultrafiltration membranes: 1) Direct-flow filtration (DFF), also known as "dead-end" filtration, applies the feed flow perpendicular to the membrane surface and attempts to allow 100% of the fluid to pass through the membrane, and 2) Tangential-flow filtration (TFF), also known as cross-flow filtration, in which the feed flow passes parallel to the membrane surface, with a portion passing through the membrane (permeate) and the remainder (residue) being recycled back into the feed container.

[0054] Preferably, the washing and concentration are performed via a tangential flow filtration (TFF) step. Tangential flow filtration (TFF), or cross-flow filtration, is a method in which the feed flow passes parallel to the membrane surface. Applied pressure forces a portion of the feed flow through the membrane (permeate), while the remainder (residue) is recycled back into the feed container.

[0055] In one embodiment, the filtrate containing at least one filtration step of the MSC-derived EV is used in the TFF concentration step of a TFF apparatus.

[0056] In a further preferred embodiment, the TFF has a molecular weight cutoff (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 MWCO of 100 kDa removes most (but not all) of particles and components with molecular weights below 100 kDa into the TFF permeate. Thus, the final composition in the permeate does not contain any free (i.e., non-EV-bound) components, elements, or substances with molecular weights below 100 kDa. These components, elements, or substances can be any particles, such as proteins or peptides typically present in or part of the culture medium.

[0057] In one embodiment, during the TFF step, the EV is bound to one or more exogenous proteins inside the TFF device or in a container fluidly connected to the TFF device and to which the EV is transferred from the TFF device. In one embodiment, the protein is replenished to the TFF device or the connected container during EV concentration. In a further embodiment, as a non-limiting example, the protein replenished to the TFF device or the connected container may be infused or injected into the TFF device, preferably in a closed system.

[0058] The protein will bind to a TFF or an EV in a container attached to a TFF. Importantly, the container should be fluidly 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 the TFF residue containing the EV can bind to the desired protein. In a non-limiting embodiment, the container can be a bag or pouch. The combined product containing the residue and protein is then transferred to the TFF.

[0059] Unbound or unassociated proteins will be removed in one or more washing steps, preferably in TFF (Thin Film Fusion). One or more washing buffers, such as salt buffers, can be used for the washing steps. The washing buffer should be compatible with EVs and proteins, and should not contain any toxic substances.

[0060] In one embodiment, the protein is selected from annexin, thioredoxin, or lactoglucosin. In a preferred embodiment, the protein is annexin V, Trx, or Mfge8, preferably recombinant annexin V, Trx, or Mfge8.

[0061] Annexin is a calcium-dependent phospholipid-binding protein that binds to EVs, increasing their anti-inflammatory properties. Some annexins, such as annexin V, are associated with pro-inflammatory activity. It has been shown that the binding of annexin V to EVs increases the anti-inflammatory activity of EVs. Annexin V has a molecular weight of approximately 37 kDa.

[0062] Thioredoxins are small redox proteins found in all living organisms. Thioredoxins (Trx) possess strong antioxidant activity, anti-inflammatory activity, and anti-fibrotic effects. Furthermore, their binding to endogenous vesicles (EVs) enhances the anti-inflammatory effects of EVs.

[0063] Milk agglutinin, also known as milk fat globule EGF factor 8 protein (Mfge8), is a secreted protein found in vertebrates, including mammals and birds. It contains a phosphatidylserine domain and an Arg-Gly-Asp binding sequence. Mfge8 binds in a calcium-dependent manner.

[0064] In a further embodiment, calcium is added to the TFF device or the fluid-connected container during the concentration of the EV. Preferably, the added calcium concentration is between about 1 mM and about 10 mM. In one example, it is between 1 mM and 9 mM; in another example, between 1 mM and 8 mM; in another example, between 1 mM and 7 mM; in another example, between 1 mM and 6 mM; in another example, between 1 mM and 5 mM; in another example, between 1 mM and 4 mM; and in another example, between 1 mM and 3 mM. Preferably, it is between 1.5 mM and 3 mM, and more preferably between 1.5 mM and 2.5 mM. Most preferably, about 2 mM of calcium is added to the TFF device during the concentration of the EV.

[0065] Calcium concentration in the human body is typically maintained within a fairly narrow range, from about 2.2 mM to about 2.7 mM. If the EV is to be administered to a (human) patient (see below), the calcium in the final product should preferably be kept below 10 mM.

[0066] Injecting calcium into the TFF device and the fluidly connected container facilitates the binding of calcium-dependent proteins to EVs, such as, but not limited to, annexin and thioredoxin. For proteins that bind in a non-calcium-dependent manner, such as Mfge8, calcium injection is not required.

[0067] In a further embodiment, the calcium and the protein are mixed in the TFF or fluid-connected container and incubated together with the EV in the TFF device or container.

[0068] In a further embodiment, the EVs bound to the protein are washed, reconcentrated, and subsequently collected outside the TFF device.

[0069] As previously mentioned, proteins that do not bind to or associate with EVs will be removed in one or more washing steps, preferably in TFF. The osmotic residue will contain EVs. One or more washing steps can be performed using a washing buffer, such as, but not limited to, a salt buffer. The washing buffer should be compatible with EVs and the proteins bound to them, in a sense, to keep the EVs and the proteins bound to them active and retain their properties.

