Method for producing MSC-derived exosomes

Abstract

A method for producing clinical-grade exosomes derived from mesenchymal stem cells (MSCs) is provided. [Solution] A method is provided for producing exosomes from mesenchymal stem cells (MSCs), comprising: (a) culturing MSCs in a functionally closed bioreactor in a medium containing human platelet lysates (PLT) to 75-95% confluence; (b) further culturing the cells in a medium essentially free of PLT; (c) collecting a conditioned medium fraction from the bioreactor; and (d) isolating exosomes from the conditioned medium fraction.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 587,408, filed November 16, 2017, which is hereby incorporated by reference in its entirety. and is hereby incorporated by reference in its entirety.

[0002] [Technical Field] The present invention generally relates to the fields of molecular biology and medicine. More specifically, the present invention relates to methods for the large-scale production of GMP (good manufacturing practice)-compliant exosomes.

Background Art

[0003] Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized intercellular communication mediators involved in several physiological processes. Specifically, exosomes are nano-sized vesicles released by cells and constitute a mode of intercellular exchange of cellular components and products, which has spurred new interest in their usefulness as therapeutic delivery agents. Unlike their artificial counterparts, these naturally produced, specialized intercellular shuttle services may offer unique advantages for the efficient delivery of therapeutic payloads. Such features of exosome production and regulatory mechanisms associated with exosome uptake remain to be further investigated. Nevertheless, the use of exosomes for the therapeutic management of diseases, including cancer, has already shown promising results. [[ID=​​​​​​​It is a promising candidate. However, its low production yield raises concerns about its potential clinical use. The potential is limited. Therefore, efficient production of exosomes that can be used for therapeutic purposes is required. Regarding methods, there are still unmet needs. [Overview of the project]

[0005] In the first embodiment, mesenchymal stem cells (MS) are cultured in a medium containing human platelet lysates (PLT). C) Confluence (e.g., 75-95% or 80-90% confluence, approximately 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or Cultivate in a functionally closed bioreactor until 90% confluence; Furthermore, the cells are cultured in a medium that is essentially free of PLT (for example, a PLT-free medium). collecting the conditioned medium fraction from the bioreactor; and extracting the exo from the conditioned medium fraction. A method for producing exosomes from MSCs, including the isolation of the exosomes, is provided.

[0006] In some embodiments, each acclimatized culture medium fraction is stored at -80°C after collection. Specific embodiments In this process, the conditioned medium fraction is thawed and pooled before isolation.

[0007] In certain embodiments, MSCs are further defined as bone marrow-derived MSCs. MSCs are further defined as adipose-derived MSCs.

[0008] In a further embodiment, the method involves culturing MSCs in a bioreactor before using a bioreactor At least 1x10 for the actor 7 Further includes sowing MSCs. In some embodiments, MSCs have approximately 400-500 cells / cm². 2 It is sown at this concentration.

[0009] In certain embodiments, a sealed bioreactor is a hollow fiber bioreactor. In this embodiment, the hollow fiber bioreactor is the Terumo cell proliferation system.

[0010] In some embodiments, PLT is present at a concentration of 5% in the medium of the MSC culture. The concentration may be approximately 2-10%, for example, 3, 4, 5, or 6%. In certain embodiments, Fresh culture medium is continuously added to the MSCs in the bioreactor. In a specific embodiment The cells are cultured in 5% oxygen. In some embodiments, the culture in step (a) is extended to 5-1 0 days (for example, 6, 7, 8, or 9 days). In a particular embodiment, step (a) The culture period is 8 days.

[0011] In certain aspects, MSCs have a confluence of 85% to 90%, for example, 85%, 86%. They are cultured to %, 87%, 88%, 89%, or 90% confluence. The sensation can be measured by monitoring glucose and lactose levels. For example, lactose levels are approximately 2-6 mmol / L, for example, approximately 2, 3, 4, 5, or It can be 6 mmol / L, and the glucose level is approximately 80-140 mg / dL, for example, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, It may be 135 or 140 mg / dL.

[0012] In some embodiments, cultivation in a medium that is essentially PLT-free is performed for 24-72 hours, for example. For example, 24-48, 36-50, 48-60, or 50-72 hours, approximately 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 This is 52 hours. In certain embodiments, the culture medium is essentially free of PLT. This is approximately 48 hours. In certain embodiments, the culture medium for the further culture step does not contain PLT. .

[0013] In certain embodiments, MSCs are cultured in PLT and are essentially PLT-free or They are washed between culture in a medium that does not contain the MSC. In some embodiments, MSCs are free The cells are cultured in a serum culture medium. In a particular embodiment, the complete method is performed under serum-free conditions. It is done.

[0014] In certain embodiments, the conditioned medium fraction is collected in a sealed bag. The acclimatized culture medium fraction is collected every 24 to 72 hours. In some embodiments, the acclimatized culture medium The ground fraction is collected every 40-50 hours. In a particular embodiment, the acclimatized culture medium fraction is collected every 48 hours. They are collected hourly.

[0015] In some embodiments, the conditioned medium fraction is 200-250 or 250-300 mL, The volume is 200-300 mL. In certain embodiments, the acclimatized medium fraction is conditioned for 10-14 days. They are collected over a period of time (for example, 10, 11, 12, 13, or 14 days). In certain embodiments, they are accustomed to The conditioned medium fraction is collected for 12 days. In certain embodiments, at least five conditioned medium fractions are collected. They are gathered. In certain aspects, this method is carried out in less than three weeks.

[0016] In some embodiments, each conditioned medium fraction is 9 × 10 11 From 50 x 10 11 of Contains exosomes. In certain embodiments, at least 10 × 10 12 The exosome , is isolated in the collected medium fraction. In certain embodiments, at least 15×10 12 exosomes are isolated in the collected medium fraction. At least 10×10 11 , 15×10 11 , 20×10 11 , 25×10 11 , 30×10 11 , 35×10 11 , 40×10 11 , 45×10 11 , 50×10 11 , 60×10 11 , 70×10 11 , 80×10 11 , 90×10 11 , 10×10 12 , 15×10 12 , 20×10 12 , or 25×10 12 exosomes can be isolated.

[0017] [[ID=4�]]In certain embodiments, the isolation includes filtration and ultracentrifugation of the pooled fraction to obtain an exosome-containing pellet and resuspending the exosome-containing pellet in a buffer. In some embodiments, the isolation is performed in a functionally closed manner using a pump and heat-sealed tubing. In some embodiments, filtration is further defined as passing the pooled fraction through a filter such as a 0.2 μm filter. In further embodiments, the isolation further includes a centrifugation step prior to filtration to remove large cell debris. In certain embodiments, the isolation is performed at 4°C.

[0018] In certain embodiments, the buffer is about 0.01 - 0.1 M, such as about 0.08, 0.09, or 0.1M, especially about 0.1-0.5M, for example, about 0.1, 0.2, or 0.3M sodium chloride Rium, approximately 0.1-0.5M, for example, approximately 0.1, 0.2, or 0.3M, especially approximately 0.23 M sodium gluconate, approximately 0.1-0.5, for example 0.1, 0.2 or 0.3M, special To approximately 0.27 M sodium acetate trihydrate, 1-10 mM, for example, about 6-7 mM, especially about 5 mM potassium chloride, and about 1-5 mM, for example, about 2-4 mM, especially about 3 mM potassium chloride Contains magnesium. In some embodiments, the buffer solution is about 6-8, for example, about 6.8, 6.9 , 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, especially around 7.4 It has a pH. In certain embodiments, the buffer solution is PLASMALYTE-A®. .

[0019] In a further embodiment, this method further encompasses the process of loading therapeutic agents into exosomes. Includes. In some embodiments, the therapeutic agent includes one or more cytokines, chemotherapeutic agents, nuclear It contains acids, small molecules, or proteins. In certain embodiments, nucleic acids include DNA and / or R Contains NA. In some aspects, RNA is siRNA, miRNA, or shRN. A. In certain embodiments, RNA is siRNA. In some embodiments, it is mounted. This includes electroporating exosomes. In certain embodiments, Electroporation is performed in PLASMALYTE-A®. In this embodiment, the method includes the steps of isolating exosomes and electroporation. The process does not include a step of washing the exosomes or a step of exchanging the buffer solution between the two steps. In particular, from the stage of isolating exosomes to the number of exosomes loaded The number of "Mu" will not decrease by more than 20%.

[0020] In another embodiment, the method of the embodiment (for example, a culture medium containing human platelet lysate (PLT)) Inside, MS in a functionally closed bioreactor up to 80-90% confluence C culture; in a medium that is essentially free of PLT (e.g., PLT-free medium) Further culturing of cells; collection of conditioned medium fraction from bioreactor; and conditioned Pharmaceuticals containing exosomes produced by isolating exosomes from culture medium fractions. A composition is provided.

[0021] In yet another embodiment, a method for treating cancer is provided, which is an embodiment Administer an effective amount of exosomes produced according to the method (for example, human platelet lysis Functionally closed in a culture medium containing the lysate (PLT) up to 80-90% confluence. Culturing MSCs in a bioreactor that does not essentially contain PLT (e.g., P Further culturing of cells in a medium (without LT); conditioned medium fraction from bioreactor This includes collecting the fraction and isolating the exosomes from the conditioned medium fraction. In some cases, the subject is human.

[0022] In a specific embodiment, electroporated exosomes are directly injected into a target. In an additional embodiment, the method further includes administering at least a second anticancer therapy. In some embodiments, at least the second anti-cancer treatment is chemotherapy, radiation therapy, and genetic therapy. This includes veterinary therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

[0023] In another embodiment, the method of this embodiment (for example, including human platelet lysate (PLT)) In a culture medium, a functionally closed bioreactor reaches 80-90% confluence. The process of culturing MSCs inside; furthermore, the cells are essentially PLT-free (e.g., PL The process of culturing in a culture medium (without T); the process of collecting the conditioned medium fraction from the bioreactor. Steps: Isolating exosomes from the conditioned medium fraction; and RN in the isolated exosomes. (Produced by a process that involves loading A and thereby producing RNA-carrying exosomes) The process includes delivering exosomes loaded with an effective amount of RNA to cells, A method of delivery is provided. In some embodiments, the cells are human cells. In particular, The cells are cancer cells or T cells.

[0024] In a further embodiment, a person who treats a disease or disorder in a person who requires it. A method is provided which includes the method of this embodiment (for example, human platelet lysate (PLT)). M in a functionally closed bioreactor until it reaches 80-90% confluence in the culture medium. Culturing SCs in a medium that is essentially free of PLT (e.g., PLT-free medium). Further culturing of cells; collection of conditioned medium fraction from bioreactor; and conditioned Effective amount of exosomes produced by isolating exosomes from the culture medium fraction. This includes administering siRNA or mi. In some embodiments, the exosomes are siRNA or mi It carries RNA. In a particular embodiment, the exosome contains KRAS siRNA. It is installed. In some embodiments, the target is a human.

[0025] In some aspects, the disease or disorder is cancer, an inflammatory disorder, or an immune-related disorder. In certain contexts, cancer is lung cancer.

[0026] In certain embodiments, exosomes are delivered orally, locally, intravenously, intraperitoneally, or intramuscularly. It is administered intramuscularly, endoscopically, percutaneously, subcutaneously, locally, or by direct injection. In some embodiments, exosomes are administered intravenously.

[0027] In additional embodiments, the method further includes administering at least a second therapeutic agent. In some embodiments, at least the second therapeutic agent is an anticancer agent. In certain embodiments, an anticancer agent This includes chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy. It is a therapy.

[0028] In another embodiment, an immune-mediated inflammatory disease is treated in a subject suffering from the aforementioned disease. A method comprising the above method (for example, a culture medium containing human platelet lysate (PLT)) applied to the subject. In a functionally closed bioreactor designed to achieve 80-90% confluence underground, M Culturing SCs in a medium that is essentially free of PLT (e.g., PLT-free medium). Further culturing of cells; collection of conditioned medium fraction from bioreactor; and conditioned A therapeutically effective amount of MSCs produced by isolating exosomes from the culture medium fraction. A method is provided which includes administering exosomes. In some embodiments, immuno Inflammatory diseases include rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and Crohn's disease. Selected from the group consisting of several. In certain embodiments, MSCs are homogeneous. In this embodiment, the exosomes are administered systemically or topically. In a particular embodiment, Exosomes are found in the rectum, nasal cavity, oral cavity, vagina, subcutaneous tissue, intradermal tissue, intravenous tissue, abdominal cavity, and intramuscular tissue. Via intra-articular, intra-synovial, intra-sternal, intramedullary, intra-lesional, or intracranial pathways, or implantable It is administered via a reservoir. In some embodiments, the exosomes are at least It is administered together with one additional therapeutic agent.

[0029] Other objects, features, and advantages of the present invention will become apparent from the detailed description below. However, However, the detailed description of the present invention, specific examples and preferred examples are provided as illustrative examples. The various changes and modifications that will become apparent to those skilled in the art through this detailed description are in accordance with the present invention. It must be understood that it is within the realm of mind and scope. [Brief explanation of the drawing]

[0030] The following drawings are included to illustrate portions of this specification and to further illustrate certain aspects of the invention. The present invention, in combination with a detailed description of specific embodiments presented herein, is shown in these drawings. This will likely be better understood by referring to one or more of the following.

[0031] [Figure 1] Figures 1A-1D: (Figure 1A) Schematic procedure designed to produce extracellular organisms (EVs) from mesenchymal stem cells (MSCs) using the Turmo Cell Expansion System (Bioreactor). (Figure 1B) Schematic diagram detailing the procedure for culturing MSCs in a bioreactor for exosome collection. (Figure 1C) Schematic diagram of the procedure for isolating exosomes from MSC conditioned medium and electroporating them. (Figure 1D) Schematic diagram showing the generation of MSC-derived exosomes.

[0032] [Figure 2] Figure 2 is a schematic diagram of a strategy for producing acclimatized media containing EVs from MSCs cultured on a bioreactor.

[0033] [Figure 3] Figure 3: Schematic diagram of the strategy for isolating EVs from MSC-conditioned medium cultured on a bioreactor.

[0034] [Figure 4] Figures 4A-4B: Identification of MSC-derived exosomes produced in the bioreactor. (Figure 4A) Flow cytometry of MSC-derived exosomes produced in the bioreactor at each collection time, showing the expression of exosome markers. (Figure 4B) Representative TEM from each collection, showing the typical morphology of exosomes.

[0035] [Figure 5] Figures 5A-5E: Quantification of exosomes produced in the bioreactor at each harvest time. (Figure 5A) Number of MSC-derived exosomes produced at quantum level as determined by microBCA and NanoSight. (Figure 5B) Particle size distribution for each harvest measure using NanoSight. (Figure 5C) Glucose and lactose levels in the bioreactor during exosome production. (Figure 5D) Number of exosomes produced per cell, isolated from MSC-conditioned medium and quantified by NanoSight at different time points. (Figure 5E) Representative flow cytometry of exosome markers on MSC-derived exosomes isolated at 24 and 48 hours.

[0036] [Figure 6] Figures 6A-6B: Quantification of MSC-derived exosomes produced in media supplemented with human platelet lysate (hPLT) or serum-free conditions. (Figure 6A) Number of exosomes per cell isolated from conditioned medium of MSCs cultured with or without hPLT, analyzed by NanoSight. (Figure 6B) Flow cytometry of MSC-derived exosomes produced using medium alone versus medium supplemented with human platelet lysate (hPLT), showing the purity of exosomes produced on serum-free medium.

