Composition containing dendritic cell exosomes and method for using said exosomes

Abstract

This specification provides a method for producing extracellular vesicles (EVs) from large-scale cultures of polarized DCs, and also describes CTLA-4 + EV and CTLA-4 neg Additional procedures are also provided for separating EVs from each other and recovering both EV populations in a physically and functionally intact state, and a composition containing the EVs obtained by the method is also provided. Also provided is CTLA-4 for treating cancer. neg The use of EVs, as well as CTLA-4 for treating GVHD and other T-cell-mediated autoimmune conditions. + The use of electric vehicles (EVs) will also be provided. TIFF2026517917000005.tif78170

Description

[Technical Field]

[0001] Reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 501,501, filed on 11 May 2023, the contents of which are incorporated herein by reference.

[0002] Statement regarding research funded by the federal government This invention was made with government support under grant numbers AI127387 and AI153326, awarded by the National Institutes of Health. The government reserves certain rights in this invention.

[0003] 1. Field The present invention relates as a whole to the fields of immunology and medicine. More specifically, the present invention relates to compositions comprising dendritic cell-derived exosomes, methods for producing said exosomes, and methods for using said exosomes. [Background technology]

[0004] 2. Explanation of related technologies Dendritic cells (DCs) are master regulators in the adaptive immune response of mammals. In this role, DCs are the T in the surrounding environment. H DCs sense signals of polarization and transmit this information, influencing the characteristics of downstream T cell responses. However, DCs are a relatively small population of circulating leukocytes. To produce sufficient numbers for therapeutic purposes, patients typically experience burdensome recruitment regimens. In addition, DC-based therapeutics are susceptible to disease-mediated immunosuppression and also carry the risk of graft-versus-host disease. Therefore, there is a need for compositions and methods that can provide the therapeutic benefits of DCs without the associated risks. [Overview of the project]

[0005] overview Based on the above, the present specification provides a therapeutic composition containing dendritic cell (DC)-derived extracellular vesicles (EVs), which can be produced from significantly fewer DCs than required for DC-based therapeutic agents, and thus reduces the need for mobilization regimens. Since DC-derived EVs are subcellular entities with unknown intrinsic capabilities in terms of both metabolism and signaling, unlike DCs, they are not affected by, for example, tumor-mediated immunosuppressive effects. In addition, due to the fact that DC-derived EVs are subcellular entities that are also not known to have cytolytic capabilities or means to drive anti-EV immunity, they can be used as off-the-shelf therapies. Therefore, it is possible to deliver allogeneic EVs without the risk of GVHD or severe graft-versus-host disease. Finally, EVs exhibit remarkable stability with respect to storage conditions. While DCs must be carefully cryopreserved and thawed to maintain their function, unlike DCs, EVs remain intact and functional over a wide temperature range (e.g., from 25°C to -80°C).

[0006] The present specification provides a method for producing extracellular vesicles (EVs) from dendritic cells (DCs), the method comprising: (i) maturing the DCs towards either T H 1 polarization or T H 2 polarization, and (ii) isolating the EVs secreted by the polarized DCs. T H The step of maturing the DCs towards 1 polarization may include maturing the DCs in the presence of IL-12 or loading the DCs with lysates and mRNA preparations derived from the same cell type. T H The step of maturing the DCs towards 2 polarization may include maturing the DCs in the presence of enterotoxin B (SEB) of S. aureus. The dendritic cells may be monocytic dendritic cells. The immature dendritic cells may be monocyte-derived dendritic cells. T H The 1-polarized DCs may be T + IL-12, T lo IL-13, and T lo CTLA-4. T HBipolar DC is IL-13 + CTLA-4 + , and IL-12 lo It is possible. H 1-polarized DC is IL-12 + , CADM1 + IL-13 lo , and CTLA-4 lo It is possible. H Bipolar DC is IL-13 + CTLA-4 + CD172a + , and IL-12 lo This may be the case. As used herein, "lo" expression refers to the expression level determined by qRT-PCR, which is up to 50% of the level observed in reverse polarized DCs, for example, T H The level of IL-13 in unipolarized DC is T H This refers to an expression level that is up to 50% of the IL-13 level in bipolar DCs.

[0007] The above method involves secreting EVs into CTLA-4 + The group and CTLA-4 neg This may further include a stage of separation into groups. CTLA-4 neg The EV group is CTLA-4 + Obtained through the depletion of EVs. CTLA-4 + The EV population is obtained by positive selection using an anti-CTLA-4 antibody bound to beads.

[0008] This specification provides compositions comprising dendritic cell-derived extracellular vesicles (EVs). Dendritic cell-derived EVs can be obtained by the methods described herein. EVs are CTLA-4 neg or CTLA-4 + It is possible. EV is T H Unipolarized DC or T H It can originate from either of the bipolarized DCs. The EV can have a diameter of approximately 30-200 nm. The EV is CD63 + CD81 + , and HLA-DR+ It is possible.

[0009] In this specification, CTLA-4 + EV and CTLA-4 neg A method is provided for separating EVs from each other in order to recover both EV populations in a physically and functionally intact state, the method comprising the steps of isolating EVs from the cell culture supernatant of mature DCs using PEG precipitation, and using CTLA-4 antibodies conjugated to beads. + Extract EV, thereby CTLA-4 + EV and CTLA-4 neg This includes the step of separating the EV from each other.

[0010] In this specification, adaptive T H 1-polarization and CD8 + A method is provided for inducing T cell activation, wherein the method involves activating T cells in CTLA-4 neg Includes the step of bringing it into contact with a DC-derived EV. CTLA-4 neg DC-derived EVs can be obtained according to the methods described herein. T cells may be present in bulk PBMCs or isolated naive T cells. T cells may be present in a subject, in which case the method includes a step of administering EVs to the subject.

[0011] In this specification, a method for treating a disease in a patient who needs it is provided, and this method is CTLA-4 neg The method includes the step of administering DC-derived extracellular viable cells (EVs) to the patient. The disease may be a viral infection or cancer. DC-derived EVs may be administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally. The method may further include the step of administering an immune checkpoint inhibitor to the patient. The immune checkpoint inhibitor may be a CTLA-4 antagonist, such as ipilimumab, pembrolizumab, or nivolumab.

[0012] In this specification, adaptive T HA method is provided to inhibit 1-polarization and T cell activation, wherein the method inhibits T cells CTLA-4 + This includes a step of contacting EVs derived from DCs. T cells are CD4 + T cells or CD8 + It could be a T cell. EV is a T H It may originate from bipolar DC. CTLA-4 + DC-derived EVs can be obtained according to the methods described herein. T cells may be present in bulk PBMCs or isolated naive T cells. T cells may be present in a subject, in which case the method includes a step of administering EVs to the subject.

[0013] This specification provides a method for treating an autoimmune condition in a patient, the method being CTLA-4 + The procedure includes administering DC-derived EVs to the patient. The autoimmune condition may be a T-cell-mediated autoimmune condition. The autoimmune condition may be post-HSCT GVHD. DC-derived EVs may be administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally.