[0070] When the unbound or unassociated proteins are thoroughly washed, the protein-bound EVs are concentrated, preferably by circulation through a TFF device. The final concentrated product containing the EVs is then collected outside the device, preferably in a collection container, such as, but not limited to, a collection bag or cryovial.

[0071] The final product can be stored below 10°C, preferably below 4°C, and more preferably by cryopreservation, wherein the composition is frozen between -20°C and -196°C, preferably between -40°C and -196°C, and more preferably between -80°C and -196°C. The freezing procedure is preferably a rapid freezing procedure or a vitrification procedure to ensure that the product retains its activity during freezing and after thawing. Another freezing procedure can be controlled-rate freezing, preferably compensating for the exothermic reaction at the crystallization point to obtain better product stability. For the latter procedure, a controlled-rate freezer can be used.

[0072] In one implementation, the culture medium used to culture and amplify MSCs before dilution and the commencement of EV collection is a cell culture medium containing purified human serum albumin.

[0073] Albumin in the final product contributes to the stability and functionality of the EV. As previously stated, in the current context, "stability" of a product (e.g., an EV) refers to the condition in which a particular formulation or product remains within predetermined ranges of physical, chemical, microbiological, toxicological, and functional specifications or characteristics under specific conditions (e.g., a container or a closed system) over a given time period. Non-limiting examples of such parameters include particle number, particle size, leakage of internal components, and activity.

[0074] Because albumin contributes to the stability and function of EVs, it is preferably present in the cell culture medium used for culturing and expansion. Therefore, the culture medium contains human albumin, which can be recombinant or purified albumin, such as albumin purified from human plasma. In a further embodiment, the concentration of albumin present in the culture medium is between 1 g / L and 5 g / L. The latter concentration has proven particularly useful for obtaining an active and stable end product.

[0075] In a second aspect, the invention also relates to a composition comprising a therapeutically effective amount of a protein-bound EV and optionally calcium. The composition is obtained through the process described above. In a preferred embodiment, the EV has a size of less than 1 μm.

[0076] In a preferred embodiment, the EV size in the composition is approximately less than 750 nm, preferably less than 500 nm, more preferably less than 400 nm, and more preferably less than 300 nm. In another or further preferred embodiment, the EV size in the composition is at least 5 nm, more preferably at least 10 nm, more preferably at least 25 nm, and more preferably at least 50 nm. In another or further preferred embodiment, the EV size in the composition is between 25 nm and 500 nm, preferably between 25 nm and 400 nm, and most preferably between about 50 nm and about 300 nm.

[0077] Optical techniques are typically used to determine the size and number of extracellular vesicles (EVs). In one embodiment of the invention, the particle size of the EVs is measured by nanoparticle tracking analysis (NTA), a preferred method for quantifying and determining the size of nanoparticles suspended in a liquid buffer. In another preferred embodiment, the particle size is measured using a tunable resistive pulse 25° sensor (TRPS), which serves as a reference for NTA. In another or further preferred embodiment, the particle size is measured using high-resolution flow cytometry.

[0078] An effective amount is the amount of a formulation that, on its own, can stimulate the desired outcome. The absolute amount depends on many factors, including the material chosen for administration, whether it is administered in a single or multiple dose, and individual patient parameters such as age, physical condition, body size, weight, and stage of disease. These factors are well known to those skilled in the art and can be resolved through routine experiments. The dosage may also vary depending on the subject administering the composition.

[0079] In one embodiment of the composition, the composition may further comprise calcium, preferably with a calcium concentration between about 1 mM and about 10 mM. In one embodiment, the calcium concentration is between 1 mM and 9 mM, or between 1 mM and 8 mM, or between 1 mM and 7 mM, or between 1 mM and 6 mM, or between 1 mM and 5 mM, or between 1 mM and 4 mM, or between 1 mM and 3 mM. Preferably, it is between 1.5 mM and 3 mM, and more preferably between 1.5 mM and 2.5 mM. Most preferably, the calcium concentration in the composition is about 2 mM.

[0080] In one embodiment, the calcium (at least partially) is bound to or associated with EV.

[0081] Calcium levels below 1 mM will reduce the binding of calcium-dependent binding proteins. Because blood calcium levels are typically maintained within a fairly narrow range, from approximately 2.2 mM to approximately 2.7 mM, the composition should be avoided in human patients as it contains calcium levels above 10 mM.

[0082] In one embodiment, the composition of the present invention will further comprise human albumin at a concentration between 10 g / L and 30 g / L, more preferably between 10 g / L and 20 g / L, and even more preferably between 15 g / L and 20 g / L.

[0083] The albumin is derived from the cell culture medium of MSCs. Although albumin can theoretically be considered a contaminant in the production process since it is not produced by MSCs, it has been surprisingly found that albumin is actually necessary to ensure the stability and activity of the final product, thereby ensuring the stability and activity of the compositions of the present invention. Albumin at the desired concentration acts as a drug stabilizer and activity enhancer. It has been found that forced removal of albumin reduces product activity (Hyungtaek Jeon et al., 2020).