[0037] [Figure 7] Figure 7: Evaluation of MSC-derived exosome electroporation using 16 different nucleofactor programs and three different nucleofactor solutions. The efficiency of electroporation was evaluated 48 hours later by apoptosis induced by siRNA delivery by MSC-derived exosomes in recipient cells.

[0038] [Figure 8] Figures 8A-8C: Evaluation of MSC-derived exosome electroporation using five different solutions. (Figure 8A) The efficiency of electroporation was evaluated by apoptosis induced by siRNA delivery by MSC-derived exosomes on recipient cells after 48 hours. (Figure 8B) Representative transmission electron micrographs of MSC exosomes after electroporation using either research buffer (RB) or clinical buffer (CB; i.e., platelet lysate (PLT)), demonstrating the maintenance of exosome integrity after electroporation. (Figure 8C) Silencing of gene transcription in recipient cells induced by siRNA delivery using MSC-derived exosomes electroporated using a Lonza apparatus and PLASMALYTE-A® solution.

[0039] [Figure 9] Figure 9: Number of exosomes before and after ultracentrifugation. Electroporation of exosomes using research buffers includes a second washing step, which can result in the loss of at least 50% of the sample.

[0040] [Figure 10]Figures 10A-10D: In vivo distribution of pre-labeled MSC-derived exosomes produced in a bioreactor and injected into mice. Fluorescence of DIR-labeled MSC exosomes 6 hours after intraperitoneal (Figures 10A, 10B) or intravenous (Figures 10C, 10D) administration of 8 × 10⁹ labeled exosomes in WT nude mice. (A, C) Dissected organs. (B, D) Dissected organs without spleen and liver. [Modes for carrying out the invention]

[0041] In order to formulate exosomes for human therapeutics, several aspects of their manufacture may be GM Exo compliant with P (good manufacturing practice) standards Large-scale production of somes requires careful consideration. Therefore, this study is GMP compliant. This concerns innovative procedures for generating xosomes and related systematic analyses. .

[0042] Therefore, in certain embodiments, this disclosure may be functionally closed off, such as in the Terumo cell proliferation system. Using chained bioreactors, exosomes and other components are extracted from mesenchymal stromal cells (MSCs). This provides efficient and clinically relevant strategies for producing EVs. The method involves a short period of time, such as about 3 weeks, with at least 10 x 10 12 It is possible to produce EV Yes, it is possible. The method of the present invention allows for the large growth of clinical-grade extravasation cells in a functionally closed serum-free system. This includes large-scale production and isolation. EVs are manufactured from bone marrow-derived MSCs, etc., in clinically appropriate doses. It will be done.

[0043] In a preferred embodiment, the entire method is serum-free, and therefore, exosomes in serum or The contamination of these MSC-derived EVs is essentially absent. Specifically, this method is used in bioreactors. This may include the use of human platelet lysates (PLT) in the initial culture of MSCs. The researchers found that when MSCs are cultured to about 80-90%, for example, about 85% confluence, E We found that efficient production of V can be obtained. Therefore, MSCs are then acclimatized to include EV. Before collecting the culture medium, allow it to stand for approximately 24-72 hours (for example, approximately 24 or 48 hours) with PLT. The medium may be switched to a non-acclimated medium. The acclimatization medium fraction is collected approximately every 48 hours for the isolation of extracellular viable cells (EVs). It may be frozen at approximately -80°C or similar temperatures. The acclimatization medium fraction can be refrigerated approximately 4 to 10 times, for example, about 5 It can be collected 6, 7, or 8 times, especially about 6 times. Therefore, to the bioreactor The period from sowing to final harvesting of MSCs can range from approximately 15 to 30 days, for example, around 20 days. Each conditioned medium fraction contains at least 1 × 10 11 exosomes, for example, at least 9 × 1 0 11 , especially about 3 x 10 12 It can contain exosomes.

[0044] Preferably, before isolating EVs, the collected culture medium fraction is thawed and pooled. The isolation of the compound is performed by the first centrifugation step (e.g., 1,000 g), followed by ultracentrifugation (e.g., approximately It may contain 100,000g. 3 rounds are required to produce the exosome pellet. Any multiple rounds of ultracentrifugation may be performed. In some embodiments, exosome pellets The net is resuspended in a GMP-compliant buffer such as PLASMALYTE-A(registered trademark). The exosomes may also be subjected to further filtration, such as through a 0.2 μm filter, before ultracentrifugation. Good. This method is 9-10 x 10 12 or more exosomes, for example, 15 x 10 1 2This can lead to the generation of total exosomes.

[0045] Furthermore, exosomes contain therapeutic agents (e.g., cytokines, chemotherapy agents, or nucleic acids). It may be included. Therefore, exosomes produced by this method can be included in PLASMAL By electroporation in an FDA-approved buffer such as YTE-A(registered trademark) A method for mounting is provided. Electroporation is a flow-through electroporation. Poration systems (e.g., 4D-Nucleofactor LV Large) This can be done using a Scale Transfection System (Lonza). Each electroporation run must be at least 2 × 10 12 Contains exosomes It is possible. Exosomes can carry nucleic acids such as siRNA. Buffering The solution is FDA approved for use in patients, is sterile, and is non-pyrogenic. Because it can be injected directly into the body, a washing step to replace the buffer solution before administration to the patient is not necessary. Therefore, conventional methods can result in the loss of approximately 50% of exosomes. There is no exosome loss from the additional washing step. This buffer is electroporated This can maintain the integrity of exosomes after the initial reaction.

[0046] Furthermore, siRNA is used for the treatment of immune-related diseases and cancer in patients. Methods for using exosomes, such as the exosomes described herein, are provided herein.

[0047] [I.Definition] When used herein, “essentially does not contain” with respect to a particular ingredient is used herein. None of the specific components are intentionally included in the composition, and / or are contaminants. It is used to mean that it exists only or in trace amounts. Therefore, the intent of the composition The total amount of specific components resulting from the absence of contamination is well below 0.05%, preferably 0.0 It is less than 1%. The most preferable method is to detect the amount of a specific component using standard analytical methods. It is not a suitable composition.

[0048] Here, "a" or "an" can mean one or multiple, but here In the context of claims, when used in conjunction with "includes," "a" or "an" means one or more. It may happen.

[0049] The use of the term "or" in the claims explicitly indicates that it refers only to substitutes. Unless otherwise indicated, it is used to mean "and / or", but this disclosure does not imply substitutes and While supporting the definition that refers only to "and / or", the term "another" as used herein means at least The latter or more may also be used, and the terms "about," "substantially," and "almost" are all related. Generally, this means plus or minus 5% of the stated value.

[0050] "Extracellular vesicles" and "EV" are classified as exosomes, exosome-like vesicles, Ectosomes (resulting from vesicle budding directly from the cell membrane), microparticles, microvesicles, secretory micromicrobes Robesicles (shedding microvesicles; SMV), nanoparticles and Furthermore, it includes (large) apoptotic vesicles or bodies (resulting from cell death) or membrane particles. These are cell-derived and cell-secreted microvesicles.

[0051] As used herein, the terms “microvesicle” and “MV” typically The diameter is approximately 100 nm to 1,000 nm, or approximately 100 nm to 400 nm in plasma. It refers to a larger extracellular vesicle or structure surrounded by a phospholipid bilayer. Robesicle / MV is regulated by cell membrane budding and bleb formation. It is formed by the release of these substances.

[0052] Within the class of extracellular vesicles, the important component is the "exosome" itself, which is directly The diameter is preferably about 40 to 120 nm (for example, 50 to 100 nm), and the film properties are Vesicles (i.e., exocytotic fusion, or "exocytes" of the polyendoplasmic reticulum (MVB)) Small cells that arise from endocytosis, surrounded by a phospholipid bilayer of endocytosis origin. Exosomes are cells. Exosomes can be isolated from any suitable biological sample of mammalian origin. This often includes whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bone marrow, and cultures. Mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and uninduced pluripotency) Stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells These include cells, hematopoietic stem cells, pancreatic stem cells, white and brown adipose tissue progenitor cells, etc. Not limited to. As those skilled in the art will understand, cultured cell samples (excluding exosomes) The cells are placed in a suitable culture medium (using serum). Exosomes have surface markers such as tetraspanins, e.g., CD9, CD37, CD 44, CD53, CD63, CD81, CD82, and CD151; Integrins, ICAM-1, EpCAM, Annexin, TSG101, ALIX Which targeting marker or membrane fusion marker; and Rab5b, HSP70, LAMP2(l (ysosome-associated membrane protein) and LI MP (lysosomal integral membrane protein) Which other exosome transmembrane proteins It includes specific surface markers not found in other vesicles.

[0053] The term “mesenchymal stem cell” or “MSC” as used herein refers to the ability to regenerate, And especially large phenotypic diversity including connective tissue, bone marrow stroma, adipocytes, dermis and muscle This refers to pluripotent somatic stem cells derived from the mesoderm that possess the ability to differentiate and produce offspring cells. MSCs are generally negative for the markers CD19, CD45, CD14, and HLA-DR. The person is sexually active and positive for markers CD105, CD106, CD90, and CD73. The MSC has a cell marker expression profile characterized by any type of combination. MSCs can be isolated from tissue. Generally, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. It is possible. In certain embodiments, MSCs are bone marrow-derived stem cells.

[0054] MSCs derived from exosomes may be autologous, homogeneous, or heterogeneous. In this case, the term "self" refers to the donor of the MSC and the exosomes derived from the said MSC. This means that the recipients of the isolated exosome population are the same subject. The term "homogeneous" refers to MSC donors and exosomes (or isolated exosomes) derived from said MSCs. This means that the recipient of the xosome population is a different subject. The term "heterogeneous" is M SC donors and exosomes (or isolated exosome populations) derived from the aforementioned MSCs. ) means that the recipient is of a different species. In a particular embodiment, MSCs derived from xosomes are allogeneic.

[0055] The terms “adipose tissue-derived stem cells” or “ASC” are used herein to mean adipose tissue-derived stem cells. This refers to MSCs derived from tissue. ASCs are defined by methods known in the field (for example, "human adipose mesenchymal stem cells"). Stem cells can be isolated from adipose tissue by the methods described below under "Stem cell isolation and proliferation". "Adipose tissue" refers to any type of adipose tissue. Adipose tissue can be found, for example, subcutaneous tissue, omentum / visceral tissue. brown or white adipose tissue originating from the breast, gonads, peristomes or other adipose tissue areas. This may be done. Preferably, the adipose tissue is subcutaneous white adipose tissue. The adipose tissue is primary cell culture It may include substances or immortalized cell lines. Adipose tissue may originate from any organism that possesses adipose tissue. In some embodiments, the adipose tissue is mammalian, and in further embodiments, the adipose tissue is It is a human being. A convenient source of adipose tissue is liposuction surgery. However, adipose tissue It is understood that neither the source nor the method of isolating the adipose tissue is important to the present invention. In certain embodiments, the ASC is isolated from the liposuctioned material of the subject.

[0056] MSC is any animal, preferably a non-primate (e.g., cattle, pigs, horses, cats, dogs, It may originate from mammals, including rats or mice and primates (e.g., monkeys or humans). In certain embodiments, the MSC is human.

[0057] The term "functionally closed" means that the entire system is hermetically sealed, or collected By providing sterile barrier filters at all connections to the system, the sterility of the fluid is ensured. This refers to a system that is sealed to guarantee safety.

[0058] The term "bioreactor" refers to a closed, sterile system that provides nutrients to cells, removes metabolites, and delivers them to cells. This refers to a large-scale cell culture system that provides a physiologically and chemically appropriate environment for cell proliferation. In this context, biological and / or biochemical processes are conducted under monitored and controlled environmental conditions. It occurs under various conditions and operating conditions (e.g., pH, temperature, pressure, nutrient supply, and waste removal). According to this disclosure, a basic class of bioreactor suitable for use with the method is , including hollow fiber bioreactors.

[0059] The term "hollow fiber" refers to a fiber that delivers nutrients (in solution) to cells contained within a bioreactor. For use in the process of removing waste materials (in solution) from cells contained within the bioreactor. To remove, hollow structures (of any shape) containing holes of a specified size, shape, and density. It is intended to include. For the purposes of this disclosure, the hollow fibers are reabsorbable or non-reabsorbable. It may be constructed from materials. Fibers include, but are not limited to, tubular structures.

[0060] As used herein, the terms “patient” or “subject” refer to a living mammalian organism. For example, humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, guinea pigs , or their transgenic species. In certain embodiments, the patient or subject is a primate. It is a type of disease. Non-limited examples of human patients include adults, adolescents, infants, and fetuses.

[0061] "Treatment" or "the act of treating" refers to (1) the subject experiencing or displaying the pathology or symptoms of a disease. or inhibiting the disease in the patient (e.g., stopping the further development of pathology and / or symptoms) (2) Improvement in the subject or patient who experiences or displays the pathology or symptoms of the disease (for example) (3) to reverse the pathology and / or symptoms of the disease, and / or to experience or express the pathology or symptoms of the disease. This includes causing any measurable reduction in the disease in the subject or patient being shown.

[0062] The term "effective" is used in this specification and / or in the claims. It means sufficient to achieve the desired, expected, or intended result. "Effective dose," "therapeutic effective dose," or "pharmaceutical effective dose" refers to the amount of a compound used to treat a patient or subject. When used in pulse therapy, when administered to a subject or patient to treat or prevent a disease. This means an amount of the compound that is sufficient to have an effect on the treatment or prevention of such a disease. ru.

[0063] As used herein, the term "cancer" refers to solid tumors, metastatic cancers, or non-metastatic cancers. It can be used for the following purposes. In certain embodiments, cancer can be found in the bladder, blood, bone, bone marrow, brain, breast, and kidneys. Intestines, esophagus, duodenum, small intestine, large intestine, colon, rectum, Anus, gums, head, kidneys, liver, lungs, nasopharynx, neck, ovaries, pancreas, prostate, skin, stomach, sperm It may originate from the nest, tongue, or uterus.

[0064] The terms "contacted" and "exposed" refer to the treatment agent being applied to target cells. To describe the process by which a substance is delivered to or directly juxtaposed with target cells, as described herein. It is used. For example, one or more drugs are used to kill cells in order to achieve cell toxicity. It is delivered to the cell in an amount effective enough to prevent cell division.

[0065] A patient's effective response or "responsiveness" means that there is a risk of disease or disability, or This refers to the clinical or therapeutic benefits provided to patients suffering from this condition. Such benefits include: Cellular or biological response, complete response, partial response, stable disease (without progression or relapse) or a response that may be accompanied by a relapse later. For example, a good response is observed in patients diagnosed with cancer. This may result in a reduction in tumor size or progression-free survival.

[0066] As used herein, “therapeutic agent” is intended to obtain therapeutic benefits for a disease or health-related condition. This refers to any drug that can be administered to the target. For example, nanoparticles containing therapeutic agents are To reduce tumor size, reduce or inhibit the local invasiveness of tumors, or reduce metastasis It may be administered to the target individual to reduce the risk of occurrence.