[0014] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are provided merely as illustrations, while illustrating preferred embodiments of the present invention, for various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0015] The following drawings form part of this specification and are included to further demonstrate certain aspects of the invention. The invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Figure 1]Figures 1A-1B. Figure 1A: Upregulation of CD8+CD161+ expression in T cells stimulated by TH1-polarized DCs and TH2-polarized DCs, as well as downregulation of T cell exhaustion markers PD-1 and Tim3, are shown. Figure 1B: In CD8+CD161+ T cells primed by TH1-polarized DCs, addition of EVs derived from TH2-polarized DCs reduces IFN-γ expression and increases Tim3 expression. [Figure 2] Whole exosomes derived from DCs were separated into CTLA-4+ and CTLA-4neg fractions, which were cultured with the described non-adherent, homogeneous whole PBMCs for 7 days and analyzed by flow cytometry as shown. When PBMCs were cultured with CTLA-4neg exosomes, CD161 expression was elevated in the CD8+ cell population, while CTLA-4+ exosomes tended to inhibit CD161 expression. Granzyme B and CD25 were substantially upregulated in CD8+CD161+ cells cultured with CTLA-4neg exosomes, whereas in most CD8+CD161+ cells cultured with CTLA-4+ exosomes, granzyme B remained low and CD25 remained negative. [Figure 3]Figures 3A-3B. Effects of DC CTLA-4+ EV secretion on downstream T cell responses. Figure 3A: Only TH1 polarized DCs secreting EVs with low levels or no CTLA-4+ are capable of generating CD8+NK1.1+ T cells. By adoptively transferring 250,000 polarized DCs to mice administered imiquimod, a TLR-7 agonist, as an adjuvant, it was demonstrated that only DCs secreting low levels of CTLA-4 could produce detectable levels of highly cytolytic CD8+NK1.1+ effector memory T cells. A tenfold increase in CD8+NK1.1+ T cells was observed only among mice adopted with CTLA-4lo TH1 polarized DCs. n = 5-10 mice per group. Figure 3B: CTLA-4hi microvesicles suppress IFN-γ+ CD8+ T cell production in vitro. CTLA-4hi microvesicles derived from the culture supernatant of TH2-polarized DCs were added to a co-culture of TH1-polarized DCs and autologous T cells as responders. The addition of these CTLA-4hi microvesicles was sufficient to reduce the number of CD8+IFN-γ+ responders by 50%. *** indicates p<0.001, one-way ANOVA with Tukey post-hoc analysis. [Figure 4A]Figures 4A-4C. CTLA-4neg EVs promote T cell polarization to TH1, while CTLA-4+ EVs inhibit T cell polarization to TH1. The DCs used for exosome isolation were TH2-polarized DCs (loaded with heterologous class I and class II peptides). Whole EVs derived from these DCs were added to non-adherent PBMCs derived from buffy coat under culture conditions. Either whole EVs or isolated CTLA-4neg EVs were used as shown. Figure 4A: Analysis of whole CD4+ cells on day 2. Figure 4B: Analysis of whole CD8+ cells on day 2. Figure 4C: Analysis of whole CD8+ cells on day 6. Flow cytometry was performed on day 6 after IL-2 was added on day 5. Bulk PBMCs were stimulated with PMA + ionomycin + brefelzin A for 5 hours before flow staining. For staining on day 6, PBMCs were maintained overnight at 4°C and then stained the following day. Statistical analysis: One-way ANOVA with Tukey post-hoc test. * represents p ≤ 0.05; ** represents p ≤ 0.01; *** represents p ≤ 0.001; **** represents p ≤ 0.0001. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A]Figures 5A-5E. CTLA4neg EVs upregulate cytotoxic CD8+CD161+ levels and cytotoxic function. DCs were polarized towards TH1 by maturation in the presence of IL-12 or towards TH2 by maturation in the presence of SEB. CTLA-4+ EVs and CTLA-4neg EVs were isolated from both TH1-polarized and TH2-polarized DCs. All four EV populations were co-cultured separately with magnetically isolated CD45RA+ naive allogeneic T cells for 5 days, after which the T cell populations were further “restimulated” with a bolus of the same EV population. Flow cytometry analysis was performed on day 13 of culture. (Figure 5A) Co-culture of EVs isolated from the supernatant of TH1-polarized DCs with naive T cells. IFNγ and granzyme B levels were measured in the CD8+CD161+ population. (Figure 5B) Co-culture of EVs isolated from the supernatant of TH2 polarized DCs with naive T cells. IFNγ and granzyme B levels were measured in the CD8+CD161+ population. (Figure 5C) Expression levels of IFN-γ and CD25 among CD8+CD161+ T cells. Top panel: EVs isolated from TH1 polarized DCs. Bottom panel: EVs isolated from TH2 polarized DCs. (Figure 5D) Expression levels of granzyme B and CD25 among all CD8+ T cells. Top panel: EVs isolated from TH1 polarized DCs. Bottom panel: EVs isolated from TH2 polarized DCs. (Figure 5E) Expression levels of IFN-γ and CD25 among all CD8+ T cells. Top panel: EVs isolated from TH1 polarized DCs. Bottom panel: EVs isolated from TH2 polarized DCs. No functional differences were observed between CTLA-4+ EV populations, whether isolated from TH1-polarized DCs or TH2-polarized DCs, nor were any functional differences observed between CTLA-4neg EV populations. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 5E] See the explanation in Figure 5A. [Figure 6A] Figures 6A–6G. Extravesicular CTLA-4 expression by human monocyte-derived DCs (moDCs) is regulated by maturation conditions. Human monocytes were isolated by positive CD14 immunomagnetic selection and subsequently cultured with IL-4 and GM-CSF for 6 days. On day 6, immature DCs matured and were polarized as described. After 48 hours, mature DCs were characterized by (Figure 6A) qRT-PCR for IL-12a, IL-12b, and IL-13, while EVs were characterized by (Figure 6E) Western blot analysis; (Figure 6B) miRNA sequencing; (Figure 6C) qRT-PCR for CTLA-4; (Figure 6G) cryo-electron microscopy; (Figure 6D) mass spectrometry; and (Figure 6F) flow cytometry. Statistical analysis by one-way ANOVA with Tukey post-hoc test. ** is p ≤ 0.01; *** is p ≤ 0.001; **** is p ≤ 0.0001. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 6F] See the explanation in Figure 6A. [Figure 6G] See the explanation in Figure 6A. [Figure 7-1]Figures 7A-7L. EVs reflect the phenotype of the parental DC that polarizes the T cell response. (Figure 7A) Allogeneic T cells were cultured for 5 days with CFSE-labeled, Th-polarized, and mature DCs. EV uptake (CFSE-positive) was assayed by flow cytometry. Allogeneic T cells were cultured for 3 days with CFSE-labeled mature Th0-polarized DCs, followed by flow cytometry to measure: (Figures 7B-7C) EV uptake by CD4 T cells and CD8 T cells, and (Figures 7D-7G) differential cytokine expression by CD4 T cells and CD8 T cells based on EV uptake status. (Figure 7H) A detailed diagram of the experimental design for the data shown in the panel of Figures 7I-7L, in which allogeneic T cells were cultured with EVs derived from the culture supernatant of Cell Trace Far Red (CTFR)-labeled Th0-polarized DCs and CFSE-labeled Th1-polarized DCs. Flow cytometry analysis was performed on the third day, and (Figure 7I) EV uptake and (Figures 7J-7L) expression of effector molecules in T cells were assayed. Statistical analysis was performed using one-way ANOVA with paired t-tests or Holm-Sidak multiple comparison tests. * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001; **** indicates p ≤ 0.0001. [Figure 7-2] See the explanation in Figure 7-1. [Figure 7-3] See the explanation in Figure 7-1. [Figure 7-4] See the explanation in Figure 7-1. [Figure 7-5] See the explanation in Figure 7-1. [Figure 7-6] See the explanation in Figure 7-1. [Figure 8]Figures 8A-8F. CTLA-4+ EVs suppress IFNγ expression by T cells. (Figure 8A) Allogeneic T cells were cultured with CFSE-labeled Th0 polarized DCs in or without anti-CTLA-4 (clone: ​​BNI3), and EV uptake was assayed by flow cytometry. (Figures 8B-8D) Allogeneic T cells were cultured with whole EVs derived from Th2 DCs, or with EVs in which the CTLA-4+ subset was depleted by immunomagnetic methods. Expression of effector molecules in T cells was analyzed by flow cytometry on day 6. (Figures 8E-8F) CFSE-labeled DCs were cultured with T cells in the presence of anti-CTLA-4, and assayed for dexosome uptake. Statistical analysis by one-way ANOVA with Dunn post-hoc test. * represents p ≤ 0.05; ** represents p ≤ 0.01; *** represents p ≤ 0.001; **** represents p ≤ 0.0001. [Figure 9A] Figures 9A–9D. DCs lacking CTLA-4 drive T cell activation, which is lethal in mice. (Figure 9A) C57BL / 6 mice were transplanted with B16-Flt3L tumors and sacrificially killed on day 12 to isolate splenocytes. CTLA-4 expression by monocyte and DC subsets was assayed by flow cytometry. (Figures 9B–9C) Splenic T cells from wild-type (WT) C57BL / 6 mice or CTLA-4fl / fl CD11ccre mice (cKO) were characterized by flow cytometry for activation status and CTLA-4 expression. (Figure 9D) H&E staining was performed in lymphoid and non-lymphoid tissues of WT or cKO mice. White arrows indicate areas of leukocyte infiltration. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 9D] See the explanation in Figure 9A. [Figure 10]EVs that have lost CTLA4 promote an antitumor T cell response. In the survival curve, at day 30, the lines from top to bottom represent OVA siCTLA4, OVA siNT, IL-12 siNT, and PBS. [Modes for carrying out the invention]

[0016] Detailed explanation Dendritic cells (DCs) are master regulators in the adaptive immune response of mammals. In this role, DCs are the T in the surrounding environment. H DCs sense signals of polarization and transmit this information to influence the characteristics of downstream T cell responses. DCs transmit this information to T cells by secreting exosomes or extracellular vesicles (EVs). H Bipolar DCs preferentially secrete EVs carrying the immunomodulatory checkpoint molecule CTLA-4, while T H 1. Polarized DCs preferentially secrete EVs lacking the protein in question. This specification provides a method for producing EVs from cultures of polarized DCs, and also describes CTLA-4 + EV and CTLA-4 neg Additional procedures are also provided for separating EVs from each other and recovering both EV populations in a physically and functionally intact state. + When cultured in vitro with EV, adaptive T H 1-polarization and CD8 + T cell activation is inhibited, while CTLA-4 neg When cultured with EV, adaptive T H 1-polarization and CD8 + T cell activation is induced. CTLA-4 neg EV also identifies important memory CD8 through the expression of CD161. + It also induces the production of T cell populations. These effects are observed in both bulk PBMCs and magnetically isolated naive T cells. By producing these EVs on a large scale and administering them directly, CD8 +It is predicted that either T cell production will be promoted, or the inhibition of activated T cells will be promoted to treat autoimmune conditions, including GVHD after HSCT. Therefore, CTLA-4 to treat cancer neg The use of EVs, as well as CTLA-4 for treating GVHD and other T-cell-mediated autoimmune conditions. + The use of EVs is provided herein.