[0084] The albumin concentration can be measured by colorimetry or by an ELISA using an anti-albumin antibody. In one embodiment, the colorimetric measurement is the bromocresol green assay.

[0085] In one embodiment, at least 90% of the albumin present in the composition binds to the EV in the composition.

[0086] Human albumin has a molecular weight of approximately 66 kDa. Therefore, albumin that is not bound to EVs is removed by TFF treatment, leaving albumin in the composition that is bound to 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 bound to EVs.

[0087] In one embodiment, the composition of the present invention comprises human albumin and calcium, preferably comprising albumin at about 25 mg to 750 mg per mg of calcium, more preferably about 25 mg to 500 mg per mg of calcium, more preferably about 50 mg to 250 mg per mg of calcium, more preferably about 50 mg to 500 mg per mg of calcium, more preferably about 75 mg to 400 mg per mg of calcium, more preferably about 100 mg to 350 mg per mg of calcium, more preferably about 125 mg to 300 mg per mg of calcium, more preferably about 150 mg to 275 mg per mg of calcium, and most preferably about 170 mg to 250 mg per mg of calcium.

[0088] In one embodiment of the composition, the protein binding to the EV is selected from annexin, thioredoxin, and lactoglucosin, preferably 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.

[0089] As previously mentioned, annexins are calcium-dependent phospholipid-binding proteins, and their binding to EVs increases the anti-inflammatory properties of EVs. Some annexins, such as annexin V, are associated with anti-inflammatory activity. However, it has been shown that the binding of annexin V to EVs increases the anti-inflammatory activity of EVs. Thioredoxins are small redox proteins known to be present in all organisms. Thioredoxin 1 (Trx) has strong antioxidant activity, exhibiting anti-inflammatory and anti-fibrotic effects. Furthermore, its binding to EVs increases the anti-inflammatory effect of EVs. Lactoglossin, also known as milk fat globule EGF factor 8 protein (Mfge8), is a secreted protein found in vertebrates, including mammals and birds. It contains a phosphatidylserine domain and an Arg-Gly-Asp binding sequence. Mfge8 binds in a calcium-dependent manner.

[0090] In one embodiment, the composition comprises EV bound to annexin V, preferably recombinant annexin V, and further comprises calcium. In one embodiment, the ratio of annexin V bound to EV to albumin is preferably between 1:90,000 and 1:3,000,000.

[0091] In one embodiment of the invention, the composition may further comprise one or more second therapeutic agents. As used herein, a therapeutic agent refers to any agent that can be used to prevent, treat, and / or control the disease discussed herein. Such agents may be intravesical (i.e., contained within the EV) or bound to the EV. Suitable therapeutic agents are known to those skilled in the art and may include non-coding RNA, miRNA, mRNA, growth factors, angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, antifibrotic molecules, antioxidant molecules, neurogenic molecules, antiviral molecules, etc. In some embodiments, isolated EVs are used together with a second agent. In some embodiments, the second 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.

[0092] Certain secondary therapeutic agents used to treat or control certain lung conditions (including but not limited to pulmonary hypertension) include oxygen, anticoagulants such as warfarin (Coumadin), and diuretics such as furosemide. or spironolactone Calcium channel blockers; potassium, for example Cardiotonic agents, such as digoxin; vasodilators, such as nifedipine. or diltiazem Endothelin receptor antagonists, such as bosentan and Anbesentan Prostacyclin analogs, such as eprostacyclin Treprostine sodium and Iloprost And PDE-5 inhibitors, such as sildenafil and Tadalafil

[0093] Preferably, the composition is formulated as a liquid. It can be stored via vials, IV bags, ampoules, cartridges, inhalers such as liquid inhalers, nebulizers, or powder inhalers, and pre-filled syringes. In addition to being the active ingredient or EV of the pharmaceutical product, the liquid formulation may also contain a variety of compounds to ensure the stability of the active pharmaceutical ingredient during subsequent storage. These include solubilizers, stabilizers, buffers, tension modifiers, fillers, viscosity enhancers / viscosity reducers, surfactants, chelating agents, and adjuvants.

[0094] In another embodiment, the composition may be lyophilized or freeze-dried. The lyophilized formulation may be stored in vials, cartridges, inhalers such as liquid inhalers, nebulizers, or powder inhalers, dual-chamber syringes, and pre-filled mixing systems. Prior to administration, the lyophilized composition is reconstituted into a liquid. This can be done by combining, mixing, and then injecting a liquid diluent with the lyophilized powder. Reconstitution typically requires a reconstitution and delivery system to ensure the drug is properly mixed and administered.

[0095] In a preferred embodiment, the composition is aqueous. In one embodiment of the invention, the EV can be formulated into a composition further comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier or diluent is selected to maintain the activity and properties of the EV of the invention. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or carrier involved in the delivery or transport of a preventive or therapeutic active agent, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation and does not cause harm to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; glycols, such as propylene glycol; polyols, such as glycerol, 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; ethanol; phosphate buffer; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0096] The composition can be stored at 4°C, more preferably by cryopreservation, wherein the composition is frozen between -20°C and -196°C, more preferably between -40°C and -196°C, and even more preferably between -80°C and -196°C. The freezing procedure is preferably a rapid freezing procedure or a vitrification procedure to ensure that the product retains its activity during freezing and after thawing. Another freezing procedure can be controlled-rate freezing, preferably compensating for the exothermic reaction at the crystallization point to obtain better product stability. For the latter procedure, a controlled-rate freezer can be used.