[0067] As used herein, “diagnostic agent” refers to an agent used to diagnose a disease or health-related condition in a subject. This refers to any drug that can be administered to a subject for a specific purpose. Diagnosis involves determining whether or not a disease is present. This includes determining whether the condition is progressing or if there are any changes in the patient's symptoms. ru.

[0068] Therapeutic or diagnostic agents include small molecules, peptides, proteins, polypeptides, antibodies, and antibody flare. It may be a compound, DNA, or RNA. In certain embodiments, the therapeutic or diagnostic agent is s It is iRNA.

[0069] As used herein, "nucleic acid" generally refers to DNA, RNA, or any other substance containing nucleic acid bases. This refers to derivative or analogous molecules (i.e., strands). Nucleic acid bases, for example, in DNA. Naturally occurring purines or pyrimidine bases can be found (e.g., adenine "A", guanine). Naturally occurring purines found in RNA ("g", thymine "T", or cytosine "C") or RNA or containing pyrimidine bases (e.g., A, G, uracil "U", or C). The term "nucleic acid" is "Oligonocytes" and "polynucleotides" are variations of the term "nucleic acid," respectively. The term "oligonucleotide" includes "othiocyanate." The term "oligonucleotide" refers to a molecule with a length of 3 to approximately 100 nucleic acid bases. It refers to a child. The term "polynucleotide" refers to a molecule with a length greater than approximately 100 nucleic acid bases. It also refers to a single molecule.

[0070] These definitions refer to single-stranded or double-stranded nucleic acid molecules. Double-stranded nucleic acids are perfectly complementary. Formed by a compound, but in some embodiments, the double-stranded nucleic acid is partially or substantially They can be formed by complementary bonds. Therefore, nucleic acids typically contain molecules, This may include double-stranded molecules containing one or more complementary chains or "complements" of a specific sequence. To use this method, single-stranded nucleic acids are denoted by the prefix "ss", and double-stranded nucleic acids by the prefix "ds". It is possible.

[0071] As used herein, "nucleotide" refers to a nucleoside that further includes a "skeletal structure". The skeletal structure generally consists of nucleotides, or nucleic acids, which are combined into another molecule containing nucleotides. It is covalently linked to another nucleotide that is formed. The "skeletal structure" typically includes a phosphorus portion covalently attached to a pentose sugar. The partial bond typically occurs at either the 3′- or 5′-position of the pentose. However, However, the nucleotides include derivatives or analogs of naturally occurring pentose or phosphorus moieties. In this case, other types of connections are known in the art.

[0072] Nucleic acids may contain nucleic acid bases, nucleic acid base linker moieties, and / Alternatively, it may include derivatives or analogues of the skeletal portion, or may be composed entirely of them. As used in the specification, "derivative" refers to a naturally occurring molecule with a different chemical composition. This refers to a modified or altered form, and is a term that is either "mimic" or "similar." The "body" (analog) may be structurally similar to a naturally occurring molecule or part, and This refers to molecules that do not necessarily have to be similar but have similar functions. A "moiety" generally refers to a smaller chemical or molecular structure of a larger chemical or molecular structure. This refers to molecular components. Nucleic acid bases, nucleosides, and nucleotide analogs or derivatives are included in this technology. It is well known in the field of technology.

[0073] The term "siRNA" (short interfering RNA) refers to a short double-stranded RNA complex (typically 19 This refers to a length of approximately 28 base pairs. In other words, siRNA is a double-stranded molecule containing two nucleotide chains. It is a nucleic acid molecule, and each chain consists of approximately 19 to 28 nucleotides (i.e., approximately 19, 20, 21, It has 22, 23, 24, 25, 26, 27, or 28 nucleotides. This complex Often, it contains a 3'-overhang. siRNA is prepared using techniques known to those skilled in the art. siRNA can be manufactured, and a wide variety of siRNAs are available in Integrated DNA Techno. From suppliers like Logies, Inc. (Coralville, Iowa) to commercial It is readily available. In one embodiment, 2′-O- Methyl-modified siRNA double helix can be incorporated into nanoparticles, and the 2′ on the antisense strand -O-methyl modification eliminates off-target effects and minimizes nonspecific immune responses, si Improves RNA stability.

[0074] MicroRNAs (miRNAs) are proteins that are coded via the 3′-UTR element. Short non-coding RNAs can target and effectively silence genes. Yes, miRNAs are approximately 21-22 nucleotides long and are non-protein-coding genes. It arises from a longer precursor that is transcribed from.

[0075] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a condition in which the immune response is impaired in the development of a disease. Alternatively, it refers to disorders that play a role in the progression of the disease. Immune-mediated diseases include autoimmune diseases and allotransfer diseases. This includes graft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0076] An "autoimmune disease" is a condition in which the immune system reacts to antigens that are part of the normal host (i.e., autoantigens). This triggers an immune response (e.g., a B-cell or T-cell response), resulting in tissue damage. This refers to a disease in which autoantigens can originate from host cells or typically form colonies on the mucosal surface. It can originate from symbiotic organisms such as microorganisms (known as symbiotic organisms).

[0077] As used herein, the term "confluence" refers to the hollow fibers of a bioreactor. Confluence refers to the percentage of cells covering the surface of a fiber or other material. This can be measured by the levels of glucose and / or lactose in the culture medium.

[0078] [II. Exosome Production] Specific embodiments of this disclosure relate to bioreactors, particularly hollow fiber bioreactors and other similar devices. Large-scale production of clinical-grade extracellular molecules, particularly exosomes, through the use of a fully closed system. Regarding the method for this, importantly, the entire process of exosome production is used for subsequent therapeutic agents. Including the inclusion of [unclear / unclear], it may be serum-free.

[0079] (A. Mesenchymal stem cells) The cells used for EV production may be MSCs such as adipose-derived or bone marrow-derived MSCs. In certain embodiments, MSCs are human MSCs that may be self-derived or allogeneic.

[0080] MSCs have approximately 100-1,000 cells / cm² 2 For example, about 150 cells / cm² 2 , about 20 0 cells / cm 2 , about 250 cells / cm 2 , about 300 cells / cm 2 For example, about 350 cells / cm 2 For example, approximately 400 cells / cm² 2 For example, approximately 450 cells / cm² 2 For example, about 500 fine cells / cm 2 For example, approximately 550 cells / cm² 2 For example, approximately 600 cells / cm² 2 For example, about 65 0 cells / cm 2 For example, approximately 700 cells / cm² 2 For example, approximately 750 cells / cm² 2 For example, about 800 cells / cm 2 For example, approximately 850 cells / cm² 2 For example, approximately 900 cells / cm² 2 ,example Approximately 950 cells / cm² 2 , or approximately 1,000 cells / cm² 2 Seed into the bioreactor at this density It can be seeded. In particular, the cells can grow at a rate of approximately 400-500 cells / cm². 2 For example, about 450 fine cells / cm 2 It can be seeded at this cell density.

[0081] The total number of cells seeded in the bioreactor is approximately 1.0 × 10⁻⁶. 6 ~Approx. 1.0×108 Cells, for example, about 1.0 × 10 6 ~5.0.0×10 6 , 5.0×10 6 ~1.0×10 7 , 1.0 × 10 7 ~5.0×10 7 , 5.0×10 7 ~1.0×10 8 It could be a cell In certain embodiments, the total number of cells seeded in the bioreactor is approximately 1.0 × 10⁻⁶. 7 ~about 3.0×10 7 For example, approximately 2.0 × 10 7 It is a cell.

[0082] The cells can be seeded in any suitable cell culture medium, many of which are commercially available. Examples of culture media include DMEM, RPMI, MEM, Media 199, and HAMS. This includes. In one embodiment, the culture medium is αMEM medium, particularly αMEM medium supplemented with L-glutamine. It is EM. The culture medium contains growth factors, cytokines, hormones, or B27, antibiotics, and One or more of the following can be added: tamine and / or low molecular weight drugs. In particular, the culture medium is free. Serum is also acceptable.

[0083] In some embodiments, cells can be incubated at room temperature. The field is heated and can have an atmosphere with approximately 5% CO2 and approximately 1% O2. In the embodiment, the CO2 concentration may be about 1-20%, 2-10%, or 3-5%. In some embodiments, the O2 concentration is about 1-20%, 2-10%, or 3-5%. That's good too.

[0084] In certain embodiments, cells are seeded and cultured in serum-free medium. The medium contains platelet lysates. In particular, it can replenish human platelet lysates (PLT). PLT is approximately 1-10%. For example, about 1-4%, 2-5%, 3-6%, 4-7%, 5-8%, or 6-10%, for example It may be present in the culture medium at a concentration of approximately 4%, 5%, or 6%, particularly about 5%.

[0085] MSC is used for approximately 5-10 days after sowing, for example, about 5, 6, 7, 8, 9, or 10 days, especially Initial culture can be performed in a bioreactor for approximately 7, 8, or 9 days. Specifically, MSC has a confluence of approximately 75-95%, for example, approximately 75-80%, 80-85%, and 8 Up to 5-90% or 90-95% confluence, especially around 85-90%, for example 85 Up to 86%, 87%, 88%, 89%, or 90% confluence, including PLT. Initial culture can be performed in culture medium. Once the cells reach the intended confluence, The cells can be transferred to a medium that does not contain PLT. Beforehand, it can be washed at least once with a buffer such as PBS.

[0086] Cell culture in a medium without PLT may be affected by exosomes that may be present in PLT. Used to prevent contamination or dilution of MSC-derived exosomes. In several embodiments This involves centrifuging the PLT to remove exosomes, and then obtaining exosome-free PLT. It can be obtained.

[0087] The cells were left in a PLT-free culture medium for approximately 8 to 100 hours, for example, 12 to 72 hours, for example. Approximately 12-15, 15-20, 20-25, 25-30, 35-40, 40-45, 45 It can be cultured for ~50, 50~55, 55~60, 65~70, or 70~72 hours. In particular, cells can be incubated in a medium without PLT for approximately 24 to 48 hours, for example, approximately 48 hours. During this time, they can be cultured before exosome collection.

[0088] (B. Bioreactor) Bioreactors include static bioreactors, stirred flask bioreactors, and rotating wall bioreactors. Includes container bioreactors, hollow fiber bioreactors, and direct perfusion bioreactors. They can be classified according to general categories. In a bioreactor, cells are migratory. They can be released or immobilized and seeded onto a porous three-dimensional scaffold (hydrogel).

[0089] Hollow fiber bioreactors can be used to promote mass transfer during culture. Hollow fiber bioreactors have a typical molecular weight cutoff of 10-30 kDa (MW). Based on hollow fibers, which are small, semi-permeable capillary membranes arranged in parallel within the CO) range. This is a D cell culture system. These hollow fiber membranes are often tubular polycarbonate cells. They are bundled and housed within the cartridge to form a hollow fiber bioreactor cartridge. The tridge contains two compartments: the capillary (IC) space within the hollow fiber and the hollow fiber There is an extracapillary (EC) space surrounding it, and these compartments have an inlet port and an outlet A tread port has also been installed.

[0090] Thus, in this disclosure, the bioreactor may be a hollow fiber bioreactor. In a hollow fiber bioreactor, cells are embedded within the lumen of the fiber, and the culture medium is located in the space outside the lumen. It can perfuse gas and culture medium through hollow fibers, or alternatively, it can provide perfusion of gas and culture medium through fine The cells can proliferate in the extraluminal space. A hollow fiber bioreactor suitable for this disclosure is This is publicly known in the art, and Cardian(Terumo)BCT Quantum A Cell Expansion System can be included, but is not limited to this. do not have.

[0091] Hollow fibers are suitable for nutrient delivery and waste removal in bioreactors. The hollow fiber may be of any shape, for example, circular or tubular. Alternatively, it may be in the form of a concentric ring. The hollow fiber is composed of an absorbent or non-absorbent membrane. It may also be done. For example, suitable components of hollow fibers include polydioxanone, polylacti Polyglycolic acid, polylactic acid, polyglycolic acid / trimethylene acid - Bonate, cellulose, methylcellulose, cellulose polymer, cellulose ester , regenerated cellulose, Pluronic®, collagen, elastin, and these A mixture is one example.

[0092] The bioreactor may be primed before seeding cells. Priming is performed using PB This may include flushing with a buffer such as S. Priming may also involve fibrous priming. By coating the bioreactor with extracellular matrix proteins such as nectin... It may also include the following. The bioreactor is then washed with PLT medium (e.g., αMEM). It is possible.

[0093] (C. Collection of acclimatization medium) The acclimatization medium from cells cultured in PLT-free medium should be changed every 8-100 hours, for example. For example, every 12 to 72 hours, for instance, approximately 12-15, 15-20, 20-25, 25-30, 3 5-40, 40-45, 45-50, 50-55, 55-60, 65-70, or 70- Samples can be collected every 72 hours. In particular, the acclimatized culture medium fraction can be collected every 24-48 hours. It can be collected.

[0094] The acclimatization medium fraction should be approximately 100-500 mL, for example, approximately 100-150 mL, 150-200 mL. 250-300, 350-400, 400-450, or 450-500 mL, especially about It can contain a volume of 250 mL. The fraction is collected in a sealed bag and the exosomes are isolated. It can be frozen and stored at temperatures between approximately -70°C and -90°C, for example, at approximately -80°C.

[0095] In a particular embodiment, the acclimatization medium is prepared at least four times for each run, for example, 4 to 10 times. Samples are collected several times, especially 5, 6, 7, 8, or 9 times, especially 6 times. Each fraction is then collected, for example, at 4°C. It can be thawed overnight, for example, at 2-6°C for about 10-20 hours.

[0096] (D. Isolation of exosomes) Next, the pooled conditioned medium fraction can be used for exosome isolation, particularly at 4°C. The acclimatization media are at approximately 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, and 18°C. It can be centrifuged at temperatures of 20°C, 22°C, 24°C, or 26°C, and in one embodiment The conditioned medium is centrifuged at a temperature of approximately 4°C. Exosome isolation is performed as follows: This can be carried out by methods known in the art. Preferably, the isolation of exosomes is Furthermore, similar to the exosome production described above, this process takes place in a closed system. This involves a pump and This can be achieved by using a sealed tube.

[0097] Isolation involves a centrifugation step to remove large fragments, followed by filtration, and one or more The isolation method may include an ultracentrifugation step. Therefore, it may be carried out multiple times (for example, three times).

[0098] The centrifugal separation process takes approximately 5 to 25 minutes for a volume of about 500 to 2,000 g (for example, about 1,000 g). This may include centrifugation (for example, for about 15 minutes). The acclimatization medium should be about 1,000 g, 2,000 0g, 4,000g, 6,000g, 8,000g; 10,0000g; 12,00 It can be centrifuged at 0g, 14,000g, 16,000g, or 18,000g. The separation period is typically 10-30 minutes, 12-28 minutes, 14-24 minutes, or 15-20 minutes. It is also permissible. As those skilled in the art will understand, a suitable commercially available laboratory centrifuge, for example, TH ERMO-SCIENTIFIC (trademark) or COLE-PARMER (trademark) It is used to perform the centrifugal separation process. In particular, centrifugal separation is used in Cobe 2991 Cel This can be done in a closed system such as a Processor (Terumo). Low-speed centrifugation The separation process is repeated at least twice to remove living cells, dead cells, and larger cellular debris. It can be done.