[0017] I. Definition Where used in the text of this specification, “one (a)” or “one (an)” may mean one or more. Where used in the claims of this specification, where used in conjunction with the word “including,” the words “one (a)” or “one (an)” may mean one or more.

[0018] The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly specified that it refers only to the options or that the options are mutually exclusive, but this disclosure supports the definitions of “options only” and “and / or” respectively. Where used herein, “another” may mean at least the second one, or any subsequent one.

[0019] Throughout this application, the term “approximately” is used to indicate that a value includes variations inherent to the apparatus, variations inherent to the method used to determine the value, variations present among the test subjects, or values ​​within 10 percent of a specified value.

[0020] As used herein, “essentially absent” with respect to a particular component means that the particular component is not intentionally included in the composition and / or is present only as an impurity or in trace amounts. Therefore, the total amount of the particular component resulting from any unintentional inclusion in the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is such that the particular component cannot be detected in quantity using standard analytical methods.

[0021] The term “essentially” should be understood to mean that a method or composition includes only certain steps or materials that do not substantially affect the fundamental characteristics of the method and composition.

[0022] "Treatment" or "to treat" includes: (1) inhibiting a disease in a person or patient experiencing or exhibiting a disease condition or overall symptoms (e.g., halting the further progression of the condition and / or overall symptoms); (2) improving a disease in a person or patient experiencing or exhibiting a disease condition or overall symptoms (e.g., recovering from the condition and / or overall symptoms); and / or (3) causing any measurable reduction of a disease in a person or patient experiencing or exhibiting a disease condition or overall symptoms.

[0023] "Prevention" or "prevention" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk of and / or predisposed to a disease but has not yet experienced or shown any or all of the symptoms or overall manifestations of the disease; and / or (2) delaying the onset of the symptoms or overall manifestations of a disease in a subject or patient who may be at risk of and / or predisposed to a disease but has not yet experienced or shown any or all of the symptoms or overall manifestations of the disease.

[0024] As used herein, the terms “patient” or “subject” refer to a living, mammalian organism, such as humans, monkeys, cattle, sheep, goats, dogs, cats, mice, rats, guinea pigs, or their transgenic species. In some embodiments, the patient or subject is a primate. Non-limiting examples of human patients include adults, adolescents, infants, and fetuses.

[0025] Where the term “effective” is used herein and / or in the claims, it means appropriate to achieve a desired, anticipated, or intended result. Where “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” is used in the context of treating a patient or subject with a compound, it means an amount of the compound that, when administered to a subject or patient to treat or prevent a disease, is sufficient to produce an effect that treats or prevents such a disease.

[0026] II. Extracellular vesicles of dendritic cells Methods for isolating, culturing, and priming dendritic cells from which DC-derived extracellular viable cells (EVs) can be isolated are well known in the art. For example, U.S. Patent No. 8,728,806, which is incorporated herein by whole reference, provides a detailed method for providing antigen-primed dendritic cells. In one aspect, dendritic cells for use according to this embodiment are isolated from an object to be treated by the method of this embodiment. In another aspect, dendritic cells may originate from another object, such as an HLA-matched donor. In one aspect, dendritic cells may originate from a dendritic cell bank from which HLA typing has been determined.

[0027] Methods for isolating cell populations enriched with dendritic cell progenitor cells and immature dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, dendritic cell progenitor cells and immature dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukocyte apheresis, by preparing a buffy coat, rosettering, centrifugation, density gradient centrifugation (e.g., using Ficol (e.g., FICOLL-PAQUE®), PERCOLL® (colloidal silica particles coated with non-dialysis-resistant polyvinylpyrrolidone (PVP) (15-30 mm in diameter)), sucrose, etc.), differential cell lysis, filtration, etc.). In one embodiment, leukocyte populations can be prepared, for example, by collecting blood from a subject, defibrinating to remove platelets, and lysing red blood cells. In other words, leukocyte populations can be prepared, for example, by defibrinizing blood samples to remove platelets and lysing red blood cells. In dendritic cell progenitor cells and immature dendritic cells, monocytic dendritic cell progenitor cells can optionally be enriched by centrifugation using, for example, a PERCOLL® gradient. In other contexts, dendritic cell progenitor cells can be selected using CD14 selection of peripheral blood mobilized by G-CSF. In yet another context, monocytic dendritic cell progenitor cells can be selected by differential centrifugation by size, sometimes referred to as elutriation.

[0028] Dendritic cell progenitor cells and immature dendritic cells can optionally be prepared in a closed, sterile system. As used herein, the terms “closed, sterile system” or “closed system” refer to a system in which exposure to non-sterile outside air or circulating air, or other non-sterile conditions, is minimized or absent. Closed systems for isolating dendritic cell progenitor cells and immature dendritic cells generally do not involve: density gradient centrifugation in open-top tubes, cell transfer in exposure to outside air, cell culture in tissue culture plates or in unsealed flasks, etc. In one typical embodiment, a closed system allows for the aseptic transfer of dendritic cell progenitor cells and immature dendritic cells from an initial collection container to a sealable tissue culture container without exposure to non-sterile air.

[0029] In one embodiment, monocyte-derived dendritic cell precursors are isolated by adhering them to a substrate that binds to monocytes. For example, a population of leukocytes (e.g., a population isolated by leukocyte apheresis) can be brought into contact with a substrate to which monocyte-derived dendritic cell precursors adhere. When the leukocyte population comes into contact with the substrate, the monocyte-derived dendritic cell precursors within the leukocyte population preferentially adhere to the substrate. Other leukocytes (including other potential dendritic cell precursors) exhibit reduced binding affinity to the substrate, thereby allowing for preferential enrichment of the monocyte-derived dendritic cell precursors on the surface of the substrate.

[0030] Suitable substrates include, for example, those with a larger surface area-to-volume ratio. Such substrates may be particulate or fibrous. Suitable particulate substrates include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other beads suitable for protein absorption. Suitable fibrous substrates include microcapillary tubes and microvilli membranes. Particulate or fibrous substrates generally allow for the elution of adhered monocytic dendritic progenitor cells without substantially reducing the viability of the adhered cells. Particulate or fibrous substrates may be substantially nonporous to facilitate the elution of monocytic dendritic progenitor cells or dendritic cells from the substrate. A "substantially nonporous" substrate is one in which at least the majority of the pores present in the substrate are smaller than cells, so that the number of cells trapped in the substrate is minimized.

[0031] Adhesion of monocytic dendritic cell progenitor cells to a substrate can optionally be enhanced by the addition of a binding medium. Suitable binding media include monocytic dendritic cell progenitor cell culture media (e.g., AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, etc.) to which, for example, the following are added individually or in any combination: cytokines (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4)), plasma, serum (e.g., human serum, e.g., autologous or allogeneic serum), purified proteins, e.g., serum albumin, divalent cations (e.g., calcium ions and / or magnesium ions), and other molecules that assist in the specific adhesion of monocytic dendritic cell progenitor cells to the substrate, or other molecules that prevent the adhesion of cells other than monocytic dendritic cell progenitor cells to the substrate. In some embodiments, plasma or serum may be thermally inactivated. The heat-inactivated plasma may be autologous or heterologous to the leukocytes.

[0032] After the monocytic dendritic cell progenitor cells adhere to the substrate, unadhered leukocytes are separated from the monocytic dendritic cell progenitor cell / substrate complex. Any suitable means can be used to separate the unadhered cells from the complex. For example, the mixture of unadhered leukocytes and the complex can be allowed to stand, and then the unadhered leukocytes and medium can be decanted or removed. Alternatively, the mixture can be centrifuged, and the supernatant containing the unadhered leukocytes can be decanted or removed, leaving the pelleted complex behind.