[0097] In one embodiment, the composition is suitable and / or formulated for administration to a patient via injection, intravenous administration, subcutaneous administration, intramuscular administration, percutaneous or transdermal administration, inhalation, endotracheal infusion, oral infusion, systemic infusion, or intranasal infusion. The composition may also be formulated for external use, alone or in combination with hydrogels, polymers, or polymer medical devices to slowly release EVs.

[0098] 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 aerosol administration. In some embodiments, the EV is administered using a nebulizer. In some embodiments, the EV is administered alone using an endotracheal cannula.

[0099] In some embodiments, the EV is applied or formulated together with a surfactant, preferably a pulmonary surfactant. The surfactant is preferably selected in a manner that does not 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. Pulmonary surfactants are lipoprotein mixtures used to maintain the patency of the lung airways (e.g., by preventing alveolar walls from sticking together). Pulmonary surfactants may consist of: phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidylglycerol (PG); cholesterol; and proteins, such as SP-A, B, C, and D. Pulmonary surfactants can be derived from naturally occurring sources, such as bovine or porcine lung tissue. For example, Alveofact... TM (from bovine lung lavage fluid), Curosurf TM (From chopped pig lungs), Infasurf TM (from calf lung lavage fluid) and Survanta TM (Derived from chopped bovine lung, containing other components including DPPC, palmitic acid, and tripalmitoylglycerol). Lung surfactants can also be synthetic. Examples include Exosurf. TM (Composed of DPPC, cetyl alcohol, and tyloxapol), Pumactant TM Or artificial lung expansion compound (ALEC) (composed of DPPC and PG), KL-4 (composed of DPPC, palmitoyl oleoyl phosphatidylglycerol, palmitic acid and a synthetic peptide mimicking SP-B), Venticute TM (Composed of DPPC, PG, palmitic acid and recombinant SP-C). Lung surfactants are available from commercial suppliers.

[0100] The present invention also includes packaged and labeled pharmaceutical products. The product or kit comprises a suitable unit dosage form in a suitable vessel or container, such as a glass vial or plastic ampoule or other sealed container. The unit dosage form should be suitable for pulmonary delivery, such as an aerosol. Preferably, the product or kit also includes instructions on how to use the product, including how to administer it. The instructions may also contain informational material advising physicians, technicians, or subjects on how to properly prevent or treat the disease or condition. In other words, the product includes instructions indicating or recommending a dosing regimen, including but not limited to actual dosage, monitoring procedures, and other monitoring information.

[0101] As with any pharmaceutical product, packaging materials and containers are designed to maintain the stability of the product during storage and transportation, and may contain desiccants to ensure stability.

[0102] This kit may contain EVs in a sterile aqueous suspension, which can be used directly or diluted with physiological saline for intravenous or nebulizer administration, or diluted or used in combination with a surfactant for intratracheal administration. Therefore, the kit may also contain diluents or thinners, such as saline or surfactants. The kit may also include lung delivery devices, such as nebulizers, or disposable components, such as mouthpieces, nosepieces, or masks.

[0103] In a final aspect, the present invention also relates to the use of the above-described compositions. More specifically, the compositions are suitable for therapeutic and preventative uses. The present invention is contemplated for the prevention and treatment of specific diseases. Disease prevention means reducing the likelihood of the disease manifesting itself and / or delaying the onset of the disease. Disease treatment 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.

[0104] The subjects are preferably human subjects, but certain aspects of the invention can be performed on any subject who may benefit therefrom, including human subjects, agricultural livestock (e.g., cattle, pigs, etc.), precious animals (e.g., horses), companion animals (e.g., dogs, cats, etc.). In a preferred embodiment, the subject is a human, preferably a human patient, who may be an adult, an infant, or a newborn.

[0105] In one embodiment, the composition of any of the above embodiments is used for the prevention or treatment of lung diseases. In one embodiment, the lung disease may be inflammatory lung disease, pulmonary vascular disease, or 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 related to sepsis or acute respiratory distress syndrome (ARDS).

[0106] These diseases also include pulmonary vascular diseases that may not have an inflammatory component. Other lung diseases that can be treated according to the invention include acute lung injury, which may be related to sepsis or ventilation. An example of the latter is acute respiratory distress syndrome.

[0107] Pulmonary hypertension is a lung disease characterized by pulmonary artery blood pressure that is much higher than normal. Symptoms include shortness of breath, chest pain (especially during physical activity), weakness, fatigue, fainting, mild headache (especially during exercise), dizziness, abnormal heart sounds and murmurs, jugular venous distension, fluid retention in the abdomen, legs and ankles, and cyanosis of the nail beds.