[0099] The filtration process removes larger fragments such as microvesicles, with a size of 0.1-0. Submicron filters such as 3-micron filters, or 0.2-micron filters. This may include the use of a filtration bag having -. The supernatant is then directly connected to a tube. Using this line, the fluid can be transferred to a tube (for example, a polycarbonate tube). Filtration can be repeated more than once. Filtration can be performed using filters of the same size, for example, 0 This can be done by passing the material through a 0.2 micron filter one or more times. , a filter of the same or decreasing size (for example, passing through a 40-50 micron filter) One or more passes, one or more passes through a 20-30 micron filter, 10-20 micron One or more passes through a ron filter, and one or more passes through a 0.2-10 micron filter. Filtration may be performed using two or more filters, such as a pass. Suitable filters for use include those with 0.45 and 0.22 micron diameters. It includes the function.

[0100] Ultracentrifugation is performed at 75,000 to 150,000 g, for example, 100,000 to 170,000 g. 00g, for example, about 100,000g, about 2-6 hours, for example, 1-3 hours, for example, This can be done in about 4 or 5 hours. To perform this step, use THERMO-SC A commercially available ultracentrifuge such as IENTIFIC or Beckman can be used. Specifically, ultracentrifugation is not limited to, but includes the Type 45 Ti rotor (Beckm This can be performed using any closed-system centrifugation method, such as an an-Coulter. The ultracentrifugation process may be optionally repeated, for example, two or more times, to improve the results. Good. Remove the exosome-containing pellet from the supernatant using established techniques, and Resuspend in a suitable physiological solution.

[0101] As those skilled in the art will understand, exosomes from either the centrifugal separation or ultracentrifugation process The pellets should be placed in a suitable physiological solution (e.g., sterile PBS, sterile 0.9% saline solution, or sterilized saline solution). The bacteria can be washed during the centrifugation process using a 0.9% physiological saline buffer containing bacterial carbohydrates. ru.

[0102] After centrifugation, the solution is removed and the exosomes are resuspended in a suitable buffer such as PBS. The pH of the buffer may be any pH that is compatible with the sample, but a typical range is 6 - 8. The buffer has a pH of 4 - 10, 4 - 6, 4 - 8, 6 - 10, 6 - 8, or 8 - 10. In particular, the exosome pellet can be resuspended in a buffer of clinical grade, for example, at a physiological pH of about 7.4 (e.g., PLASMALYTE - A®). The volume of the buffer can be from about 0.01 volume to about 0.09 volume, about 0.02 volume to about 0.08 volume, about 0.03 volume to about 0.07 volume of the precipitation solution. The collected exosomes can be used immediately for electroporation or treatment, etc., or can be frozen and stored, for example, at - 20 °C for later use.

[0103] As used herein, analysis includes any method that enables direct or indirect visualization of exosomes and may be in vivo or ex vivo. For example, analysis includes, but is not limited to, ex vivo microscopy or cytometry detection and visualization of exosomes bound to a solid substrate, flow cytometry, fluorescence imaging, etc. In an exemplary embodiment, exosomes derived from cancer cells are detected using an antibody against glypican 1, then bound to a solid substrate, and visualized using microscopy or cytometry detection. Exosomes can be analyzed by flow cytometry expression of exosome surface markers: CD63, CD47, CD9, and CD81, and / or by transmission electron microscopy (TEM). Furthermore, nanoparticle tracking analysis and micro BCA assay can be used to quantify exosomes.

[0104] Therefore, the period from seeding of MSCs into the bioreactor to final collection is about 15 - 30 days, such as 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, such as about 1 9 or 20 days. Each acclimation medium fraction contains at least 1×10 12 exosomes, such as at least 2×10 12 exosomes, particularly about 3×10 12 exosomes. This method can produce at least 10×10 12 exosomes, such as at least 11× 10 12 exosomes, such as at least 12×10 13 exosomes, such as at least 13×10 12 exosomes, such as at least 14×10 12 exosomes , such as at least 15×10 12 exosomes, such as at least 16×10 12 exosomes, such as at least 17×10 12 exosomes, such as at least 18 ×10 12 exosomes, such as at least 19×10 12 exosomes, or for example at least 17×10 12 exosomes can result in the production of all exosomes.

[0105] (E. Loading of Exosomes) The exosomes produced by the method of the present invention can be loaded with cargo such as a therapeutic or diagnostic agent. Examples of cargo that can be delivered using the exosomes of the present invention are nucleic acid molecules such as DNA (both nuclear DNA and mitochondrial DNA), mRNA, tRNA, mi RNA, and RNA such as siRNA, aptamers and other nucleic acid-containing molecules, peptides, RNA, and siRNA, aptamers, and other nucleic acid-containing molecules, peptides, Exozoites such as proteins, lipozynes, carbohydrates, polymers, therapeutic drugs, and small molecules The formula contains exogenous substances that do not exist naturally (derived from external sources), but is not limited to these. It will not be done.

[0106] In one embodiment, the isolated exosome of the present invention is a compound having a secondary structure (for example) (For example, miRNA, mRNA, proteins / peptides), as well as large compounds (e.g., 20 Base pairs exceeding a certain number, for example, more than 50 base pairs, or more than 100 base pairs, pept It is particularly useful for the delivery of nucleic acid molecules (including proteins).

[0107] Cargo uses methods established in the field for introducing cargo into cells. It can be introduced into xosomes. Therefore, for example, a voltage in the range of approximately 20-1000 V / cm The cargo can be introduced into the exosome using applied electroporation. Transfection using cationic lipid-based transfection reagents is also possible. Furthermore, it can be used to introduce cargo into exosomes. Proper transfection Examples of reagents include Lipofectamine MessengerMAXTm Tra nsfection Reagent, Lipofectamine RNAiMAX Transfection Reagent, Lipofectamine 3000 Transfection Reagent or Lipofectamine LTX Examples include Reagent with PLUSTM Reagent, but these are not limited to these. It is not determined. The appropriate amount of transfection reagent is used for cargo loading, but The amount may vary depending on the reagent, sample, and cargo. For example, Lipofecta mine MessengerMAXTm Transfection Reagent When using, use an amount in the range of approximately 0.15uL to 10uL, 100ng to 250 0 ng of mRNA or protein can be loaded into the exosome. Alternatively, for example, The use of cell-penetrating peptides for protein delivery also introduces cargo into exosomes. It can be used for that purpose.

[0108] In a specific embodiment, the cargo, such as nucleic acids like siRNA, is electroporated into the exosome. Equation (for example, in an FDA-approved buffer (e.g., PLASMALYTE-A)) It is installed using electroporation. Electroporation systems (e.g., 4D-Nucleofactor LV L) Runs on Large Scale Transfection System (Lonza) Each electroporation run is at least 2 × 10 12 Exos It may contain [unclear]. The buffer is FDA approved for use in patients and is sterile. Furthermore, since it is non-pyrogenic and can be injected directly into the patient, the buffer solution should be mixed before administration to the patient. The washing process required for replacement is unnecessary. Therefore, approximately 50% of the exosomes are lost. Exosome loss due to additional washing steps, as in conventional methods, can result from the loss of exosomes. do not have.

[0109] In certain embodiments, the therapeutic agent is siRNA, shRNA, plasmid, mRNA, mi RNA, or RNA such as ncRNA, especially siRNA or miRNA therapeutics Yes. The miRNA can be a miRNA mimic or a miRNA precursor. The RNA carried by the exosome can have a length of less than 100 nucleotides, such as less than 75 nucleotides , particularly less than 50 nucleotides. For example, the RNA can be about 10-100 nucl eotides (e.g., 20-50 nucleotides, particularly 10-20, 15-25, 20-30, 25-35, 30-40, or 45-50 nucleotides) in length.

[0110] The RNA may or may not be modified. The RNA can include modifications of one or more nucl eotides. Such modifications can include the addition of non-nucleotide substances (e.g., at the end or within the RNA (at one or more nucleotides of the RNA)). In certain embodiments , the RNA molecule includes a 3'-hydroxyl group. The nucleotides in the RNA molecules of the present disclosure can also include non-standard nucleotides, including non-natural nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide can contain a modified backbone, such as phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or can include non-natural nucleoside linkages. Further modifications of siRNA (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate nucleotide linkages, and inverted deoxyabasic residue incorporation ) are found in U.S. Patent Publication No. 20040019001 and U.S. Patent No. 6,673,611. ) It is possible (each of these is incorporated as a reference to the whole). Collectively, All of the above-mentioned modified nucleic acids or RNAs are called modified siRNAs.

[0111] Preferably, RNAi reduces protein expression by at least 10%, 20%, or 30%. Or 40%, more preferably at least 50%, 60%, or 70%, even more preferably It can be reduced by at least 75%, 80%, 90%, 95%, or more. .

[0112] The siRNA used in the methods or compositions described herein is the same as described. The portion of the mRNA sequence that is encoded by the NCBI reference sequence of the gene / protein. It may include. In one embodiment, the siRNA includes a double-stranded portion (double helix). In one embodiment, the siRNA is 20 to 25 nucleotides long. One or both strands independently have one or two nucleotide 3′ overhangs. It contains 9-21 core RNA double helixes. In one embodiment, the overhang is UU. RNA can be 5′ phosphorylated or not, and its effect against nuclease degradation To improve strength and / or resistance, it may be modified with any of the modifications known in the art. In a non-limiting embodiment, siRNA is transfected into one or more cells. It can be administered in this manner. In one embodiment, siRNA delivers double-stranded RNA, including the first and second strands. It can include, where one strand of RNA is part of the RNA transcript of the gene, 80, 85 They are 90, 95, or 100% complementary.

[0113] In one embodiment, the single-stranded component of the siRNA of this disclosure is 14 to 50 nucleotides long. In another embodiment, the single-stranded component of siRNA is 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotide lengths Yes. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 21 nucleos It is the length of the nucleotide. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 22 This is the nucleotide length. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 2 It is 3 nucleotides long. In one embodiment, the siRNA of this disclosure has 28 to 56 nucleotides. He is the chief.

[0114] Target genes are generally polynucleotides containing a polypeptide-coding region, or polynucleotides. Polypeptides have significant role in regulating replication, transcription, translation, or other processes in peptide expression. A nucleotide region, or a region encoding a polypeptide, and a polypeptide that regulates expression This refers to a polynucleotide that includes both a operably linked region and a target gene. It can be chromosomal (genome) or extrachromosomal. It may also be endogenous to the cell. In some cases, it may be an exogenous gene (introduced gene). Exogenous genes are incorporated into the host genome. They may exist on extrachromosomal genetic constructs such as plasmids or cosmids. Furthermore, the target gene may originate from a pathogen, such as a virus, bacteria, fungus, or protozoan. These can infect organisms or cells. The target gene is a retroviral gene. Viral and proviral genes that do not induce an interferon response like those found in genes It's possible. The target gene could be a protein-coding gene, or ribosomal RNA, or a splinter gene. Non-protein encoding genes such as those encoding lysosomal RNA and tRNA. It may also be a gene.

[0115] Any gene expressed within a cell can be targeted. Preferably, the target gene Whether the child is involved in the progression of cellular activity that is important to the disease or of particular interest as a research subject, It is related. Therefore, as an example, the following are used to regulate or reduce the expression of target genes. A class of possible target genes that may be used in the methods of this disclosure: developmental genes (e.g.) Adhesion molecule, cyclin kinase inhibitor, Wnt family member, Wing Ed Helix family member, Hox family member, cytokine / phosphorus Hokines and their receptors, growth factors or differentiation factors and their receptors, neurotransmitters (and its receptor), tumor suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2) b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTEN Rb, Utegrobin, Skp2, BRCA-1, BRCA-2, CHK2, CHKN 2A, DCC, DPC4, MADR2 / JV18, MEN1, MEN2, MTS1, NF 1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1, ras, MMAC1, FCC, MCC, FUS1, Gene 26(CACNA2D2) , PL6, Beta*(BLU), Luca-1(HYAL1), Luca-2(HYA L2), 123F2(RASSF1), 101F6, Gene 21(NPRL2) or Genes encoding SEM A3 polypeptides, pro-apoptotic genes (e.g., C D95, Caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hi d, Bak, MKP-7, PARP, bad, bcl-2, MST1, bbc3, Sax BIK and BID), cytokines (e.g., GM-CSF, G-CSF, IL-1) α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL -15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-2 8, IL-29, IL-30, IL-31, IL-32, IFN-α, IFN-β, IF N-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, TNF-β, PDGF , and mda7), oncogenes (e.g., ABLI, BLC1, BCL6, CBFA1, C BL, CSFIR, ERBA, ERBB, EBRBB2, ETS1, ETS1, ETV6 ,FGR,FOX,FYN,HCR,HRAS,JUN,KRAS,LCK,LYN,M DM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML , RET, SRC, TAL1, TCL3 and YES), and enzyme (ACP-desaturates) -ase and hydroxylase, ADP-glucose pyrophyllase, ATPase, alcohol Dehydrogenase, amylase, amyloglucosidase, catalase, cellulase, Cyclooxygenase, decarboxylase, dextrinase, esterase, DNA and RNA polymerase, galactosidase, glucanase, glucose oxidase, GTPase, helicase, hemicellulase, integrase, invertase, sandworm Lase, kinase, lactase, lipase, lipoxygenase, lysozyme, pectin Stellase, peroxidase, phosphatase, phospholipase, phosphorylase, po Ligalacturonase, proteinase and peptidase, planase (pullanas Examples include es), recombinase, reverse transcriptase, topoisomerase, and xylanase. .

[0116] As those skilled in the art will understand, the exosome of the present invention is used before or after loading cargo. To enhance its usefulness as a vehicle for cargo delivery, it includes a targeted portion. It can be further modified by [doing something]. In this regard, exosomes can be [doing something] in certain cells. It can be manipulated to incorporate entities that specifically target a particular tissue type. This target-specific entity ( For example, peptides that have affinity for receptors or ligands on target cells or tissues are For example, fusion to exosome membrane markers using well-established methods in the field. This allows it to be incorporated into the exosome membrane.

[0117] [III. How to use] In some embodiments, the present disclosure is intended for the delivery of therapeutic agents such as RNAi to cells. The document provides instructions for the use of exosomes. Additional immune cells that can be targeted include dendritic cells, NK cells, and / or B cells. In some embodiments, the therapeutic agent delivered by the exosomes of the present disclosure is a small molecule, It may be a peptide, vaccine, or antigen. The cells may be in vivo or ex vivo. In one embodiment, this involves administering an effective amount of exosomes containing RNAi to cells. A method is provided for delivering RNA to cells. The cells are immune cells such as T cells, or KR cells. Even cancer cells like AS-positive cancer cells can be cancerous.

[0118] In a further embodiment, an effective amount of RNA-encapsulating exosomes is administered to the target. A method is provided for immunostimulating an organism, including the RNA being immunomodulatory RNA. Another embodiment may include administering an effective amount of the exosomes of this disclosure. A method for treating a subject suffering from an illness or disability is provided. In some embodiments, Use of the exosomes of this disclosure for the treatment of disease or disorder, or for immunostimulation of a subject. Use is provided.