[0033] Isolated dendritic cell progenitor cells may be cultured ex vivo for differentiation, maturation, and / or expansion. Where used herein, isolated, immature dendritic cells, dendritic cell progenitor cells, T cells, and other cells refer to cells that exist by human hands, away from their natural environment, and therefore are not natural products. Isolated cells may exist in a purified form, a semi-purified form, or in a non-natural environment. Briefly, ex vivo differentiation typically involves culturing dendritic cell progenitor cells, or a cell population containing dendritic cell progenitor cells, in the presence of one or more differentiation-inducing agents. Suitable differentiation-inducing agents may be, for example, cell growth factors (e.g., cytokines, e.g., (GM-CSF), interleukin-4 (IL-4), and / or combinations thereof). In one embodiment, monocytic dendritic cell progenitor cells are differentiated to form monocytic-derived immature dendritic cells.

[0034] Dendritic cell progenitor cells can be cultured and differentiated under appropriate culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, etc. To promote cell differentiation, serum, amino acids, vitamins, cytokines, such as GM-CSF and / or IL-4, divalent cations, etc., may be added to the tissue culture medium. In one embodiment, dendritic cell progenitor cells can be cultured in serum-free medium. Such culture conditions are optional and may exclude any animal-derived products. A typical cytokine combination in a typical dendritic cell culture medium is approximately 500 units / ml each of GM-CSF (50 ng / ml) and IL-4 (10 ng / ml). When dendritic cell progenitor cells differentiate to form immature dendritic cells, they have a phenotype similar to Langerhans cells of the skin. Immature dendritic cells are typically CD14 neg and CD11c + CD80 and CD83 expression are absent, and soluble antigens can be captured via specialized endocytosis. Immature DCs express high levels of CD86.

[0035] Mature dendritic cells are formed by the maturation of immature dendritic cells. Mature DCs have lost the ability to take up antigens and exhibit upregulation of the expression of costimulatory molecules and various cytokines on their cell surface. Specifically, mature DCs express higher levels of MHC class I and II antigens than immature dendritic cells, and mature dendritic cells express CD80 + , CD83 + CD86 + , and CD14 neg It is generally identified as follows: When MHC is more highly expressed, the antigen density on the DC surface increases, and at the same time, the T cell activation signal is enhanced via counterpart co-stimulatory molecules, such as CD28 on T cells, through the upregulation of the costimulatory molecules CD80 and CD86.

[0036] For example, mononuclear cells (MNCs) are separated from recruited (e.g., recruited by granulocyte colony-stimulating factor (G-CSF)) peripheral blood progenitor cells by centrifugation at 450xg for 20 minutes on a Histopaque-1077 (Sigma, St. Louis, MO) gradient. After separation, CD14 + MNC can be isolated in a magnetic separation column using CD14 microbeads, according to the manufacturer's (Miltenyi Biotec) instructions for use. + The cells were placed in a humidified chamber at 37°C and a 5% CO2 atmosphere, in a 2x10⁶ chamber. 6 Cells may be cultured for 6 days at a concentration of cells / ml in AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with: 10% human AB serum (Atlanta Biologicals, Lawrenceville, GA), 50 μg / ml streptomycin sulfate (Invitrogen), 10 μg / ml gentamicin sulfate, 2 mM L-glutamine (Invitrogen), 50 ng / ml GM-CSF (Amgen, Thousand Oaks, CA), and 10 ng / ml IL-4 (R&D Systems, Minneapolis, MN). On day 3, the culture medium may be removed and replaced with the same amount of fresh medium. After 6 days of culture in the presence of GM-CSF and IL-4, immature DCs are harvested using enzyme-free cell dissociation buffer. Immature DCs are determined by flow cytometry to have CD11c + , CD80 neg , CD83 neg CD86 + CD209 + , and HLA-DR + This shows the phenotype.

[0037] Immature DCs may be loaded with individual recombinant proteins and / or may be transduced with any vector expressing them. For example, immature DCs may be loaded with either tumor lysate or tumor mRNA, or both. When lysate is loaded, an exemplary protocol is as follows: Immature DCs are cultured at 37°C for 3 hours at a concentration of 5x10 6 cells / ml in a 4-fold dilution of cell lysate. After loading the lysate, the cells are pelleted, washed once to remove remaining lysate, and then matured at a density of 1 - 2x10 6 cells / ml for 36 - 48 hours in: AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 10% human AB serum (Atlanta Biologicals, Lawrenceville, GA), 50 μg / ml streptomycin sulfate (Invitrogen), 10 μg / ml gentamicin sulfate, 2 mM L-glutamine (Invitrogen), 50 ng / ml GM-CSF (Amgen, Thousand Oaks, CA), 10 ng / ml IL-4 (R&D Systems, Minneapolis, MN), and ITIP [10 ng / ml IL-1β (R&D Systems), 10 ng / ml TNF-α (R&D Systems), 15 ng / ml IL-6 (R&D Systems), and 1 μg / ml PGE2 (Sigma)] (see Decker et al., Vaccine 24:3203-3216, 2006). For the purpose of stimulating DC T H polarization, for example, 10 ng / ml of SEB is further added to the medium (see Halpert et al., Stem Cells and Development 25:774-787, 2016). T H polarized DCs exhibit a phenotype of expressing low levels of IL-12 and high levels of IL-13, GATA3, CTLA-4, and SIRP-α (CD172a) when measured by qPCR. DC T HFor the purpose of stimulating polarization, for example, 1 - 2 ng / ml of IL-12 is further added to the medium. T H Polarized DC exhibits a phenotype of expressing low levels of IL-13 and CTLA-4, as well as high levels of IL-12 and CADM1 when measured by qPCR.

[0038] An exemplary electroporation protocol for loading mRNA is as follows: Immature DC is suspended in Viaspan (Barr Laboratories, Pomona, NY) at a concentration of 4x10 7 cells / ml, mixed with tumor mRNA at a concentration of 1 μg mRNA / 10 6 cells, and incubated on ice for 10 minutes in an electroporation cuvette with a 0.4 cm gap (Biorad, Hercules, CA). The cells are then electroporated using a GenePulser Xcell (Biorad) at 300 V, 150 μF, and Ω=∞. After electroporation, the cells are promptly introduced into culture and treated in the same manner as the lysate-loaded DC. The survival rate of DC after electroporation typically exceeds 75%.

[0039] Doubly loaded DC may be given lysates and mRNA preparations from the same cell type, thereby inducing H polarization. When both mRNA and lysate are loaded, the cells may be given either the lysate first and then the mRNA, or the mRNA first and then the lysate. Preferably, when both mRNA and lysate are loaded, the cells are given the mRNA first and then the lysate to avoid exposing the mRNA to any RNase that may be present in the lysate.

[0040] After loading and maturation, DC is + CD11c + CD80 +CD86 ++ CD209 + , and HLA-DR ++ This phenotype is observed. Note that as the organism matures, markers involved in co-stimulation or antigen presentation are upregulated (CD80, CD83, CD86, HLA-DR), while markers involved in adhesion are downregulated (CD11c, CD209).

[0041] The mature dendritic cells of the present invention can be prepared (i.e., matured) by contacting immature dendritic cells with an effective amount or effective concentration of a nucleic acid composition and an antigenic composition of homologous cells. The effective amount of nucleic acid composition is typically in the following ranges per culture dish or per cell: up to 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng, or up to 0.01, 0.1, 1, 5, 10 mg to 10, 15, 20, 50, 100 mg of nucleic acid, including all values ​​and ranges between them, or at least 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng, or at least 0.01, 0.1, 1, 5, 10 mg to 10, 15, 20, 50, 100 mg of nucleic acid, or about 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng of nucleic acid, or approximately 0.01, 0.1, 1, 5, 10 mg, to 10, 15, 20, 50, 100 mg. The effective amount of the cell lysate antigenic composition is typically in the following ranges per culture dish or per cell: up to 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng, or up to 0.01, 0.1, 1, 5, 10 mg to 10, 15, 20, 50, 100 mg of protein, or at least 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng, or at least 0.01, 0.1, 1, 5, 10 mg to 10, 15, 20, 50, 100 mg of protein, or about 0.01, 0.1, 1, 5, 10 ng to 10, 15, 20, 50, 100 ng, or about 0.01, 0.1, 1, 5, 10 From mg to 10, 15, 20, 50, and 100 mg of protein. In some cases, 0.001 ng of tumor antigen / cell to 1 μg of tumor lysate antigen / million cells may be used. The cell lysate antigenic composition may optionally be thermally inactivated, irradiated, or treated (e.g., exposed to proteases) before contact with the DCs.