[0108] Bronchopulmonary dysplasia (BPD) is a condition that afflicts newborns or premature newborns who require oxygen or are on a ventilator, especially very premature newborns (e.g., those born before 32 weeks of gestation). It is also known as neonatal chronic lung disease. Causes of BPD include mechanical injury (e.g., from ventilation), oxygen toxicity (e.g., from oxygen therapy), and infection. Over time, the disease may progress from non-inflammatory to inflammatory. Symptoms include cyanosis, chronic cough, tachypnea, and shortness of breath. Subjects with BPD are more susceptible to infections such as respiratory syncytial virus (RSV). Subjects with BPD may develop pulmonary hypertension.

[0109] Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome, is a condition caused by lung injury or acute illness. Lung injury can be caused by ventilation, trauma, burns, and / or aspiration. Acute illness can be infectious pneumonia or sepsis. It is considered a severe acute lung injury and is often fatal. It is characterized by lung inflammation, impaired gas exchange, release of inflammatory mediators, hypoxemia, and multiple organ failure. ARDS can also be defined as a ratio of arterial oxygen partial pressure (PaO2) to inhaled oxygen (FiO2) of less than 200 mmHg in the presence of bilateral infiltrates on chest X-ray. A PaO2 / FiO2 ratio of less than 300 mmHg with bilateral infiltrates indicates acute lung injury, which is often a precursor to ARDS. Symptoms of ARDS include shortness of breath, tachypnea, and confusion due to hypoxemia.

[0110] Idiopathic pulmonary fibrosis is characterized by unexplained scarring or thickening of the lungs. It most commonly occurs in people aged 50 to 70. Its main symptoms include shortness of breath, frequent cough (usually dry), chest pain, and decreased activity levels.

[0111] In some cases, prevention and / or treatment may include the use of EVs alone or in combination with one or more second agents or active ingredients. Mechanical interventions may also be administered to the subject, such as ventilation with or without external oxygen supply.

[0112] Subjects may be those suffering from lung diseases (or conditions) suitable for treatment with the EV of this invention, or those at risk of developing such diseases (or conditions). Subjects include newborns, particularly those born at low gestational age. As used herein, a human newborn refers to a person from birth to approximately 4 weeks of age. As used herein, a human infant refers to a person from approximately 4 weeks of age to approximately 3 years of age. As used herein, low gestational age refers to birth (or delivery) earlier than the normal gestational age for a given species. A full human gestation period is approximately 40 weeks, and can range from 37 weeks to more than 40 weeks. Low gestational age (similar to preterm birth) in humans is defined as birth before 37 weeks of gestational age, including birth at shorter gestational ages (e.g., before 36 weeks, 35 weeks, 34 weeks, 33 weeks, 32 weeks, 31 weeks, 30 weeks, 29 weeks, 28 weeks, 27 weeks, 26 weeks, or 25 weeks of gestation). Such preterm infants are typically treated as newborns; however, this invention contemplates treating them beyond the newborn stage, reaching childhood and / or adulthood. Some subjects may have a genetic predisposition to certain forms of lung disease (e.g., pulmonary hypertension), and these subjects may also be treated according to the present invention.

[0113] For newborns, especially those of low gestational age, this invention considers administering EVs at 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 after birth. In some critical cases, MSC exosomes are administered within 1 hour of birth.

[0114] This disclosure further considers the administration of EVs even in the absence of symptoms of lung disease (e.g., but not limited to BPD).

[0115] In one embodiment, the EV-containing composition of the present invention can be used to treat COVID-19, more specifically, COVID-19-induced pneumonia or acute pneumonia. COVID-19 is a novel infectious disease caused by severe acute respiratory syndrome that attacks the human respiratory system and lung epithelial tissue. It has been reported that a subset of patients are at higher risk of developing more severe symptoms of COVID-19. Major complications include pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, septic shock, and death.

[0116] It has been shown that COVID-19 (also known as SARS-CoV-2) involves different mechanisms of lung infection, which can develop into acute respiratory distress syndrome (ARDS) potentially triggered by cytokine storm, multiple organ failure, septic shock, and thrombosis. In contrast, bacterial pneumonia, a common lung infection in which air sacs throughout or in part of the lung become inflamed and filled with fluid, pus, and cellular debris, is primarily caused by viruses, fungi, or bacteria and is usually treated with antibiotics. In other cases, cardiovascular complications, elevated liver enzymes reflecting liver damage, and neurological manifestations may occur. In children, if the infection progresses, it can develop into pediatric multisystem inflammatory syndrome, with symptoms similar to Kawasaki disease, which can be fatal. Based on current data, children account for a small percentage of reported cases, with approximately 1% in children under 10 years of age and 4% in children between 10 and 19 years of age.

[0117] The composition containing EV provides multi-target therapeutic effects, with its primary mode of action being the inhibition of the inflammatory process.

[0118] EV targets multiple lung injury mechanisms, including high inflammatory response and cytokine storm, fibrosis, oxidative stress induced by (mechanical) ventilation, and epithelial cell apoptosis due to viral activity and inflammatory response.

[0119] In another embodiment, the EV-containing composition is used as adjunctive therapy for COVID-19, more specifically, COVID-19-induced pneumonia or acute pneumonia.