[0119] In vivo cells can exist in any subject, such as mammals. For example, the subject could be humans, mammals. Morons, rats, rabbits, dogs, cats, cows, horses, pigs, goats, sheep, primates, or birds It could be a type. In a particular embodiment, the subject refers to a human being. For example, a human being may have a disease. These may include inflammatory diseases, hyperproliferative diseases, infectious diseases, or degenerative diseases. It may be any disease that afflicts the subject. In certain embodiments, the disease may be a disease such as cancer. It is a hyperproliferative disorder. For example, cancers include breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer cells, and liver cancer. , cervical cancer, colon cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, malignancy It may be lymphoma, gastric cancer, pancreatic cancer, testicular cancer, intestinal cancer, lymphoma, or leukemia. Specific implementation Morphologically, the cancer is ovarian cancer.

[0120] Another embodiment includes administering a therapeutically effective amount of the exosomes of this disclosure to the subject. Furthermore, a method for treating immune-mediated inflammatory diseases in subjects suffering from the aforementioned disease is provided. It can be done.

[0121] Cancer can be, but is not limited to, the following histological types: Neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant Large and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphocellular carcinoma; basal cell carcinoma; piloetein carcinoma ;transitional cell carcinoma;papillary transitional cell carcinoma;adenocarcinoma;gastrinoma, malignant;cholangiocarcinoma;hepatocellular carcinoma;liver Combination of cell carcinoma and cholangiocarcinoma; cord-like carcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial Sexual polyposis; solid carcinoma; carcinoid tumor, malignant; acinar cell adenocarcinoma; papillary adenocarcinoma; pigment adenomatous dysplasia sexual carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary Follicular and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; lymphoid carcinoma; sebaceous carcinoma; testicular adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous sac Adenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammation Symptomatic cancer; Paget's disease, breast; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; egg Focal stromal tumor, malignant; thecoma; granulosa cell tumor, malignant; male germ cell tumor (androblastoma), malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant Lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, pheochromocytoma; malignant melanoma Melanin-deficient melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; above Dermocyte melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; lipid Sirosarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed Combined tumor, malignant; Müllerian duct mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner Tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; fetal carcinoma; teratoma, malignant; ovary Goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hematopoietic disease Extraductal dermactoroma, malignant; lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma, malignant; mesenchymal cartilage Tumor; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; supraenamel sarcoma; supraenamel Malignant skin tumors; amelophyte fibrosarcoma; malignant pineal gland tumors; chordomas; malignant gliomas; ependymomas ;astrocytoma; plasma astrocytoma; fibrillar astrocytoma; astroblastoma; glioblastoma; oligodendroglioma Oligodendroglioblastoma; Primitive neuroectoderm; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Olfactory neurogen Malignant tumors; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma tumor; Hodgkin's disease; Hodgkin's granuloma; small lymphocytic lymphoma; diffuse large cell malignant lymphoma Lymphoma; follicular lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant Histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia Disease; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia Diseases; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; And hair cell leukemia.

[0122] In some embodiments, an effective amount of exosomes carrying the therapeutic agent is provided as needed. A method is provided for treating a disease or disorder in a subject, which includes administering the drug to the subject. This disease may be an immune-related disorder such as an autoimmune disease. Examples include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, and parasymptoms. Autoimmune diseases of the kidneys, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, and testicular diseases. Inflammation, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac disease. Spurt dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polycystic neoplasm Lopathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutination Lupus palsy, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - Fibromyalgia, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic hematology Thylem-pneous purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus. Diabetes, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes Diseases such as myasthenia gravis, nephrotic syndrome (minimal change disease, focal segmental glomerulosclerosis, or membranous kidney disease) (e.g., pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, Polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary Biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, Lucoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, systemic lupus erythematosus -Death, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (polyarteritis nodosa, hypertension) Arteritis of the genitals (such as temporal arteritis / giant cell arteritis or herpetiform cutaneous vasculitis), vitiligo, and Wegener's disease. Granulomatous diseases are one example. Therefore, treatment using the methods disclosed herein is possible. Some examples of autoimmune diseases that can cause this include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythema. Todes, type 1 diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing This includes, but is not limited to, spondylitis, vasculitis, or psoriasis. It also includes conditions such as asthma. They may have allergic diseases.

[0123] Treatment outcomes can be predicted and monitored, and / or benefits can be obtained from such treatment. Patients who can be obtained may be identified or selected through the methods described herein.

[0124] Regarding the treatment of neoplastic conditions, the treatment of neoplastic conditions is carried out according to the stage of the neoplastic disease, One or more treatment methods, such as surgery to remove genital tissue, radiation therapy, and chemotherapy. It is performed in conjunction with other treatment regimens, such as the administration of anticancer drugs, for example, therapeutic compositions and chemotherapeutic agents. They can be combined. For example, a patient being treated with such anticancer drugs can also receive radiation therapy. You may undergo medical treatment and / or surgery.

[0125] For the treatment of the disease, the appropriate dose of the therapeutic composition is as defined above. The type of disease, the severity and course of the disease, the patient's medical history and response to medication, and the main treatment It will depend on the doctor's judgment. The medication should be administered appropriately to the patient, either as a single dose or over a series of treatments. It is administered to them.

[0126] Therapeutic and preventive methods and compositions are provided in combination amounts effective in achieving the desired effect. Tissue, tumor, or cells may be provided as one or more compositions or drugs containing one or more agents. It can be brought into contact with a physical preparation, or tissue, tumor, and / or cells in two or more separate locations. It can be brought into contact with the composition or formulation. Combination therapy can be used in combination with chemotherapy, radiotherapy, surgery, or immunotherapy. It is thought that...

[0127] Concomitant administration includes simultaneous administration of two or more drugs in the same dosage form, and simultaneous administration of drugs in different dosage forms. This may include administration and separate administration. That is, the therapeutic composition of the present invention and another therapeutic agent are the same They may be prescribed together in medication form and administered simultaneously. Alternatively, they may be part of the therapeutic composition in question. And another therapeutic agent can be administered simultaneously, where both drugs are present in separate formulations. In another alternative, the therapeutic agent may be administered immediately after another therapeutic agent, or vice versa. It is possible. In a separate administration protocol, the therapeutic composition in question and another therapeutic agent may be administered for several minutes. It can be administered at intervals, at intervals of several hours, or at intervals of several days.

[0128] The exosomes described herein may be used in therapeutic, research, and diagnostic applications. For example, the exosomes described below enhance one or more phenotypic characteristics of a cell. Therefore, it can be added to cell cultures. The exosomes of the present invention possess one or more phenotypic properties of cells. It can be added to cell cultures to inhibit. The exosomes of the present invention can be added to cell cultures. When added, it can provide new phenotypic characteristics to cells.

[0129] (A. Pharmaceutical composition) Certain methods described herein involve the pharmaceutically appropriate treatment of the exosome-containing composition of this disclosure. This relates to a method that includes administering an effective amount.

[0130] As used herein, “pharmaceutically acceptable carrier” is known to those skilled in the art. Sea urchin, any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives ( For example, antibacterial agents, antifungal agents, isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels Materials such as binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof This includes combinations (Remington's, 1990). Any conventional carrier and the active ingredient Except insofar as it is incompatible, it is intended for use in therapeutic or pharmaceutical compositions. Whether the composition used in this context should be administered in solid, liquid, or aerosol form. , and depending on whether it needs to be sterilized for administration routes such as injection, different It may contain a carrier of a certain type.

[0131] The use of such media and agents for pharmaceutically active substances is well known in the field. Except in cases where the conventional medium or drug is incompatible with the active ingredient, the therapeutic composition Intended for use. Co-active ingredients can also be incorporated into the composition, which are described in more detail below. Let's discuss this in detail. For administration to humans, preparations are required by FDA Biomedical Standards. Therefore, it is preferable that the product meets standards for sterility, pyrogenicity, general safety, and purity.

[0132] Compositions containing exosomes may, if necessary, remove unwanted low molecular weight molecules. For wider dialysis and / or easier formulation to the desired vehicle, freeze-drying is possible. It can be dried. In particular, the compositions constituting the exosomes of this disclosure (e.g., PLASMALY TE-A) requires no processing whatsoever and can be injected directly into the target. Next, Sexual compounds are generally prescribed for administration by any known route (e.g., parenteral administration). The method of administration will be discussed in more detail below.

[0133] This disclosure relates to intravascular injection, or any other method as discussed in more detail below. This method intends to use a composition that is a sterile solution for application by road. Those skilled in the art will Proficient in techniques for producing sterile solutions for injection or application via other routes of intent. Sterile injection solutions are prepared by adding the required amount of active compound in a suitable solvent, using species well known to those skilled in the art. It is prepared by incorporating it together with other components.

[0134] The formulation of the composition may vary depending on the route of administration. For parenteral administration in aqueous solution, For example, the solution should be properly buffered, and the liquid diluent should first be sufficiently physiologically charged. It should be made isotonic with saline or glucose. In connection with this, sterile water can be used. The medium will be known to those skilled in the art in light of this disclosure.

[0135] Other pharmaceutically acceptable forms include those prepared for parenteral administration, such as intravenous or intramuscular injection. In addition to formulated compounds, formulations for administration via implantable drug delivery devices, and any other forms thereof. This disclosure includes nasal drops or sprays, aerosols or inhalants. You can also use medication.

[0136] Oral formulations include, for example, pharmaceutical-grade mannitol, lactose, starch, and stearic acid. Commonly used ingredients include magnesium, sodium saccharin, cellulose, and magnesium carbonate. These compositions contain excipients. These compositions are available in the form of solutions, suspensions, tablets, pills, capsules, and sustained-release formulations. Or it may take the form of a powder. Those skilled in the art are familiar with the well-known techniques for preparing oral formulations. It is likely.

[0137] In certain embodiments, the pharmaceutical composition contains at least about 0.1% by weight of the active agent. The substance may, for example, contain about 0.01%. In other embodiments, the pharmaceutical composition may, for example, , about 2% to about 75% by weight of the composition, or about 25% to about 60% by weight of the composition, It includes any range that can be derived within that range.

[0138] The pharmaceutical composition may contain various antioxidants to slow down the oxidation of one or more components. Furthermore, parabens (e.g., methylparaben, propylparaben) can prevent the action of microorganisms. ), chlorobutanol, phenol, sorbic acid, thimerosal, or combination thereof This is due to various preservatives, including but not limited to antimicrobial and antifungal agents. This composition may be stable under manufacturing and storage conditions, and may be free from microorganisms (e.g., It can be preserved against contamination by bacteria and fungi. Exotoxin contamination can be kept at a safe level, for example. It is understood that the protein level should be kept to a minimum of less than 0.5 ng / mg.

[0139] In embodiments where the composition is in liquid form, the carrier is water, ethanol, polyol (for example) (For example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids ( For example, this includes triglycerides, vegetable oils, liposomes, and combinations thereof. These may be solvents or dispersion media, but are not limited to these. Often, for example, sugars, sodium chloride, etc. It is preferable to include an isotonic agent such as um or a combination thereof.

[0140] In other embodiments, the Disclosure may use a nasal spray, aerosol, or inhalant. It may be used. Nasal spray is water designed to be administered intranasally by drip or spray. A solution may also be used.

[0141] The sterile injection solution contains, if necessary, the required amount of nanoparticles, as well as the various other nanoparticles listed above. It is prepared by incorporating the components into a suitable solvent, followed by sterilization.

[0142] At the time of prescription, exosomes are administered in a manner compatible with the drug prescription and are therapeutically effective. It is administered in such a quantity.

[0143] Nanoparticles can be administered to a target using any method known to those skilled in the art. For example, , a pharmaceutically effective amount of composition containing exosomes is administered intravenously, intracerebrally, intracranially, intrathecally, substantia nigra or Within the substantia nigra region, intradermal, intraarterial, intraperitoneal, intralesional, intratracheal, nasal, locally, intramuscular, abdominal Intracavitary, subcutaneous, oral, topical, partial, inhalation (e.g., aerosol inhalation), injection, infusion Continuous injection, direct local perfusion of target cells, via catheter, via washing, cream In a lipid composition (e.g., liposomes), or by other methods known to those skilled in the art, Or it can be administered by any combination of the above (Remington's (1990). In certain embodiments, the composition is administered to a subject using a drug delivery device. It can be done.

[0144] In other embodiments, exosomes are delivered orally, intranasally, enterally, topically, sublingually, intraarterially, orally. By routes including, but not limited to, internal, intrathecal, inhalation, ocular, percutaneous, vaginal, or rectal routes Formulated for administration and containing an appropriate carrier in each case. For example, exo for topical application. The somal composition may be prepared including a suitable carrier. Creams, lotions, and ointments are available. It can be prepared for topical application using a suitable base such as a triglyceride base. Such creams, lotions, and ointments may also contain surfactants. Aerosol The formulation may also be prepared so that an appropriate propellant adjuvant is used. Jubant can also be added to the composition, regardless of how it is administered, for example. For example, antimicrobial agents, antioxidants, and other preservatives prevent microbial growth and Alternatively, it may be added to the composition to prevent decomposition.

[0145] The pharmaceutically effective amount of nanoparticles is determined based on the intended target, such as inhibition of cell death. The amount administered will be determined according to both the number of treatments and the dosage, and will be the target of treatment. It depends on the condition, the desired protection, and the route of administration. The exact amount of medication is also at the discretion of the practicing physician. They are dependent and unique to each individual.

[0146] For example, the dosage of the medication is approximately 0.0001 milligrams to approximately 1.0 milligrams per dose. M, or approximately 0.001 milligrams to approximately 0.1 milligrams, or approximately 0.1 milligrams to approximately 1 It can be 0.0 milligrams, or even around 10 milligrams. Repeated administration is also possible. In some embodiments, the dose is at least about 0.0001 milligrams. In a further embodiment, the dose is at least about 0.001 milligrams. In further embodiments, the dose is at least 0.01 milligrams. In the embodiment, the dose is at least about 0.1 milligrams. In this embodiment, the dose may be at least 1.0 milligram. The dose may be at least 10 milligrams. In a further embodiment, the dose It is at least 100 milligrams or more.

[0147] In other non-limiting examples, the dose is also approximately 1 microgram / kg body weight, or approximately 5 micrograms. micrograms / kg body weight, approximately 10 micrograms / kg body weight, approximately 50 micrograms / kg body weight Weight, approximately 100 micrograms / kg body weight, approximately 200 micrograms / kg body weight, approximately 350 micrograms / kg body weight, approximately 500 micrograms / kg body weight, approximately 1 milligram / kg Body weight, approximately 5 mg / kg; Body weight, approximately 10 mg / kg; Body weight, approximately 50 mg / kg g body weight, approximately 100 mg / kg body weight, approximately 200 mg / kg body weight, approximately 350 mg grams / kg body weight, approximately 500 milligrams / kg body weight, approximately 1000 mg / kg body weight, or so This may include more than or equal to the dose, and any range that can be induced therein. Enumerated herein In non-limiting examples of the range that can be derived from the number, it is approximately 5 mg / kg / body weight to approximately 100 mg / kg ranges such as g / kg / body weight, approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight. The number of doses may be administered based on the above figures.

[0148] The dosage can be repeated as determined by those skilled in the art. Therefore, in this specification In some embodiments of the methods described, a single dose is intended. Other embodiments In this case, two or more doses are intended. When two or more doses are administered to the subject, the dose The time interval between doses can be any time interval determined by those skilled in the art. For example, between doses. The time intervals are approximately 1 hour to 2 hours, 2 hours to 6 hours, 6 hours to 10 hours, and 1 0 hours to approximately 24 hours, approximately 1 day to approximately 2 days, approximately 1 week to approximately 2 weeks, or longer, or these It can be any time interval that can be derived from any of the enumerated ranges.