[0042] Immature DCs are typically exposed to an effective amount of nucleic acid composition and cell lysate antigenic composition for the following periods: up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 minutes to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 minutes, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 minutes to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days. Immature DCs can be cultured and matured under appropriate maturation culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, etc.To promote cell maturation, amino acids, vitamins, cytokines, such as GM-CSF and / or IL-4, and divalent cations may be added to the tissue culture medium.

[0043] Dendritic cell maturation can be monitored by methods known in the art. Cell surface markers can be detected by assays commonly used in the art, such as flow cytometry and immunohistochemical analysis. Cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immunoassays). Dendritic cell progenitor cells, immature dendritic cells, and mature dendritic cells can be cryopreserved for later use, whether or not they have been primed with antigens. Methods for cryopreservation are well known in the art, for example, U.S. Patent No. 5,788,963, which is incorporated herein by reference in its entirety.

[0044] To isolate EVs from mature DCs, the cell culture supernatant is subjected to continuous centrifugation at room temperature for 10 minutes at 400xg, followed by 30 minutes at 2000xg, to remove pelletized cells and cell debris from the preparation. EVs present in the resulting supernatant are precipitated using PEG precipitation. For example, the resulting supernatant is incubated with cooled Total Exosome Isolation Reagent (Invitrogen, catalog number: 4478359) for 16–18 hours. Subsequently, this mixture is spun down at 10,000xg for 1 hour at 4°C. The resulting pellet is resuspended in PBS and then subjected to CTLA-4 by applying a strong magnetic field. + To facilitate the isolation / depletion of EVs, they are incubated at 4°C for 16-18 hours with anti-CTLA-4 bound to magnetic beads. + EV recovery is performed by elution in a high-salt buffer at room temperature for 2 hours. Isolated EVs may have a diameter of 30–200 nm when measured by cryo-EM. Regardless of the state of CTLA-4, EVs are CD63+ CD81 + , and HLA-DR + This exhibits the following phenotype.

[0045] III. Compositions and methods for treatment Some aspects of this embodiment involve CTLA-4 isolated from mature dendritic cells. + EV or CTLA-4 neg Related to EV administration. In particular, CD8 to treat cancer. + For the purpose of producing T cells, CTLA-4 neg EV will be administered. On the other hand, to treat T cell-mediated autoimmune conditions, such as GVHD after HSCT, CTLA-4 is used to inhibit activated T cells. + EV will be administered.

[0046] With regard to the treatment of T cell-mediated autoimmune conditions, such conditions that can be treated according to the methods described herein include, but are not limited to, post-HSCT graft-versus-host disease (GVHD), psoriasis, lichen planus, vitiligo, autoimmune type 1 diabetes mellitus (T1DM), rheumatoid arthritis, multiple sclerosis, celiac disease, and MHC-related inflammatory eye diseases (e.g., idiopathic uveitis, scatter chorioretinitis (BSR), and sympathetic ophthalmia). Furthermore, examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, Henoch-Schönlein purpura, post-streptococcal nephritis, and erythema nodosum. Nodosurn, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis obliterans, primary biliary cirrhosis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal fistula, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrous alveolitis.

[0047] With regard to the treatment of cancer, cancers that can be treated by the methods described herein include, but are not limited to, cancers of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. In some aspects, cancer can be: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; hair matrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; cord-like adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyps; adenocarcinoma within familial adenomatous polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; Basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; unencapsulated sclerotic carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant andro Blastomas; Sertoli cell carcinoma; Malignant Leydig cell tumor; Malignant lipid cell tumor; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Glomus angiosarcoma; Malignant melanoma; Apigmented melanoma; Superficial spreading melanoma; Malignant melanoma within a giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian duct mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant Mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; fetal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastoma; malignant ameloblastoma; ameloblastoma; malignant pineal glandoma; chordoma; malignant glioma; ependymoma; astrocytoma;Plasma astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granulocyte tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lateral granuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma Parkinson's disease; mycosis fungoides; other identified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basocytic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In a further context, cancers include brain cancer (e.g., glioma), prostate cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., ductal adenocarcinoma), acute myeloid leukemia (AML), melanoma, renal cell carcinoma, or chronic lymphocytic leukemia.

[0048] In some cases, the extracellular viable (EV) for use in this method is appropriately contained in a pharmaceutically acceptable carrier. The carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the EV. The EV may be formulated as a preparation for local delivery (i.e., delivery to a specific location in the body, such as skeletal muscle or other tissue) or as a preparation for systemic delivery. Suitable carriers for injectables, parenteral delivery via infusion or irrigation, and for local delivery include distilled water, phosphate-buffered saline, ordinary Ringer's solution or lactated Ringer's solution, dextrose solution, Hanks' solution, or propanediol. In addition, sterile non-volatile oils may be used as solvents or suspension media. Any biocompatible oil may be used for this purpose, including synthetic monoglycerides or diglycerides. In addition, fatty acids, such as oleic acid, are used in the preparation of injectables.

[0049] Solutions of pharmaceutical compositions can be prepared in water that is appropriately mixed with a surfactant, such as hydroxypropyl cellulose. The compositions of this disclosure may include glycerol, liquid polyethylene glycol, and mixtures thereof. Under normal conditions for storage and use, these preparations contain preservatives to prevent microbial growth.

[0050] In certain situations, it is beneficial for pharmaceutical compositions to be administered in the form of an injectable composition, either as a liquid solution or a suspension. Typical compositions for such purposes include pharmaceutically acceptable carriers. For example, a composition may contain the following amounts of human serum albumin per milliliter of phosphate-buffered saline: less than 10 mg, 25 mg, or 50 mg; or 10 mg, 25 mg, or 50 mg; or more than 10 mg, 25 mg, or 50 mg; or up to approximately 100 mg. Other pharmaceutically acceptable carriers include aqueous solutions and non-toxic excipients, which include salts, preservatives, buffers, etc.

[0051] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, physiological saline, and parenteral media such as sodium chloride and dextrose-added ringer. Intravenous media include fluids and nutrient supplements. Preservatives include antimicrobial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH of the pharmaceutical composition and the exact concentrations of the various components are adjusted according to well-known parameters.

[0052] In a further context, pharmaceutical compositions containing EV may include classical pharmaceutical preparations. In some embodiments, the compositions may be administered to a subject by any method known to those skilled in the art. Examples of such methods include: intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intratumoral, peritumoral, intramuscular, subcutaneous, intravesicularly, mucosal, injection, infusion, serial infusion, catheter, or by any other method or any combination thereof known to those skilled in the art. In some contexts, administration of a pharmaceutical composition containing EV may be via any common route available to the target tissue. Administration may be by injection, intradermal, subcutaneous, intramuscular, intratumoral, peritumoral, intraperitoneal, or intravenous. Such compositions may be administered as pharmaceutically acceptable compositions, typically comprising pharmaceutically acceptable carriers, buffers, or other excipients. In some aspects of this embodiment, EV is administered to a site of lymphoid tissue proximal to the diseased cell population in a subject. The site of lymphoid tissue may be lymphoid tissue flowing in from the surrounding tissue of the diseased cell population. For example, in some aspects, EV is administered to lymph nodes flowing in from the surrounding tissue of the diseased cell population. In some aspects, EV is administered to tissue flowing into a site of lymphoid tissue proximal to the diseased cell population in a subject.

[0053] The effective dose of a pharmaceutical composition is determined based on the intended target. The terms "unit dose" or "dosage" refer to a physically separated unit suitable for use in the subject, where each unit contains a predetermined amount of the pharmaceutical composition, calculated to produce the desired response discussed above, in conjunction with its mode of administration, i.e., the appropriate route and treatment regimen. The amount administered varies depending on the desired protection or effect, taking into account both the number of treatments and the unit dose.

[0054] The exact amount of a pharmaceutical composition also varies depending on the physician's judgment and is unique to each individual. Factors influencing the dosage include: the patient's physical and clinical condition, the route of administration, the intended purpose of the treatment (e.g., symptom relief or cure), and the efficacy, stability, and toxicity of the specific therapeutic substance used. In some cases, the actual dosage of a composition administered to a patient or subject may be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, past or present therapeutic interventions, patient idiopathy, and route of administration. In any case, the physician administering the treatment will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0055] In one embodiment, the pharmaceutical composition may contain, for example, at least about 0.1% (w / v) of an active substance, such as isolated EV. In another embodiment, the EV may constitute, for example, about 2% (w / v) to about 75% (w / v) of a unit, or about 25% (w / v) to about 60% (w / v), and any range that can be derived therefrom. In other non-limiting examples, doses may also include: per single dose, from approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body 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 milligrams / kg / body weight, approximately 10 milligrams / kg / body weight, approximately 50 milligrams / kg / body weight, approximately 100 milligrams / kg / body weight, approximately 200 milligrams / kg / body weight, approximately 350 milligrams / kg / body weight, approximately 500 milligrams / kg / body weight, up to approximately 1000 mg / kg / body weight or more, and any range that can be derived within those amounts. In non-limiting examples derived from the values ​​listed herein, doses may be administered in ranges such as approximately 5 micrograms / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight.