[0120] In another embodiment, the composition of any of the above embodiments is used for the prevention or treatment of inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis. IBD is a group of diseases that cause inflammation of the gastrointestinal tract (GI), the two most common being ulcerative colitis (UC) and Crohn's disease (CD). UC is a disease that causes chronic inflammation and ulceration in the inner lining of the deepest part of the colon and rectum. CD can develop anywhere in the digestive tract and can penetrate deep into the affected tissues. A symptom of CD is the development of anal fistulas. Both diseases are similar in that they both cause abdominal pain, severe diarrhea, fatigue, and weight loss.

[0121] The compositions of the present invention have been shown to contain a therapeutically effective amount of EV bound to annexin V, preferably recombinant annexin V, which is particularly effective for the treatment and prevention of inflammatory bowel disease, especially for Crohn's disease and ulcerative colitis, preferably for Crohn's disease.

[0122] In cases of pulmonary disease, the preferred administration method is intratracheal infusion or inhalation. In cases of neurological disease, systemic infusion, intranasal infusion, or inhalation are preferred. In cases of Crohn's disease fistula, ulcer, or cartilage repair, local injection is preferred. In cases of treating wound healing, burns, and / or ulcers, EV is preferably administered externally to the body, preferably in conjunction with a hydrogel or polymer medical device for slow EV release.

[0123] The EV of the present invention is administered in an effective amount. An effective amount refers to the amount of the formulation that, when used alone, produces the desired result. This absolute amount depends on many factors, including the material chosen, whether it is administered in a single or multiple dose, and individual patient parameters including age, physical condition, body size, weight, and stage of disease. These factors are well known to those skilled in the art and can be resolved through routine experiments. The dosage may also vary depending on the subject administering the composition.

[0124] In one embodiment of the invention, the EV is 10 9 EVs / kg up to 10 12 The dosage administered to the patient is preferably 10 EVs / kg. 10 EVs / kg up to 10 12 The dosage for the patient is EVs / kg. In a further embodiment, the dosage for children (aged 0 months to 12 years) ranges from 10 EVs / kg. 9 EVs / kg up to 10 11 EVs / kg. The range of doses for adolescents and adults is 10 in adults. 9 EVs / kg up to 10 12 EVs / kg.

[0125] In one embodiment of the invention, the EV is approximately 10 per patient. 10 To about 10 12 EV dosage is administered to treat CD anal fistula.

[0126] This disclosure also considers repeated administration of the EV, including two, three, four, five or more administrations. In some cases, the EV can be administered continuously. Depending on the severity of the disease being treated, repeated or continuous administration can occur over several hours (e.g., 1 to 2 hours, 1 to 3 hours, 1 to 6 hours, 1 to 12 hours, 1 to 18 hours, or 1 to 24 hours), several days (e.g., 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, or 1 to 7 days), or several weeks (e.g., 1 to 2 weeks, 1 to 3 weeks, or 1 to 4 weeks). If repeated but not continuous administration is used, the time between two administrations can be several hours (e.g., 4 hours, 6 hours, or 1 hour), several days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or several weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks). The time between two administrations can be the same or different. For example, if the symptoms of the disease seem to be worsening, EVs can be administered more frequently, and once the symptoms stabilize or improve, the frequency of EV administration can be reduced.

[0127] In some cases, low-dose EVs can be repeatedly administered intravenously. Therefore, this disclosure contemplates both repeated administration of low-dose EVs and single administration of high-dose EVs. The low-dose range can be 10... 10 Up to 10 11 EV per kilogram or per local infusion, but not limited to this, while the range of high doses can be 10. 11 Up to 10 12 EV per kilogram or per local infusion. It should be understood that single or repeated administration of low or high doses of EV should be considered depending on the severity of the disease, the subject's health status, and the route of administration.

[0128] EVs can be administered via any route of delivery to the lungs or gastrointestinal tract. Systemic administration routes such as intravenous bolus and continuous infusion are suitable. More direct routes such as intranasal administration, intratracheal administration (e.g., via intubation), and inhalation (e.g., aerosols via the nose or mouth) are also considered in this invention and may be more suitable in certain cases where rapid action is required. As used herein, an aerosol is a liquid suspension dispersed in a gas as small particles, and includes fine mists or sprays containing such particles. As used herein, aerosolization is the process of generating an aerosol by converting a liquid suspension into small particles or droplets. This can be done using aerosol delivery systems such as pressurized packs or nebulizers. Nebulizers include air jet nebulizers (i.e., pneumatic), ultrasonic nebulizers, and vibrating mesh nebulizers, using suitable propellants such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In addition to nebulizers, other lung delivery devices include, but are not limited to, metered-dose inhalers (MDIs) and dry powder inhalers (DPIs). Capsules and vials made from gelatin, for example, for use in inhalers or blowpipes, can be formulated to contain lyophilized exosomes and a suitable powder matrix, such as lactose or starch.

[0129] When whole-body delivery of EVs is required, the EVs can be formulated for parenteral administration via injection, such as by bolus or continuous infusion. Injectable formulations can be in single-dose form, such as ampoules or multi-dose containers, with or without preservatives.