[0149] In certain embodiments, this method can provide continuous supply of a pharmaceutical composition to a patient. This can be achieved by catheterization followed by continuous administration of the therapeutic agent. Administration is performed during the procedure or This can occur post-operatively.

[0150] (B. Combination therapy) Certain embodiments of this disclosure are one or more secondary forms of treatment for the treatment or prevention of disease The administration or application is provided. For example, the disease may be a hyperproliferative disease such as cancer.

[0151] Secondary treatment involves the administration of one or more secondary pharmacological agents applicable to the treatment or prevention of cancer. You may give it.

[0152] If the second-line treatment is a pharmacological formulation, it should be administered before, concurrently with, or after the administration of nanoparticles. It may be permitted.

[0153] The interval between exosome administration and secondary treatment is any interval determined by those skilled in the art. This is possible. For example, the interval may range from a few minutes to several weeks. In terms of administration methods, generally, each therapeutic agent still exerts an effect when combined favorably for the target. To enable this, ensure that no long period of time elapses between each delivery. For example, The interval between the drugs may be approximately 12 hours to approximately 24 hours from each other, and more preferably between each other. The treatment period may be extended to approximately 6 to 12 hours. However, in some situations, the treatment period may be extended. It can be extended, in which case several days (2, 3, 4, 5, 6, or 7) may be taken between each dose. Several weeks (1, 2, 3, 4, 5, 6, 7, or 8) have passed. In some embodiments... The timing of administration of the second-line treatment is determined based on the target's response to the nanoparticles. It can be done.

[0154] Various combinations can be used. In the following example, the exosome composition is "A". Therefore, anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / B A / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / B B / A / A / AA / B / A / AA / A / B / A

[0155] Administration of any compound or treatment described herein to a patient should take into consideration the toxicity of the drug (if any). Then, follow the general protocol for administering such compounds. Therefore, several In that embodiment, there is a step of monitoring toxicity resulting from the combination therapy. It is expected that the cycle can be repeated. In addition, various standard therapies and surgical treatments are available. The interventions described can be applied in combination with the therapies.

[0156] In certain aspects, standard therapy may include chemotherapy, radiotherapy, immunotherapy, surgical therapy, or This includes gene therapy, and as described herein, inhibitors of gene expression therapy, It can be used in combination with both anticancer therapy or gene expression therapy inhibitors and anticancer therapy. This is intended.

[0157] (1.Chemotherapy) According to this embodiment, a wide variety of chemotherapy agents can be used. The term "chemotherapeutic agent" refers to the use of drugs to treat cancer, while "chemotherapeutic agent" refers to the use of drugs in the treatment of cancer. It is used to mean the compound or composition that is administered. These drugs or medications are, for example... If so, then, whether they affect the cell cycle, and at what stage they affect it. They are classified according to the mode of their activity within the cell. Alternatively, drugs can directly bridge DNA. The ability to bridge, insert into DNA, or influence nucleic acid synthesis, thereby affecting chromosomes They can be characterized based on their ability to induce mitotic abnormalities.

[0158] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; bus Alkyl sulfonates such as ruphan, improsulfan, and biposulfan; benzo Aziridines such as dopa, carbocon, metredopa, and uredopa; altretamine, tri Ethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide, and Ethyleneimines and methylamemines containing methylolomellamine; acetogenins (especially) , bratacin and bratacinone); camptothecin (including synthetic analog topotecan); b Riostatin; calistatin; CC-1065 (its adzeresin, karzeresin, and Including bizeresin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 1). Putophycin 8); Dorastatin; Duocalmycin (synthetic analogues, KW-2189 and (Includes CB1-TM1); eleuterobin; pancratistatin; sarcodicin; Pongistatin; chlorambucil, chlornafadin, chlorophosphamide, estrum Sutene, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphala N, Novemubicin, Fenesterine, Prednimustine, Trophosphamide, Urasirma Nitrogen mustard such as Stard; carmustine, chlorozotocin, fotem Nitrosoureas such as tin, lomustine, and nimustine; enegyoin antibiotics (calcarema) Antibiotics such as Icin, especially calicheamicin gamma I and calicheamicin omega I1) Quality; Dynemicin containing Dynemicin A; Bisphosphonates such as chlordronate; S Peramycin; similarly, neocardinostatin chromophores and related chromophores, neocardinos Tatin chromophore, actinomycin, actinomycin, autoralnicin, azacerin, Bleomycin, Cactinomycin, Carabicin, Carminomycin, Cardinophilin chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5 -Oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomol Ruhorino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin (including), epirubicin, esorubicin, idarubicin, marcelomycin, maitomycin Syn C, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Pochi Romycin (potfiromycin), buromycin, keramycin, rhodoruby Syn, streptonigrin, streptozosin, tubercidine, ubenimex, Zinosta Mitomycins such as tin and zolubicin; methotrexate and 5-fluorouracil Antimetabolites such as (5-FU); denopterin, pteropterin, and trimethrexer Folic acid analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioglycolate; Anine and other purine analogs; ancitabine, azacitidine, 6-azauridine, kalmov Phloxacin, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxacin Pyrimidine analogs such as uridine; carsterone, dronanonone propionate, epithio Androgens such as stanol, mepiciostane, and testactone; mitotane, trilos Anti-adrenal agents such as tan; folic acid supplements such as floric acid; acegraton; aldofosphamide Lycoside; aminolevulinic acid, enyluracil; amsacrin; Bestlovesil; Bisan Tren; Edatrexate; Defofamine; Demecolsin; Elfor mithine; eriptinium acetate; epotilon; etoglucide; galu nitrate Mu; hydroxyurea; lentinan; ronidynin; meitansin and anthamitocin Which meitansinoids; mitogazone; mitoxantrone; mopidammol; nitraeri? Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxa Ntron; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysulfate Kaleidone complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenure Zon acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecene ( In particular, T-2 toxin, beraclin A, loridine A, and angiidine; urethane; vindesine; Dacarbaidin; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoid, for example Paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopri Platinum-coordinated complexes such as cisplatin, oxaliplatin, and carboplatin; Vinbla Styn; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxanthrone; Pink Listine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Dounomycete N; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-1) 1) Topoisomerase inhibitor RFS 2000; Difluoromethyl hydroxylnithine (DMF O); Retinoids (retinoic acid, etc.); Capecitabine; Carboplatin, Procarbazin n, precomycin, gemcitabine, navelbine, farnesyl protein transfer Gelase inhibitors, transplatinum, and any pharmaceutically acceptable salt or acid of the above. Examples include derivatives.

[0159] (2. Radiation therapy) Other factors that cause DNA damage and are widely used include gamma rays, X-rays, and / or This includes what is known as targeted delivery of radioisotopes to tumor cells. Wave and proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287) Other forms of DNA damage factors, such as UV irradiation, are also intended. These factors are All of these processes include DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. It is highly likely to affect the extent of damage. The X-ray dose range is long-term (3 to 4 weeks). From a daily dose of 50-200 roentgens to a single-dose dose of 2000-6000 roentgens. The dose range of radioactive isotopes varies widely, depending on the half-life of the isotope and the intensity of the emitted radiation. It depends on the type and uptake by tumor cells.

[0160] (3. Immunotherapy) Those skilled in the art will know that further immunotherapies can be used in combination with or in combination with the methods of the embodiments. It is important to understand that this is possible. In relation to cancer treatment, immunotherapies target and destroy cancer cells. This may depend on the use of immune effector cells and molecules. Rituximab (RITUX AN(registered trademark) is an example of this. Immune effectors, for example, can affect the surface of tumor cells. It may be an antibody specific to several markers on the surface. The antibody alone may not be a therapeutic effector. - It may serve as a means to actually influence cell killing by mobilizing other cells. Antibodies can also be drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, etc.). It can bind to (such as cough toxin) and serve as a targeting agent. Alternatively, the effect T is a lymphocyte that carries surface molecules that interact directly or indirectly with tumor cell targets. Yes, that's fine. Various effector cells include cytotoxic T cells and NK cells.

[0161] Antibody-drug conjugates (ADCs) are monoclonal antibodies covalently bound to a cytotoxic drug. This approach includes antibodies (MAb). This approach utilizes the high specificity of MAb against their antigen targets. By combining this with a very potent cytotoxic drug, the payload (drug) is abundant. This results in "armed" MAbs that deliver antigens at a high level to tumor cells. Targeted drug delivery is also This minimizes exposure to normal tissue, resulting in reduced toxicity and improved therapeutic index. Two ADC treatment drugs, ADCETRIS® (brentuximab vedotin) In 2011, KADCYLA (registered trademark) (trastuzumab emtansine or T-DM) was introduced. The FDA's approval of 1) in 2013 validated the validity of this approach. This approach combines the high specificity of MAbs with the antigen. Currently, There are more than 30 ADC drug candidates in clinical trials at various stages for cancer treatment. As antibody engineering and linker-payload optimization become increasingly mature, new A The discovery and development of DCs involves the identification and confirmation of new targets suitable for this approach, as well as target It is becoming increasingly dependent on MAb production. Two criteria for ADC targets are development in tumor cells. This represents an upregulation / high-level and robust internalization of the current system.

[0162] In one form of immunotherapy, tumor cells are easy to target, i.e., large amounts of other cells. It can carry several markers that are not present in the area. Many tumor markers They exist, and any of them may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), and gp68. TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, Lami This includes nin receptors, erb B, and p155. Alternative forms of immunotherapy are used to enhance anticancer effects. The goal is to combine it with an immunostimulatory effect. Immune stimulatory molecules include IL-2, IL-4, and IL- 12. Cytokines such as GM-CSF, γ-IFN, MIP-1, MCP-1, IL- This includes chemokines such as 8, and growth factors such as FLT3 ligands.

[0163] Examples of immunotherapies currently under investigation or in use include immunoadjuvants, such as Mycobact. Plasmodium falciparum, the parasite that causes tropical malaria rum), dinitrochlorobenzene, and aromatic compounds; cytokine therapy, for example, Intraferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy, e.g. For example, TNF, IL-1, IL-2, and p53; as well as monoclonal antibodies, for example, These are anti-CD20, anti-ganglioside GM2, and anti-p185. One or more anticancer therapies are used. It is intended to be used in conjunction with the antibody therapies described herein.

[0164] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Epidemic checkpoints either increase the signal (e.g., a co-stimulatory molecule) or suppress the signal. It is a molecule in the immune system that suppresses immune function. It can be a target for immune checkpoint blockade. Sex checkpoint molecules include the adenosine A2A receptor (A2AR) and B7-H3 (CD). Also known as 276), B and T lymphocyte attenuators (BTLA), cytotoxicity Sex T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), India Alamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR) Lymphocyte activation gene-3 (LAG3), programmed DES-1 (PD-1), T cell immunotherapy Epidemic globulin domain, mucin domain 3 (TIM-3), and T cell activation V domain There is an Ig inhibitor (VISTA). In particular, immune checkpoint inhibitors affect the PD-1 axis and It targets / or CTLA-4.

[0165] Immune checkpoint inhibitors are drugs that exist in various forms, such as small molecules, ligands, or recombinant receptors. It may be a substance, or in particular an antibody such as a human antibody (for example, an international patent publication Publication No. WO2015016718; both are incorporated herein by reference). Immunology Known inhibitors of checkpoint proteins or their analogues may be used, in particular Chimeric forms, humanized forms, or human forms of antibodies may be used. As those skilled in the art will know, Alternative and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also an alternative and equivalent to MK-3475 and pembrolizumab. It is known that it is known by its name.

[0166] In some embodiments, a PD-1-binding antagonist binds PD-1 to its ligand. It is a molecule that inhibits binding to its partner. In certain embodiments, it binds to PD-1 ligand. The partners are PDL1 and / or PDL2. In another embodiment, PDL1 Synthetic antagonists are molecules that inhibit the binding of PDL1 to its binding partner. In a specific configuration, the PDL1 binding partner is PD-1 and / or B7-1. In this embodiment, the PDL2-binding antagonist transmits PDL2 to its binding partner. It is a molecule that inhibits the binding of [substance]. In certain embodiments, the PDL2 binding partner is PD-1. Antagonists are antibodies, their antigen-binding fragments, immunoadhesins, and fusion tannins. It may be a protein or oligopeptide. An example antibody is described in U.S. Patent No. . 8,735,553, 8,354,509 and 8,008,449 (all by reference) (as incorporated herein) is used in the methods provided herein. Other PD-1 axis antagonists for use are specified in U.S. Patent Application No. 20140294898. , and No. 2014022021 and No. 20110008369 (all as specified herein) As described in (referenced for reference), it is publicly known in the field.

[0167] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., These are human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, they are anti-PD-1 The antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesian (for example, PDL1 or PDL2 fused to a normal region (e.g., the Fc region of an immunoglobulin sequence) It is an extracellular immunoadhesin (containing a PD-1 binding moiety). In some embodiments, The PD-1 binding antagonist is AMP224. Nivolumab is MDX-1106- 04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO Also known as (registered trademark), it is described in WO2006 / 121168 as an anti-P It is a D-1 antibody. Pembrolizumab is also known as MK-3475, Merck 3475, and Lan. Brolizumab, also known as KEYTRUDA® and SCH-900475, W This is an anti-PD-1 antibody described in O2009 / 114335. CT-011, hB Also known as AT or hBAT-1, it is an antibody described in WO2009 / 101611. It is a PD-1 antibody. AMP-224 is also known as B7-DCIg, and WO2 PDL2-Fc as described in 010 / 027827 and WO2011 / 066342 It is a fusion-soluble receptor.

[0168] Another immune checkpoint that may be targeted in the methods provided herein is C Cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as D152. The complete cDNA sequence of human CTLA-4 is Genbank accession number L1. It has 5006. CTLA-4 is found on the surface of T cells and on the surface of antigen-presenting cells. When coupled to CD80 or CD86, it functions as an "off" switch. CTLA4 is disabled It is a member of the globulin superfamily, expressed on the surface of helper T cells, and suppresses T cell activity. It transmits regulatory signals. CTLA4 is similar to CD28, a T cell costimulatory protein. Both molecules are found on antigen-presenting cells, and CD80 and CD86 (B7-1 and B7, respectively) CD28 (also called -2) binds to CTLs. CD28 transmits stimulant signals, whereas CTLs A4 transmits inhibitory signals to T cells. Intracellular CTLA4 is also found in regulatory T cells. Therefore, it is considered important for its function. T cell receptors and CD28-mediated T cell Activation increases the expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0169] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (for example) (For example, human antibodies, humanized antibodies, or chimeric antibodies), their antigen-binding fragments, immunoadhesins, It is a fusion protein or oligopeptide.