[0056] The above method may involve administering a composition containing (or a composition containing) the following: about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4. 1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 9, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 6 70, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 92 5, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2 800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8 000, 9000, 10000 nanograms (ng), or approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1. 4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 310 0, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms (meg), or approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4 0.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8. 0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14 0.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 2 90, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 47 0, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725 ,730,740,750,760,770,775,780,790,800,810,820,825,830,840,850,860,870,875,880,890,900,910,920,925,930,940,950,960,970,975,980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3 400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg), or approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100 , 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 grams, exosomes, at least about 0.01, 0.02, 0 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3 , 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8 0.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5,17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970、975、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、2600、2700、2800、2900、3000、3100、3200、3300、3400、3500、3600、3700、3800、3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 nanograms (ng), Or at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 , 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8 0.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135 ,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 7 30, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 99 0, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 340 0, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms (meg), or at least approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2. 0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970、975、980、990、1000、1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg), or at least about 0.0 1, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8 0.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16 0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 ,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 5 00, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 76 0, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1 200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3 Exosomes in amounts of 700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, and 10000 grams, or up to approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1 400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3 900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 nanograms (ng), or up to approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2. 7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970、975、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、2600、2700、2800、2900、3000、3100、3200、3300、3400、3500、3600、3700、3800、3900、4000、4100、4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms (meg), or up to approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0. 06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 8 10, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600 , 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100 , 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg), or up to approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2. 9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 , 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、445、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970、975、980、990、1000、1100、1200、1300、1400、1500、1600、1700、Exosomes of 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, and 10000 grams, or any range that can be derived within them. The above figures may also represent the dosage administered to the patient based on their weight, and this dosage may be expressed as ng / kg, mg / kg, or g / kg, or as any range deriveable from the above figures.

[0057] Alternatively, the composition may have the following exosome concentrations: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 0.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, ​​5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 ,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,410,420,425,430,440,441,450,460,470,475,480,490,500,510,520,525,530,540,550,560,570,5 75, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 ng / ml, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、410、420、425、430、440、441、450、460、470、475、480、490、500、510、520、525、530、540、550、560、570、575、580、590、600、610、620、625、630、640、650、660、670、675、680、690、700、710、720、725、730、740、750、760、770、775、780、790、800、810、820、825、830、840、850、860、870、875、880、890、900、910、920、925、930、940、950、960、970、975、980、990、1000 μg / ml、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 ,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,410,420,425,430,440,441,450,460,470,475,480,490,500,510,520,525,530,540,550,560,570,5 75, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 mg / ml, or 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 ,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,410,420,425,430,440,441,450,460,470,475,480,490,500,510,520,525,530,540,550,560,570,5 75, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 g / ml, or any range that can be derived from these.

[0058] The composition may be administered to (or ingested by) a patient in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, or in any range that can be derived therefrom, and the composition may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or every 1, 2, 3, 4, 5, 6, 7 days, or every 1, 2, 3, 4, 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or in any range that can be derived therefrom. The composition is particularly intended to be administered once, twice, three, four, five, or six times a day (or any range that can be derived therefrom) and / or as needed by the patient. Alternatively, the composition may be administered to or by the patient every two, four, six, eight, twelve, or 24 hours (or any range that can be derived therefrom). In some embodiments, the patient receives the composition over a period of time or in a certain number of doses after experiencing symptoms of demyelinating disorder.

[0059] IV. Combination Therapy To enhance the efficacy of dendritic cell-derived extravasation therapy, it may be desirable to combine these compositions with other active ingredients that are effective in treating the disease of interest.

[0060] As a non-limiting example, cancer treatment may be carried out using the primed dendritic cell composition of this embodiment together with other anticancer agents. The “anticancer” agent has the ability to negatively affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or extending the lifespan of a subject with cancer. More generally, these other compositions may be provided in a total amount effective in killing or inhibiting the proliferation of cancer cells. This process may involve simultaneously contacting cells with an anticancer peptide or nanoparticle complex and an active substance or multiple factors. This can be achieved by contacting cells with one composition or pharmaceutical preparation containing both active ingredients, or by simultaneously contacting cells with two different compositions or pharmaceutical preparations, one of which comprises a dendritic cell composition and the other comprises a second active ingredient.

[0061] Treatment with the dendritic cell composition may be performed before or after treatment with other active agents, with an interval ranging from several minutes to several weeks between treatments with other active agents. In embodiments where other active agents and the dendritic cell composition are applied to the subject separately, it is generally advisable to avoid long intervals between the delivery times so that the active agents and the dendritic cell composition can continue to exert beneficial synergistic effects on the cells. In such examples, it is intended that the cells may come into contact with each of the modalities within approximately 12 to 24 hours, and more preferably within approximately 6 to 12 hours. In some situations, it may be desirable to significantly extend the treatment period by including a drug-free interval of several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0062] When dendritic cell-derived MV therapy is "A" and the second active agent, such as radiotherapy, chemotherapy, or an anti-inflammatory agent, is "B", the following various combinations may be used. In one embodiment, the administration of the dendritic cell therapy of this embodiment to a patient is carried out in accordance with a general protocol for administering chemotherapy, taking into consideration, in some cases, the toxicity of the vector. The treatment cycle is expected to be repeated as needed. It is also intended that various standard treatments and surgical interventions may be applied in combination with the described therapies for hyperproliferative cells.

[0063] A. Chemotherapy Cancer treatments also include various combination therapies. In some aspects, the dendritic cell-derived MV composition of this embodiment is administered (or formulated) together with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor, which is, for example, an inhibitor of EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF. Non-exclusive examples of protein kinase inhibitors include: afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, hostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23 451, vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridahorolimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0064] Further combination chemotherapy includes, for example: alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocon, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog, topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs, adzeresin, karzeresin, and bizeresin); and cryptophycin. (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eloiterobin; pankratistan; sarcodictiin; spongstatin; nitrogen mustard, such as chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimus Examples include thin, trophosphamide, and uracil mustard; nitrosureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as engine antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1); dynemycin, including dynemycin A; bisphosphonates such as clodronate; esperamycin;Furthermore, neocardinostatin chromophores and related pigment proteins, enediin antibiotic chromophores, aclasinomycin, actinomycin, authrarnnycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- Oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, such as mitomycin C, mycophenolic acid, nogalarnycin, olibomycin, peplomycin, potfiromycin, puro Antibiotics such as mycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, and thiamipri , thioguanine, etc.; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal agents such as mitotane, trilostane, etc.; folic acid supplements such as frolinic acid, etc.; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecolsin; diazicon;Elformithine; eriptinium acetate; epotilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; mayansinoids such as maytansine and anthamitosine; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2''-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A) A) Loridine A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids such as paclitaxel and docetaxel, and gemcitabine; 6-thioguanine; Mercaptopurine; Platinum complexes such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In one embodiment, the compositions provided herein may be used in combination with gefitinib. In another embodiment, this embodiment may be carried out in combination with Gleevac (for example, about 400 to about 800 mg / day of Gleevac may be administered to the patient). In one embodiment, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.

[0065] B. Immunotherapy Those skilled in the art will understand that immunotherapy may be used in combination with or in conjunction with the methods of this embodiment. In general, immunotherapeutic agents, in relation to the treatment of cancer, require the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Immune effectors can be antibodies, for example, that are specific to some marker on the surface of tumor cells. Antibodies may act alone as effectors of therapy, or antibodies may mobilize other cells that actually carry out the cell killing. Antibodies may also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) or simply act as targeted agents. Alternatively, the effector may be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0066] In one aspect of immunotherapy, tumor cells need to carry some marker suitable for targeting, i.e., a marker not present in most other cells. Many tumor markers exist, and any of them may be suitable for targeting in this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy is combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0067] Examples of immunotherapies currently under investigation or in use include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapies such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapies such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0068] In some embodiments, immunotherapy may involve immune checkpoint inhibitors. Immune checkpoints either enhance or weaken signals (e.g., costimulatory molecules). Immune checkpoint proteins that can be targeted in immune checkpoint inhibition include: adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), and B and T lymphocyte attenuation factors. T-cell immunoreceptors (T-cell immunoreceptors) (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), HLA-DRB1, ICOS (also known as CD278), HLA-DQA1, HLA-E, indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, OX40 (also known as CD134), programmed death 1 (PD-1), programmed death ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T-cell immunoreceptors (T-cell immunoreceptors) having an Ig domain and an ITIM domain. These include immunoreceptors with Ig and ITIM domains (TIGIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), V-domain Ig suppressor of T-cell activation (VISTA, also known as C10orf54), and 4-1BB (CD137). In particular, immune checkpoint inhibitors target the PD-1 system and / or CTLA-4.