[0130] The composition may be in the form of an aqueous suspension, solution, or emulsion on an oily or aqueous carrier, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Suitable lipophilic agents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase solubility. Additionally, exosomes may be in lyophilized or other powder or solid form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to use.

[0131] The EVs and compositions described herein are preferably obtained by the methods described above.

[0132] The invention will now be described in more detail with reference to non-limiting embodiments.

[0133] Description of embodiments and / or accompanying drawings

[0134] Example 1 - Separation of EVs from MSC

[0135] Umbilical cord (UC)-derived MSCs obtained from umbilical cord tissue or Wharton's jelly are amplified in an amplification unit comprising a stirred bioreactor (2) fluidly connected to a cell culture medium supply container (1). (See reference...) Figure 1 The following numbers represent:

[0136] 1. Cell culture medium container

[0137] 2. Stirred bioreactor

[0138] 3. Cell supernatant container

[0139] 4. Refrigerator

[0140] 5a, 5b, 5c. Peristaltic pump

[0141] 6. First Filtering Unit

[0142] 7. Second Filtering Unit

[0143] 8. TFF box

[0144] 9. Final Product

[0145] Cells proliferated in the presence of three-dimensional microcarriers in the bioreactor (2) and in a heterologous and serum-free culture medium containing 3 g / L of purified human albumin. The culture medium also contained 60 mg / L of recombinant purified transferrin.

[0146] MSCs were amplified and grown in bioreactor (2) until they reached a density of at least 40 x 10⁻⁶ in bioreactor (2). 6The minimum cell / L concentration and the maximum MSC confluence were 80%. Cell concentration was measured by cell counting and fluorescent labeling of MSCs attached to microbeads. At this stage, the cells were ready to begin secreting EVs. For this purpose, fresh serum-free and heterologous medium was diluted 1:10 from basal medium (DMEM / high glucose / phenol red-free / glutamax, Thermo Scientific) and added to the cell medium. After 24 hours, the system was ready to begin collecting EVs. For this purpose, conditioned cell medium or cell supernatant from bioreactor (2) was pumped into cell supernatant container (3), which was placed in a refrigerator (4) set at 4°C. The supernatant contained EVs produced by MSCs. The supernatant collected in container (3) was then pumped to a first filtration unit (6) with a 2-micron filter via a peristaltic pump (5c). The supernatant was filtered by dead-end filtration in a closed system with a peristaltic pump (5c) providing a constant flow rate of 100 ml / min through the filter (6). The flow-through liquid from the first filtration step was then filtered in a second filtration unit (7), which was fluidly connected to the first filtration unit (6) and had a 0.2-micron filter. Filtration was carried out in a closed system using dead-end filtration with a peristaltic pump (5c) providing a constant flow rate of 100 ml / min through the filter (7). The flow-through liquid containing EV was collected in a 1-liter bag and then stored at 4°C.

[0147] In the final step, the flow-through solution is introduced into a TFF box with a molecular weight cutoff of 100 kDa, which is fluidly connected to dead-end filter units (6, 7). The EV-containing residue in the TFF box (8) is washed with saline buffer and concentrated to a final volume of 10 ml using a peristaltic pump (5c) at a speed of 300 rpm. The final product (9) is collected from the TFF (8) and frozen below -80°C or stored in cryovials, bags, or other suitable containers at 4°C.

[0148] The final product is analyzed to ensure its quality. Quality control includes measuring the concentration of albumin bound to EVs, and particle analysis using techniques such as colorimetric analysis and ELISA, nanoparticle tracking analyzer (NTA), tunable resistive pulsed light sensor (TRPS), electron microscopy, and / or RAMAN microscopy to ensure the purity and stability of the product. The product sample is tested for the presence of endotoxins and / or mycoplasma. Other quality control measures include qualitative chromatography, mass spectrometry, ELISA, sequencing, qRT-PCR, and activity assays, such as in vitro T-cell, B-cell, and macrophage polarization.

[0149] Under the above culture conditions, during the MSC culture period of 18 to 24 hours, the MSCs will produce at least 0.25 x 10⁻⁶ ppm of culture medium per ml. 9 Each EV. This experiment was performed using MSCs derived from UC, but can also be replicated using MSCs derived from other tissues or sources, including but not limited to mammary gland, bone marrow, Wharton's jelly, (umbilical cord) blood, peripheral blood, amnion, adipose tissue, dental pulp, fallopian tubes, liver tissue, and lung tissue.

[0150] Example 2 - Protein binding to EV

[0151] EVs were produced as described in Example 1 until collection after the second filtration step. The fluid collected through the filtration step was then transferred to a TFF box with a molecular weight cutoff of 100 kDa for labeling / binding of EVs with annexin V, vesicle washing, and concentration. All steps were performed in a closed system.

[0152] The flow-through collected during the filtration step was recirculated and concentrated in a TFF cartridge using a peristaltic pump at 300 rpm until a final volume of 15 ml was obtained. The EV remained in the permeate, which was then discarded as waste. The calcium concentration in the permeate was then measured, and calcium was replenished until the concentration reached 2 mM.

[0153] Next, 0.5 mg of annexin V was added to each milliliter of osmotic fluid in the TFF cassette, and the product was then recirculated at room temperature for 15 minutes. A small sample was taken to check the concentration of annexin V using an ELISA test.