[0170] Anti-human CTLA-4 antibodies (or VH and / or VH derived therefrom) suitable for use in this method The VL domain can be generated using methods well known in the art. In this technology, anti-CTLA-4 antibodies recognized in this field can be used. For example, National Patent No. 8,119,129, International Publication No. 01 / 14424, International Publication No. 98 / 42 Publication No. 752; International Publication No. 00 / 37504 (CP675,206, as tremelimumab) Also known as; formerly known as tisilimmab, disclosed in U.S. Patent No. 6,207,156 The anti-CTLA-4 antibody can be used in the method disclosed herein. The lessons learned from the above publications are included here for reference only. (Conclusion to CTLA-4) For compatibility, antibodies that compete with any of these technically recognized antibodies can be used. Yes, for example, the humanized CTLA-4 antibody is subject to international patent application WO2001014424. As described in WO2000037504 and U.S. Patent No. 8,017,114, All references are incorporated herein by reference.

[0171] An example of an anti-CTLA-4 antibody is ipilimumab (10D1, MDX010, MDX10) 1, and also known as Yervoy (registered trademark) or its antigen-binding fragment and It is a variant. In other embodiments, the antibody is ipilimumab heavy and light chain CDR or V It contains R. Therefore, in one embodiment, the antibody is CDR1 of the VH region of ipilimumab. CDR2 and CDR3 domains, as well as CDR1 and CDR2 domains of the VL region of ipilimumab. , and includes the CDR3 domain. In another embodiment, the antibody is the same CTLA- 4 competes for binding to and / or binding to the epitope on 4. In another embodiment, anti The body has at least approximately 90% variable region amino acid sequence identity with the above antibodies (e.g., ipilin). It has at least approximately 90%, 95%, or 99% variable region identity with mumab.

[0172] Other molecules for modulating CTLA-4 include U.S. Patent No. US5844905, U S5885796, International Patent Application Numbers WO1995001994 and WO199804 This includes CTLA-4 ligands and receptors as described in 2752. All references incorporated herein and described in U.S. Patent No. US 8329867 are used herein. Immunoadhesion such as (as incorporated herein by reference).

[0173] (4.Surgery) Approximately 60% of people with cancer undergo prophylactic, diagnostic, or staging, curative, and palliative surgery. They undergo some kind of surgery. Curative surgery involves the physical removal of all or part of the cancerous tissue. They are removed, excised, and / or destroyed, and used in the treatment, chemotherapy, radiotherapy, and hormone therapy of this embodiment. It can be used in combination with other therapies such as MON therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgery may be performed Treatment options include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (micro-surgery). This includes surgery.

[0174] When cancerous cells, tissue, or part or all of a tumor are removed, cavities may form within the body. Treatment can be achieved by perfusion, direct injection, or local application in the area accompanied by additional anticancer therapy. Such treatments may be administered, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, or 4 days. and every 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months These treatments can be repeated. These treatments can also be administered in various dosages.

[0175] (5. Other medications) To improve the therapeutic effect of the treatment, other drugs are combined in a specific manner according to this embodiment. It is intended that it may be used. Therefore, further examples can be considered. These additional drugs affect the upregulation of cell surface receptors and gap junctions. For drugs that inhibit cell proliferation and differentiation, inhibitors of cell adhesion, and agents that induce apoptosis. This includes drugs that increase the sensitivity of overproliferating cells, or other biological agents. Increased intercellular signaling by increasing the number of connections affects adjacent hyperproliferating cells. This likely increases the anti-overgrowth effect on the population. In other embodiments, cell proliferation In order to improve the anti-overgrowth efficacy of the inhibitor or differentiation agent, in a specific aspect of this embodiment, They can be used in combination. Cell adhesion inhibitors are intended to improve the efficacy of this embodiment. As illustrated, examples of cell adhesion inhibitors include local adhesion kinase (FAK) inhibitors and lovastatin. Furthermore, the sensitivity of hyperproliferating cells to apoptosis, such as that of the antibody c225. Other drugs that increase may be combined with certain embodiments of this embodiment to improve therapeutic efficacy. It is further intended that it may be used in this way.

[0176] [kit] In various embodiments of the embodiment, a drug containing a therapeutic agent and / or other therapeutic agents and a delivery agent is included. A set is assumed. In some embodiments, this embodiment is an exoso This invention is intended to be a kit for preparing and / or administering a medical composition. The kit is intended to be a medical composition of this embodiment. This kit may include one or more sealed vials containing any of the drug compositions. , exosomes, and reagents for preparing, formulating and / or administering the components of the embodiment, This may include reagents for carrying out one or more steps of the method of the present invention. Several embodiments The kit also includes Eppendorf tubes, assay plates, syringes, bottles, Alternatively, it may include a suitable container that does not react with the components of the kit, such as tubes. The container may be made from a sterilizable material such as plastic or glass.

[0177] The kit outlines the steps of the procedure of the method described herein. The procedure is substantially the same as the one described, or is known to those skilled in the art, and may include further instructions. Good. The instructions state that when executed using a computer, a pharmacopoeially effective amount of the drug will be dispensed. Computer-readable instructions, including machine-readable instructions, that cause the display of actual or virtual procedures to be delivered. It may be included in reading materials. [Examples]

[0178] [V. Examples] The following examples are included to demonstrate preferred embodiments of the present invention. The inventors have found that the methods disclosed herein function adequately in carrying out the present invention. The presented technology is represented and, therefore, will be understood by those skilled in the art to function well in the practice of the present invention. It should be understood. However, a person skilled in the art would consider the disclosure of the present invention and disclose... In certain embodiments in which similar results are obtained, there is a departure from the spirit and scope of the present invention. It should be understood that many changes are possible without any effort.

[0179] [Example 1 - Exosome Production and Characterization] To generate large quantities of exosomes from MSCs, a bioreactor for bone marrow-derived MSCs. The culture was adapted to allow for the collection of 250 mL of sample from the conditioned medium. The mo Quantum Cell Expansion system uses GMP (Good Manufacturing Practice) technology to improve cell GMP. This is an automated hollow fiber cell culture platform designed for conformity-based cell production.

[0180] MSCs are approximately 450 cells / cm². 2 At this concentration, approximately 2.0 × 10 7 Biolia with MSC The cells were seeded in a cadmium. For MSC proliferation and adhesion, L-glutamine + human platelets were used. The cells were cultured for 8 days in 5% oxygen using αMEM medium supplemented with lysate (PLT).

[0181] The unique step in the present invention's method, which has not been reported by others, is in the bioreactor. For optimal MSC confluence, the first use of platelet lysates, followed by bio... Once the MSCs in the reactor reach a confluence of 85% or more, the growth medium is changed to serum-free medium. This step includes replacing the acclimatization medium. This step is necessary because large amounts are found in the platelet lysate, and Otherwise, the final product will be contaminated with exosomes that dilute MSC-derived exosomes. To avoid this, leave the acclimatized medium in the bioreactor for 48 hours, then for EV purification. Samples were collected (Figure 1). The bioreactor produced EVs including six samples (one every 48 hours). Production continued for 12 consecutive days (Figure 2).

[0182] Using this approach, the system will be able to sequentially collect EVs from approximately 600 million MSCs. Optimized. After 21 days, the conditioned medium fraction was collected every 48 hours and stored at -80°C. The samples were thawed, pooled, and filtered in a functionally closed system (Figure 3).

[0183] The pump (Baxter) is used after the heat sealing of the bag tube for this process. It is used for filtration, which is required for GMP compliance in clinical cell therapy procedures. This matched. XE-90 Ultracentrifuge (Beckman Coul Using ter, EV is obtained by ultracentrifugation at 4°C for 4 hours with 100,000g. They were isolated as exosomes from the conditioned medium using a Baxter pump and then heat-sealed. Using tubing, ultracentrifugation is performed in a functionally closed manner for the highest quality clinical products. Exosomes were removed from the aircraft after takeoff.

[0184] Exosomes are concentrated by filtration and ultracentrifugation, and the number of exosomes per collection is The range was between 900 billion and 4.5 trillion. The total number of exosomes generated was... The range is 9.8 to 15.1 trillion per reactor run. Six bioreactor experiments The mode size of exosomes from all harvested samples showed a characteristic peak at approximately 170 nm. The exosome protein content was determined by NanoSight analysis. This approximated the number of soms. In particular, the metabolic harvest of the conditioned medium. s) remained constant, supporting the conclusion that MSCs remained viable.

[0185] Purified exosomes are identified by exosome surface markers: CD63, CD47, CD9 and flow cytometry expression of CD81; and transmission electron microscopy (TEM) They were identified using nanoparticle tracking analysis and micro-BCA assay. Exosomes were quantified (Figure 5). The yield from one bioreactor run was 100 Equivalent to the approximate exosome yield from a T-225 flask, minimum 10 × 10 12 workman It was a xosome. Therefore, the method of the present invention provides efficient clinical-grade extraction of exosomes. Enables production.

[0186] [Example 2 - Electroporation of exosomes] The 4D Nucleofactor system is similar to the exosome derived from Example 1. , 2.5×10 10 ~2.5×10 12 Closed, effective large exosome number range To enable efficient and scalable in vitro transfection, LV Uni The 4D Nucleofactor device was compatible with three different nuclear factors. It contains the liquids SE, SF, and SG, each of which is derived from MSCs and special Heterogenous siRNAs were tested in combination with 16 different nuclear factor programs. The efficiency of each condition for efficiently incorporating siRNA into MSC-derived exosomes is measured using si Apoptosis of recipient cells induced by RNA-containing MSC exosomes The results were evaluated by assay (Figure 7).

[0187] Previously, a specified electroporation buffer (research buffer, "RB") was used. Then, siRNA was introduced into the exosome, but this was because the buffer was suitable for human use. Because it is not approved. An exosome washing step is required before treating cells or mice. The washing process is associated with the loss of exosomes, which leads to the transfer of siRNA to the exosomes. This enables successful electroporation and can be directly administered to cells or mice. This can be mitigated by the use of diluents. Clinical buffer (PLASMALYTE-A (registered product) (Standard), "CB"), FDA-approved diluent for human use (this is a multiplier for MSCs and patients) We tested electroporation (which is used for injecting many other cell products). Following electroporation of MSC-derived exosomes, electron microscopy analysis revealed the most The initially prescribed research buffer (RB) or PLASMALYTE-A® (Figure 8) The presence of complete exosomes was confirmed using one of the methods in B).

[0188] [Example 3 - Optimization of conditions in exosome production] To determine the best time for exosome production from MSC cultures, acclimatization culture Soil samples were collected at different times (Figure 5D). Data obtained from nanosites (i.e., The number of particles) is used in the data obtained from flow cytometry (i.e., exosomes). This was combined with the percentage of markers. The results showed that the number of particles from MSCs was highest in 24 hours. It was shown that high levels were reached and maintained for up to 48 hours. After that, the number of particles decreased significantly. The flow data showed that the percentage of exosome markers was the percentage over 24 hours. Compared to the tage, it was shown that concentration occurred in 48 hours (Figure 5E). 24 hours vs. 48 hours Since the number of particles between these points did not differ significantly, the 48-hour point in time was chosen as the collection point. I selected it.

[0189] Next, to determine the ideal conditions for producing exosomes from MSCs in culture... Furthermore, regardless of whether or not the medium contains PLT, after 24 hours of incubation, MS was performed on the conditioned medium. C-derived exosomes were isolated. The result was that the number of particles isolated from the medium containing PLT was The levels were higher than in serum-free medium (Figure 6A). However, the exosome markers Flow cytometry is likely due to the large number of proteins and lipids in PLT. The percentage of sosomes was low (Figure 6B).

[0190] Therefore, electroporation of nucleic acid or protein subsequences into exosomes Rothes uses a sterile solution (i.e., PLASMALYTE-A®) (Figure 8A). Optimized for use, this can be directly injected into humans, minimizing manipulation, material loss, and treatment. This reduced the cost (Figure 9). PLASMALYTE-A® is the optimal electric Before demonstrating that it produces tropolysis results, five different research buffers were tested. (Figure 8A). Furthermore, the MSC-derived exosomes produced by this strategy The ability to target several organizations was demonstrated in vivo (Figure 10).

[0191] Therefore, this study aims to suppress the in vitro expression of target RNA in recipient cells. Efficient large-scale production of MSC-exosomes containing KRAS siRNA that can be used This was demonstrated (Figure 8C). This approach yields a larger number of EVs compared to previous methods. It generates more than one logarithmically. Furthermore, the method of the present invention is operated without washing or other operations. This enables the direct injection of exosomes.

[0192] [Example 4 - Materials and Methods] Cells: Cell Therapy Laboratory, MD Anderson Cancer Center MSCs (passage 3) derived from y were treated with 1% L-glutamine and 5% human platelet lysate. The cells were cultured in αMEM (complete medium) supplemented with 1% penicillin-streptomycin. We evaluated MSCs from different donors, and then, based on their high exosome production yield... A single donor was selected. For in vitro transfection, a 6-well plate was used. 250,000 Panc-1 cells were seeded overnight per well. Exosome processing Before processing, wash the monolayer twice with 1 ml of PBS, then add 1 ml of serum-free medium (1% penicillin). In each assay, exosomes in RPMI supplemented with syrin-streptomycin were used. I processed it as instructed, as described above.

[0193] Clinical buffer (PLASMALYTE-A® pH 7.4 or "CB") is 0.09M sodium chloride, 0.23M sodium gluconate; 0.27M sodium acetate It is composed of methyl trihydrate, 5 mM potassium chloride, and 3 mM magnesium chloride. It contains an antibacterial agent. No. Adjust the pH to 7.4 with sodium hydroxide.

[0194] Clinical-grade exosomes are available at Cell Thera, MD Anderson Cancer Center. Prepared strictly from MSCs cultured in py Laboratory's GMP facility. The ntum bioreactor culture system (Terumo BCT) is cultured in 1L of 1×PBS. Mixing (automated process), 5 mg of human fibrillation diluted in 250 ml of 1x PBS. Coated with Ronectin (BD Biosciences, Germany) for 24 hours. Next Then, the bioreactor was filled with 1% L-glutamine, 5% human platelet lysate, and 1% penicillin. Wash with 500 ml of cultured αMEM supplemented with syrin-streptomycin (complete medium). Then, diluted in 25 ml of complete medium, 20 × 10 6 Load MSC (passage 3) and complete cultivation The cells were grown for 9 days using soil. Fresh complete medium was continuously added to the cells, and daily glucose was administered. The injection rate was adjusted as determined by the measurement of phosphate and lactate. After 9 days, the cells were When approximately 80% confluence is reached (confirmed by glucose and lactose measurements) Wash the cells in 2 L of 1×PBS and transfer them to a complete medium without PLT (1% L- The αMEM was replaced with one supplemented with glutamine and 1% penicillin-streptomycin. Next, the bioreactor-conditioned medium (250 ml) was placed in a sealed bag (closed system) for 48 hours. Samples were collected at intervals, for a total of six collections (retrievals). During these 12 days, the cultures were collected every The exosomes did not expand based on a constant glucose level measured daily. The harvested material was collected continuously every 48 hours for 12 days. They were stored at -80°C. Each harvested product was tested for sterility (negative for anaerobic and aerobic bacteria). It was confirmed that endotoxin (<1 EU / ml) and mycoplasma (PCR, A negative test was performed. Next, the collected samples were thawed overnight at 4°C, pooled, and Cobe 29 91 Closed-system odor using Cell Processor (Terumo BCT) Then, 1,000g was centrifuged for 15 minutes. Centrifugation (1,000g, 4°C for 15 minutes) After removing large cell debris, the acclimatization medium is filtered through a 0.2 μm filter (Terum The solution was filtered in a closed system using a BCT filter bag. Then, 600 ml of the supernatant was used. Direct connection to syringe and polycarbonate tubing (Beckman Coulter). Using the lines, six clear polycarbonate tubes (each tube) are connected in a semi-closed system. Transfer to a 100ml capacity container, seal, and place in a 45-type Ti rotor (Beckman). -Coulter) was used to centrifuge at 100,000g for 3 hours. All collected samples were centrifuged. This process was repeated three times until it was completed (totaling 1500 ml). Next, connect to the pump. 16G syringe (BD Biosciences, catalog number 14-826-18) The supernatant was aspirated using B). The exosome pellet was collected in an 18G syringe (BD, Catalogue). Using (number 408360), manually refill into 4 ml (per tube) of clinical buffer. It was suspended and then transferred to a sterile glass container (APP Pharma, 30 ml capacity). The mixture was maintained at 4°C for up to 72 hours until all centrifugation was complete. When completed, the final pool volume of the resuspended exosomes was 72 ml. The extracted MSC exosomes were processed using NanoSight™ (0.5 ml) and flow cytometry. Analysis was performed using Lee (1 ml), and endotoxin (pooled sample 0.5 ml). (using l) and sterile (using 1 ml of pooled sample as detailed above) The following was tested using (2 ml each). Finally, the exosome (final volume 69 ml) was divided into two parts (2 ml each). The contents were divided into cryoglass vials and stored at -80°C. For the manufacture of future clinical products. Furthermore, exosomes are directly processed for large-scale electroporation (for details) (See below for details), then dispensed and stored at -80°C.