[0069] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant ligands or receptors, or antibodies such as human antibodies (e.g., International Publication No. 2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-264, 2012; both publications are incorporated herein by reference). Known inhibitors or analogues of immune checkpoint proteins may be used, in particular chimeric antibodies, humanized antibodies, or humanized antibodies. As those skilled in the art will understand, several antibodies referred to in this disclosure may be known by their aliases and / or synonyms. Such aliases and / or synonyms are interchangeable in the context of this disclosure. For example, lambrolizumab is known to be also known by its alias and synonym MK-3475 and pembrolizumab.

[0070] In some embodiments, a PD-1-binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In one particular aspect, the ligand-binding partner of PD-1 is PD-L1 and / or PD-L2. In another embodiment, a PD-L1-binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In one particular aspect, the binding partner of PD-L1 is PD-1 and / or B7-1. In another embodiment, a PD-L2-binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In one particular aspect, the binding partner of PD-L2 is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 antagonists for use in the methods provided herein are also known in the art, such as those described in U.S. Patent Publications 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0071] In some embodiments, the PD-1-binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1-binding antagonist is an immunoadhesin (e.g., an immunoadhesin containing the extracellular portion or PD-1-binding portion of PD-L1 or PD-L2, fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1-binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody and is described in International Publication No. 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody and is described in International Publication No. 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody and is described in International Publication No. 2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor and is described in International Publication Nos. 2010 / 027827 and WO2011 / 066342.

[0072] Another immune checkpoint protein that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is analogous to CD28, a T cell co-stimulatory protein, and both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells, which may be important for the function of these cells. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0073] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human CTLA-4 antibody (or a VH domain and / or VL domain derived therefrom) suitable for use in this method can be prepared using methods well known in the art. Alternatively, an anti-CTLA-4 antibody recognized in the art may be used. For example, the anti-CTLA-4 antibodies disclosed below may be used in the methods disclosed herein: U.S. Patent No. 8,119,129; International Publication Nos. 01 / 14424, 98 / 42752, and 00 / 37504 (CP675,206, also known as tremelimumab; formerly known as tisilimmab); U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA, 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology, 22(145): Abstract No. 2505 (Antibody CP-675206); and Mokyr et al. (1998) Cancer Res, 58:5301-5304. The teachings of each of the above publications are incorporated herein by reference. With regard to binding to CTLA-4, antibodies that compete with any of the aforementioned antibodies recognized in the Art may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Publication Nos. 2001 / 014424, 2000 / 037504, and U.S. Patent No. 8,017,114; all of these are incorporated herein by reference.

[0074] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, International Publication No. 01 / 14424). In other embodiments, the antibody comprises the CDR or VR of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on CTLA-4 as the antibody described above. In another embodiment, the antibody has at least about 90% amino acid sequence identity in the variable region with respect to the antibody described above (for example, at least about 90%, 95%, or 99% identity in the variable region with respect to ipilimumab). Other molecules for modulating CTLA-4 include ligands and receptors for CTLA-4, such as those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Publication Nos. 1,995,001,994 and 1,998,042,752, all incorporated herein by reference, and immunoadhesins, such as those described in U.S. Patent No. 8,329,867, also incorporated herein by reference.

[0075] Another immune checkpoint protein that may be targeted in the methods provided herein is lymphocyte-activating gene 3 (LAG-3), also known as CD223. The complete protein sequence of human LAG-3 has Genbank accession number NP-002277. LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an "off" switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 activates both effector T cells and inhibitory regulatory T cells. In some embodiments, the immune checkpoint inhibitor is an anti-LAG-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. Anti-human LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in these methods can be prepared using methods well known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art may be used. Exemplary anti-LAG-3 antibodies include relatrimab (also known as BMS-986016) or its antigen-binding fragment and variants (see, for example, International Publication No. 2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, for example, International Publication No. 2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in International Publication No. 2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in International Publication No. 2017 / 220569.

[0076] Another immune checkpoint protein that may be targeted in the methods provided herein is the V-domain Ig suppressor of T cell activation (VISTA), also known as C10orf54. The complete protein sequence of human VISTA has Genbank accession number NP_071436. VISTA is found in leukocytes and inhibits the effector function of T cells. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human VISTA antibody (or the VH domain and / or VL domain derived therefrom) suitable for use in these methods can be prepared using methods well known in the art. Alternatively, an anti-VISTA antibody recognized in the art may be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as ombachilimab) (see, for example, International Publication Nos. 2015 / 097536, 2016 / 207717, 2017 / 137830, and 2017 / 175058). VISTA is also inhibited by the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, for example, International Publication Nos. 2015 / 033299 and 2015 / 033301).

[0077] Another immune checkpoint protein that may be targeted in the methods provided herein is indoleamine 2,3-dioxygenase (IDO). The complete protein sequence of human IDO has Genbank accession number NP_002155. In some embodiments, the immune checkpoint inhibitor is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and napoximod (GDC-0919).

[0078] Another immune checkpoint protein that may be targeted in the methods provided herein is CD38. The complete protein sequence of human CD38 has Genbank accession number NP_001766. In some embodiments, the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human CD38 antibody (or its VH and / or VL domain) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, an anti-CD38 antibody recognized in the art may be used. An exemplary anti-CD38 antibody is daratumumab (see, for example, U.S. Patent No. 7,829,673).

[0079] Another immune checkpoint protein that may be targeted in the methods provided herein is ICOS, also known as CD278. The complete protein sequence of human ICOS has the Genbank accession number NP_036224. In some embodiments, the immune checkpoint inhibitor is an anti-ICOS antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human ICOS antibody (or the VH and / or VL domain derived therefrom) suitable for use in the methods herein can be prepared using methods well known in the art. Alternatively, an anti-ICOS antibody recognized in the art may be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, for example, International Publication Nos. 2016 / 154177 and 2018 / 187191) and GSK3359609 (see, for example, International Publication No. 2016 / 059602).

[0080] Another immune checkpoint protein that may be targeted in the methods provided herein is the T cell immune receptor (TIGIT), which has an Ig domain and an ITIM domain. The complete protein sequence of human TIGIT has Genbank accession number NP_776160. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human TIGIT antibody (or a VH domain and / or VL domain derived therefrom) suitable for use in the methods can be prepared using methods well known in the art. Alternatively, an anti-TIGIT antibody recognized in the art may be used. An example anti-TIGIT antibody is MK-7684 (see, for example, International Publication Nos. 2017 / 030823 and 2016 / 028656).

[0081] Another immune checkpoint protein that may be targeted in the methods provided herein is OX40, also known as CD134. The complete protein sequence of human OX40 has the Genbank accession number NP_003318. In some embodiments, the immune checkpoint inhibitor is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human OX40 antibody (or the VH and / or VL domain derived therefrom) suitable for use in the methods herein can be prepared using methods well known in the art. Alternatively, an anti-OX40 antibody recognized in the art may be used. An exemplary anti-OX40 antibody is PF-04518600 (see, for example, International Publication No. 2017 / 130076). ATOR-1015 is a bispecific antibody that targets CTLA4 and OX40 (see, for example, International Publication Nos. 2017 / 182672, 2018 / 091740, 2018 / 202649, and 2018 / 002339).

[0082] Another immune checkpoint protein that may be targeted in the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), also known as TNFRSF18 and AITR. The complete protein sequence of human GITR has Genbank accession number NP_004186. In some embodiments, the immune checkpoint inhibitor is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human GITR antibody (or its VH and / or VL domain) suitable for use in the methods herein can be prepared using methods well known in the art. Alternatively, an anti-GITR antibody recognized in the art may be used. An exemplary anti-GITR antibody is TRX518 (see, for example, International Publication No. 2006 / 105021).

[0083] Another immune checkpoint protein that may be targeted in the methods provided herein is T-cell immunoglobulin and mucin-domain containing-3 (TIM3), also known as HAVCR2. The complete protein sequence of human TIM3 has Genbank accession number NP_116171. In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human TIM3 antibody (or its VH and / or VL domain) suitable for use in the methods herein can be prepared using methods well known in the art. Alternatively, an anti-TIM3 antibody recognized in the art may be used. Exemplary anti-TIM3 antibodies include LY3321367 (see, for example, International Publication No. 2018 / 039020), MBG453 (see, for example, International Publication No. 2015 / 117002), and TSR-022 (see, for example, International Publication No. 2018 / 085469).