[0154] To subsequently remove unbound annexin or unwanted contaminants <100 kDa, wash the osmotic fluid (approximately 15 ml) with saline wash buffer, circulate it in TFF at a low speed of 200 rpm, and concentrate it again (200 rpm) to approximately 15 ml. Repeat this washing step twice more, checking the waste (osmotic fluid) each time before discarding it to determine the amount of unbound annexin V (ELISA). If the third wash osmotic fluid contains only very little unbound annexin V, it indicates that most of the unbound annexin has been washed away. Stop the peristaltic pump and then collect the final concentrated product in a collection syringe. The product can be aliquoted before storage, or frozen directly below -80°C, or stored in cryovials, bags, or other suitable containers at 4°C.

[0155] The experiment of binding annexin V to EV has been described; however, the experiment was repeated in order to obtain EV bound to thioredoxin and EV bound to lactogrin.

[0156] Example 3: EV activity in an in vivo model of Crohn's disease

[0157] This experiment aimed to demonstrate the activity of EVs that bind to annexin V, which were produced in an in vivo model of Crohn's disease in a manner similar to Example 2. Briefly, the experiment was performed on female mice. Colitis was induced by applying 3% sodium lauryl sulfate (SDS) to drinking water, administered randomly for 5 days. This is a well-established and widely used animal model, cited hundreds of times in PubMed (review for Kawada et al., 2007). All animals were handled according to appropriate ethical guidelines. The animals were subdivided into three experimental groups.

[0158] Group 1 (normal control): 0.2 ml of PBS (carrier only) was injected intraperitoneally (ip) daily from day 1 to day 5;

[0159] Group 2 (Induced Colitis): Same as Group 1, but with 3% SDS added to drinking water.

[0160] Group 3 (Induction of colitis and treatment with EV): Same as Group 2, but MSC-EV suspended in 0.2 ml PBS was added via the IP route.

[0161] On day 6, animals were euthanized using CO2. The colon was removed, a portion of the tissue was fixed in formalin for subsequent histological analysis, and another portion was immediately frozen in liquid nitrogen and then stored at -80°C for subsequent RNA extraction.

[0162] Dosage and route of administration of EV

[0163] EVs are separated and applied according to the above procedure.

[0164] Administration route: From day 0 to day 5, administer EV suspended in 0.2 ml PBS daily via intraperitoneal (ip) route.

[0165] Intestinal injury assessment

[0166] animal weight

[0167] Disease activity index (fecal assessment; see Tanaka F, 2008)

[0168] - Histopathological markers of inflammation (Flogosis)

[0169] - Titration of inflammatory mediator expression in colon tissue extracts: TNFα, IL6, IL-1β and Cox2 were detected by RT-PCR.

[0170] Statistical analysis of results

[0171] All data are expressed as mean ± SD. Differences between groups were analyzed by t-test, and p < 0.05 was considered statistically significant.

[0172] result

[0173] In summary, the results indicate that the annexin V-bound EV of this invention is particularly suitable for reducing the inflammatory response in experimental colitis, resulting in a reduction in the disease activity index and improvement of common symptoms. The annexin V-bound EV improved clinical presentation and inflammatory response in animal models of intestinal inflammatory disease.

[0174] This invention is by no means limited to the embodiments described and / or shown in the accompanying drawings. Rather, the methods of this invention can be implemented in many different ways without departing from the scope of the invention.

Claims

1. A method for manufacturing protein-bound extracellular vesicles (EVs) derived from MSCs, the method comprising the following steps: - Purify EVs from a cell culture medium containing MSCs, wherein the purification is performed by at least one filtration step in the culture medium; next - A step of concentrating the filtrate from the at least one filtration step, wherein the EV is concentrated by tangential flow filtration TFF in a tangential flow filtration TFF device; and - During the TFF step, the EV binds to one or more proteins inside the TFF device or in a container fluidly connected to the TFF device and transferring the EV from the TFF device to it. The protein is replenished to the TFF device or connected container during the concentration of the EV.

2. The method of claim 1, wherein, Calcium is added to the TFF device or connected container during the concentration of the EV.

3. The method of claim 2, wherein, The calcium and the protein are mixed in the TFF or connected container and incubated together with the EV present in the TFF device or connected container.

4. The method of any one of claims 1 to 3, wherein, The EVs bound to the protein were washed, reconcentrated, and subsequently collected outside the TFF device.

5. The method of any one of claims 1 to 3, wherein, The protein is selected from annexin, thioredoxin, and lactoglucosin.

6. The method of any one of claims 1 to 3, wherein, The protein in question is annexin V, Trx, or Mfge8.

7. The method of any one of claims 1 to 3, wherein, The protein in question is recombinant annexin V, Trx, or Mfge8.

8. The method according to any one of claims 1 to 3, wherein, The TFF device has a molecular weight cutoff of 100 kDa.

9. The method according to any one of claims 1 to 3, wherein, The MSCs were cultured and amplified in a cell culture medium containing purified human serum albumin.

10. The method of claim 9, wherein, The concentration of albumin in the culture medium is 1 g / L to 5 g / L.

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