[0195] Measurement of particle size and concentration distribution by NTA: The isolated exosome suspension was subjected to Na noSight TM Analysis was performed using the LM 10 instrument (NanoSight Ltd). We optimized the analysis settings, kept them constant between samples, and analyzed each video to determine each particle size. The mean, mode, median, and estimated concentration were obtained.

[0196] Exosome quantification by microBCA assay: MSC exosomes resuspended in CB Wash the soms with 1x PBS and then use a SW 41 type Ti tortoise (Beckman Coultre). The MSC exosoleum was centrifuged at 100,000g for over 3 hours. Then, the washed MSC exosoleum was used. The microBCA protein was measured again using NanoSight™. Using the quality assay reagent kit (Thermo Scientific), the manufacturer's specifications The total protein content was analyzed according to the specifications.

[0197] Exosome electroporation: 0.5 × 10¹² in total 12 Individual MSC-derived extract Sosomes and 0.5 mg of siRNA source 2 (Avecia) in 20 ml of clinical buffer. They were mixed in solution. These exosomes were electroporated in a closed system using a 4D Nucleofator LV unit (Lonza). The LV Nucleocuvette cartridge is a new cuvette system that enables electroporation up to 20 ml. The cartridge is connected to two reservoir bags TM (inlet and outlet) and a peristaltic pump that fills the cartridge at 1 ml per hour. During all procedures, the outlet bag was maintained on ice and it took about 10 minutes to complete the procedure. After electroporation, the exosomes were analyzed by NanoSight and tested for endotoxin and sterility (as detailed above), aliquoted into cryovials, and stored at −80 °C. Next, these exosomes were thawed on ice and used for subsequent in vitro and in vivo experiments. For in vitro experiments, the exosomes were diluted for downstream applications detailed below. For in vivo experiments, 1 × 109 electroporated exosomes were diluted in 100 μL of research buffer or clinical buffer. Electron microscopy: Specimens fixed at optimal concentration were placed on 300 mesh carbon / formvar coated TM grids and absorbed in formvar for at least 1 minute. The grids were rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes. After rinsing with PBS and distilled water, the grids were dried and stained for contrast using uranyl acetate. The samples were examined with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR, USA) at 120 kV. USA) at 120 kV. After that, they were used for subsequent in vitro and in vivo experiments. For in vitro experiments, the exosomes were diluted for downstream applications detailed below. For in vivo experiments, 1 × 109 electroporated exosomes were diluted in 100 μL of research buffer or clinical buffer. Electron microscopy: Specimens fixed at optimal concentration were placed on 300 mesh carbon / formvar coated grids and absorbed in formvar for at least 1 minute. The grids were rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes. After rinsing with PBS and

[0198] Electron microscopy: Specimens fixed at optimal concentration were placed on 300 mesh carbon / formvar coated grids and absorbed in formvar for at least 1 minute. The grids were rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes. After rinsing with PBS and distilled water, the grids were dried and stained for contrast using uranyl acetate. The samples were examined with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR, USA) at 120 kV. distilled water, the grids were dried and stained for contrast using uranyl acetate. The samples were examined with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR, USA) at 120 kV. distilled water, the grids were dried and stained for contrast using uranyl acetate. The samples were examined with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR, USA) at 120 kV. grids and absorbed in formvar for at least 1 minute. The grids were rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 minutes. After rinsing with PBS and Observe with sboro (OR), and the image is taken with an AMT CCD camera (Advanced Microwave OCR). The images were taken using roscopy Techniques (Danvers, MA).

[0199] Flow cytometry analysis of exosomes: Exosomes from MSCs as shown above Isolation and resuspending in 200 μl of PBS. Aldehyde / sulfate beads (10 μl, Li fe Technologies) is added to the solution, and the beads and exosome mixture is added. The mixture was mixed at room temperature for 15 minutes using a benchtop rotator. Then, PBS (600 μl) was dissolved in it. Add to the solution, continue mixing at 4°C overnight, add 1M glycine (400 μl), and mix at room temperature. This was continued for 1 hour. Next, the mixture was spun down at 8,000 g for 1 minute. Then, precipitation was performed. The substance was resuspended in 100 μL of PBS with 10% BSA and mixed at room temperature for 45 minutes. The mixture was spun down at 8,000g for 1 minute, and the supernatant was aspirated. Then, the exosomes attached to the mixture were removed. The beads (pellets) were resuspended in 20 μl of 2% BSA in PBS, and then CD4 7. CD63, CD81, CD9, CD29, CD90 or isotype control Immunolabeling was performed for the following: Exosomes bound to beads were treated with 1 μl of anti-CD47 antibody (e Biosciences, catalog number 14-0479) or 1 μl of anti-CD63 (BD Biosciences, catalog number 556019), or 1 μl of anti-CD-81 anti Body (BD Biosciences, catalog number 555675), or 1 μl of anti-CD fluid. 9 antibody (Sigma, catalog number SAB4700092) or 1 μl anti-CD29 immunization (Biolegend, catalog number 303001), or 1 μl of anti-CD90 immunoglobulin ( Biolegend (catalog number 328101), or 1 μl of mouse IgG1, κA Isotype-controlled immunology (BD Biosciences, catalog number 5557) 46) Incubate in 20 μl volume and mix at room temperature for 30 minutes. Next, mix the mixture 8 Centrifuge at 0,000g for 1 minute, aspirate the supernatant, and add the pellet to PBS with 2% BSA2. It was then resuspended in μl. Next, 1 μl of secondary antibody (Invitrogen, catalog number A) was added. 21202) was added to the sample and isotype control. Then all samples The mixture was mixed at room temperature for 1 hour. Then, the sample was centrifuged at 8,000 g for 1 minute, and the supernatant was removed. The pellet was aspirated and resuspended in 200 μl of 2% BSA in PBS. The beads were bound to the pellet. The exosomes were washed three times with 2% BSA in PBS. Exosome marker (C D9, CD63 and CD81, CD47, and mesenchymal markers (CD29 and CD90) The expression of was analyzed using an LSR Fortessa X-20 cell analyzer. The data was analyzed using FlowJoR software (TreeStar Inc.). Flow cytometry data were obtained for both the isotype control and the samples in each experiment. They were obtained by arranging them side by side. Flow cytometry experiments were performed independently using the same exosome preparation. And they repeated it twice.

[0200] All methods disclosed and claimed herein are subject to the same regulations as, in light of this disclosure, excessive experimentation. It can be manufactured and executed without performing [the necessary steps]. The composition and method of the present invention are preferred in [the necessary steps]. Although the methods of application have been described, without departing from the concept, spirit, and scope of the present invention, Modifications can be applied to the method and steps or sequences of steps of the method described in the specification. This will be obvious to those skilled in the art. More specifically, specific chemical and physiologically related It is reasonable to assume that the same or similar results can be achieved by substituting the drugs described herein with other drugs. It will be understood that all such similar substitutions and modifications are obvious to those skilled in the art. It is deemed to fall within the spirit, scope, and concept of the invention as defined by the claims of the invention.

[0201] [References] The following references are illustrative procedures or other references that supplement those shown herein. To the extent that it provides further details, it is incorporated herein by reference. International Publication WO 00 / 37504 International Publication WO 01 / 14424 International Publication WO 98 / 42752 International Publication No. WO1995001994 International Publication No. WO1998042752 International Publication No. WO2000037504 International Publication No. WO2001014424 International Publication No. WO2006 / 121168 International Publication No. WO2009 / 101611 International Publication No. WO2009 / 114335 International Publication No. WO2010 / 027827 International Publication No. WO2011 / 066342 International Publication No. WO2015016718 U.S. Patent No. 4,870,287 U.S. Patent No. 5,760,395 U.S. Patent No. 5,844,905 U.S. Patent No. 5,885,796 U.S. Patent No. 8,008,449 U.S. Patent No. 8,017,114 U.S. Patent No. 8,119,129 U.S. Patent No. 8,329,867 U.S. Patent No. 8,354,509 U.S. Patent No. 8,735,553 U.S. Patent Publication No. 20110008369 U.S. Published Patent Number 2014022021 U.S. Patent Publication No. 20140294898

Claims

1. Mesenchymal stem cell (MSC)-derived exosomes: (a) Culturing MSCs in a functionally closed bioreactor in a medium containing human platelet lysates (PLT); (b) Further culturing the cells in a medium that is essentially free of PLT; (c) Collecting one or more conditioned medium fractions from the bioreactor; and (d) Isolating exosomes from the conditioned medium fraction. Obtained by a method including, The mesenchymal stem cell-derived exosome expresses CD63, CD47, CD9, and CD81.

2. The mesenchymal stem cell-derived exosome according to claim 1, wherein the MSCs are cultured to 75-95% confluence in step (a).

3. The mesenchymal stem cell-derived exosome according to claim 1, wherein the functionally closed bioreactor is a hollow fiber bioreactor.

4. The mesenchymal stem cell-derived exosome according to claim 1, wherein the culture medium of step (b) contains 0 to 0.01% PLT.

5. The mesenchymal stem cell-derived exosome according to claim 1 or 2, wherein the mesenchymal stem cell-derived exosome has a most frequent particle size of about 100 to 400 nm, or about 170 nm, as determined by nanoparticle tracking analysis.

6. The mesenchymal stem cell-derived exosome according to claim 1, wherein the MSCs are bone marrow-derived MSCs or adipose-derived MSCs, and / or each conditioned medium fraction is stored at -80°C after collection, and the conditioned medium fraction is thawed and pooled before step (d).

7. A mesenchymal stem cell-derived exosome according to any one of claims 1, 2, 3, or 6, satisfying at least one of the following: (a) The method further comprises seeding at least 1 × 10⁷ MSCs into the bioreactor prior to step (a) of claim 1, wherein the MSCs are optionally seeded at a density of about 400 to 500 cells / cm²; (b) The hollow fiber bioreactor is a Terumo cell proliferation system; (c) The PLT is present in the culture medium of step (a) of claim 1 at a concentration of 5%; (d) Fresh culture medium is continuously added to the MSCs in the bioreactor; (e) The cells are cultured in 5% oxygen; (f) The culture in step (a) is 5 to 10 days, and optionally, the culture in step (a) of claim 1 is 8 days; (g) The MSCs are cultured to 80-90% or 85-90% confluence; (h) The culture in step (b) of claim 1 is 24 to 72 hours, and optionally, the culture in step (b) of claim 1 is 48 hours; (i) The culture medium of step (b) of claim 1 is free of PLT; (j) The MSC is washed between steps (a) and (b) of claim 1; (k) The MSCs are cultured in serum-free standard medium; (l) Steps (a) to (d) of claim 1 are carried out under serum-free conditions; (m) The conditioned medium fraction is collected in a sealed bag; and / or (n) The conditioned medium fraction is collected every 24 to 72 hours, optionally every 40 to 50 hours, and optionally every 48 hours.

8. A mesenchymal stem cell-derived exosome according to any one of claims 1, 2, 3, or 6, satisfying at least one of the following: (a) Each of the conditioned medium fractions has a volume of 200 to 300 mL; (b) The conditioned medium fraction is collected for 10 to 14 days; (c) The conditioned medium fraction is collected for 12 days; (d) At least five conditioned medium fractions are collected; (e) Steps (a) to (d) of claim 1 are carried out in less than three weeks; (f) Each conditioned medium fraction contains 9 × 10¹¹ to 50 × 10¹¹ exosomes; (g) In step (d) of claim 1, at least 10 × 10¹² exosomes are isolated; and / or (h) In step (d) of claim 1, at least 15 × 10¹² exosomes are isolated.

9. The isolation comprises at least one of the following mesenchymal stem cell-derived exosomes according to any one of claims 1, 2, or 6: (a) Filtering and ultracentrifugation of the pooled fraction to obtain an exosome-containing pellet, and resuspending the exosome-containing pellet in a buffer; (b) The isolation is carried out in a functionally closed manner using a pump and a heat-sealed tube; (c) The filtration is further defined as passing the pooled fraction through a 0.2 μm filter; (d) The isolation further includes a centrifugation step before filtration to remove large cell debris; (e) The isolation is carried out at 4°C; or (f) The buffer solution comprises 0.09 M sodium chloride, 0.23 M sodium gluconate, 0.27 M sodium acetate trihydrate, 5 mM potassium chloride, and 3 mM magnesium chloride, optionally having a pH of 7.4, and further optionally being PLASMALYTE-A®; or (g) The buffer solution has a pH of 7.4, and optionally, the buffer solution is PLASMALYTE-A®.

10. Mesenchymal stem cell-derived exosome according to any one of claims 1, 2, 6, or 9, further comprising at least one of the following: (a) loading a therapeutic agent into the exosome, wherein the therapeutic agent comprises one or more cytokines, chemotherapeutic agents, nucleic acids, small molecules, or proteins, and optionally the nucleic acid comprises DNA and / or RNA, and optionally the RNA comprises siRNA, miRNA, or shRNA; and / or (b) The loading includes electroporating the exosomes, optionally the electroporation is performed in PLASMALYTE-A®, and optionally the method does not include washing the exosomes or exchanging the buffer between step (d) of claim 1 and the electroporation, and optionally the number of loaded exosomes relative to the number of exosomes obtained from step (d) of claim 1 is not reduced by more than 20%.

11. A pharmaceutical composition comprising mesenchymal stem cell-derived exosomes according to any one of claims 1, 2, 3, or 6.

12. A pharmaceutical composition according to claim 11 for use in the treatment of a disease or disorder in a subject requiring treatment, wherein the disease or disorder is cancer, an inflammatory disorder, an immune-related disorder, or an immune-mediated inflammatory disease, optionally the cancer is lung cancer, and optionally the exosome is equipped with KRAS siRNA.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is administered orally, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, locally, or by direct injection, and / or at least a second therapeutic agent is further administered to the subject, wherein the at least second therapeutic agent is an anticancer therapy including chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

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