[0084] Other immune checkpoint proteins that may be targeted in the methods provided herein are 4-1BB, also known as CD137, TNFRSF9, and ILA. The complete protein sequence of human 4-1BB has Genbank accession number NP_001552. In some embodiments, the immune checkpoint inhibitor is an anti-4-1BB antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. An anti-human 4-1BB antibody (or its VH and / or VL domain) suitable for use in the methods herein can be prepared using methods well known in the art. Alternatively, an anti-4-1BB antibody recognized in the art may be used. An exemplary anti-4-1BB antibody is PF-05082566 (utomirumab; see, for example, International Publication No. 2012 / 032433).

[0085] In one embodiment, immunotherapy may be adoptive immunotherapy, which involves the transfer of antigen-specific autologous T cells produced ex vivo. T cells used in adoptive immunotherapy can be produced either by expanding antigen-specific T cells or by redirecting T cells through genetic engineering. Isolation and transfer of tumor-specific T cells have shown success in the treatment of melanoma. Genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) has successfully induced novel specificity in T cells. CARs are synthetic receptors consisting of a targeting moety to which one or more signaling domains are bound within a single fusion molecule. Typically, the binding moety of a CAR consists of the antigen-binding domain (scFv) of a single-chain antibody, which includes a light chain fragment and a variable fragment of a monoclonal antibody linked by a flexible linker. The use of binding moeties based on receptor domains or ligand domains has also been successful. The signaling domains of first-generation CARs are derived from the cytoplasmic region of the CD3ζ chain or the Fc receptor γ chain. CARs have successfully redirected T cells to antigens expressed on the surface of tumor cells from various malignant tumors, including lymphomas and solid tumors.

[0086] In one embodiment, the present application provides a combination therapy for treating cancer, wherein the combination therapy comprises adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells target a tumor antigen. The engineered hSDH enzyme may be administered to the patient before and / or concurrently with the administration of the adoptive T cell therapy. In another aspect, the autologous and / or allogeneic T cells may be engineered to express the engineered hSDH enzyme.

[0087] C. Radiation therapy Other widely used factors that cause DNA damage include those commonly known as gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of factors that damage DNA are also intended, such as microwave and UV irradiation. All of these factors are very likely to cause widespread damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50-200 roentgens for long-term exposure (3-4 weeks) to a single dose of 2000-6000 roentgens. The dose range for radioisotopes is highly variable and changes depending on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0088] When applied to cells, the terms “contact” and “exposure” are used herein to describe a process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to or positioned directly adjacent to target cells. For the purpose of achieving cell elimination or quiescence, both agents are delivered to the cells in a total amount of a combination effective in killing the cells or preventing their division.

[0089] D. Gene therapy In yet another embodiment, the second treatment is gene therapy, in which the therapeutic polynucleotide is administered before, after, or concurrently with the therapeutic composition. Viral vectors for expressing gene products are well known in the art and include eukaryotic expression systems such as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papillomaviruses, and also include SV40. Alternatively, the administration of the expression construct can be achieved using liposomes or lipid-based vectors such as DOTAP: cholesterol vesicles.

[0090] E. Surgical procedures Approximately 60% of people with cancer undergo some type of surgical procedure, including surgical procedures for prevention, diagnosis or staging, treatment, and palliative care. Surgical procedures for treatment are cancer treatments that may be used in conjunction with other therapies, such as the procedures provided herein, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0091] Surgical procedures for treatment include excision, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor excision refers to the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs procedure). This embodiment is further intended to be used in conjunction with the removal of superficial cancer, precancerous tumors, or amounts of normal tissue that occur incidentally. In some cases, after tumor removal, the dendritic cell composition of this embodiment is administered to lymphoid tissue flowing from the site where the tumor was previously located.

[0092] V. Kit Several embodiments relate to kits, such as diagnostic kits and therapeutic kits, as well as kits for preparing and / or delivering exosomes or MVs. For example, a kit may include one or more pharmaceutical compositions described herein and, optionally, instructions for use thereof. A kit may also include one or more devices for achieving the administration of such compositions. For example, the kit in question may include a pharmaceutical composition and a catheter for achieving direct administration of the composition to a patient having or at risk of demyelinating disorder. In other embodiments, the kit in question may include ampoules pre-filled with isolated exosomes, which are optionally formulated as pharmaceuticals or lyophilized for use with a delivery device. The techniques described herein also include kits for producing MVs from dendritic cells.

[0093] The kit may include labeled containers. Suitable containers include, for example, bottles, vials, and test tubes. Containers may be made from a variety of materials, such as glass or plastic. Containers may hold compositions containing antibodies effective for therapeutic or non-therapeutic uses, such as those described above. Labels on the containers may indicate that the composition is for use in a particular therapy or for a particular non-therapeutic use, and may also indicate instructions for either in vivo or in vitro use, such as those described above. In some embodiments, the kit includes the containers described above, as well as one or more other containers containing materials desirable from a commercial and user perspective, such as buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. [Examples]

[0094] VI. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments are representative of the techniques that the inventors have found to be sufficiently functional for practicing the present invention, and can therefore be considered to constitute a preferred mode for practicing the present invention. However, those skilled in the art will understand that, in light of this disclosure, many modifications are possible in the particular embodiments disclosed, and in such cases, similar or equivalent results can be obtained without departing from the spirit and scope of the present invention.

[0095] Example 1 In summary, the data included herein is based on DC's CTLA-4 + Extracellular vesicles and CTLA-4 neg It has been demonstrated that both extracellular vesicles are expressed and secreted. H Unipolarized DC is CTLA-4 + CTLA-4 at a higher rate than EV neg EV secretes and T H Bipolar DC is CTLA-4 + CTLA-4 at a lower rate than EV neg EVs secrete; however, both types of EVs are DC T H It can be recovered regardless of its polarization state, and similarly, its functional properties appear to be present regardless of the polarization state of the DC. CTLA-4 neg EVs can turn T cells into T cells. H It has the ability to induce a unipolar phenotype, inducing upregulation of IFN-γ, granzyme B, CD25, and CD161 expression, and inducing a higher CD8 to CD4 ratio in proliferating T cell blasts. CTLA-4 + EV produces the opposite effect, T H This induces unipolarization and downregulation of activation markers, as well as upregulation of fatigue markers such as PD-1 and Tim3. These data are from CTLA-4. +This indicates that EV or "dexosomes" can be administered on a case-by-case basis in a manner useful for improving autoimmune conditions, parenchymal organ transplant rejection, or GVHD after HSCT. In contrast, CTLA-4 neg EV / dexosomes are used in T, including for chronic viral infections or cancer immunotherapy. H 1. It may be administered in situations where enhancement of the immune response is required.

[0096] Example 2 Dendritic cells (DCs) are crucial mediators of immunity, bridging the gap between the innate and adaptive immune responses. Dendritic cells not only participate in frontline defense against pathogens and tumors (innate immunity), but also drive subsequent adaptive immune responses, specifying the strength and nature of such responses. How dendritic cells help to modulate adaptive immune responses to ongoing attacks is an area that has been thoroughly investigated, as the mechanism involves the accumulation of external and internal signals that drive the differentiation and maturation pathways of DCs, particularly with respect to surface receptors and soluble inflammatory mediators expressed by DCs (Cella et al., 1999; Sallusto et al., 1995; Sallusto et al., 1994). In addition, DCs release extracellular vesicles (EVs), and it has been reported that EVs themselves influence the immune response in various ways (Segura et al., 2005; Thery et al., 2002; Wakim et al., 2011).

[0097] As described in other reviews (Thery et al., 2009), extracellular vesicles (EVs) are membrane-bound entities, 30–1000 nm in size, that carry cargo and are released from cells both under steady state and upon stimulation. These vesicles can be classified based on their size, mode of biosynthesis, ultracentrifugation characteristics, and contents (Thery et al., 2009). In the case of exosomes (30–150 nm), parental cell proteins and nucleic acids (mainly miRNAs) become targets of the endosomal membrane, which then invaginates to encapsulate the targeted cargo, resulting in the formation of a multivesicular body (MVB) with vesicles present within the endosome. These vesicles are released into the extracellular space when the MVB membrane fuses with the plasma membrane (Babst et al., 2002; Odorizzi et al., 1998; Saksena et al., 2007; Stuffers et al., 2009). Alternatively, budding of the plasma membrane to the outside gives rise to microvesicles (100–1000 nm), ectosomes (50–200 nm), and apoptotic bodies (50–500 nm), which can be distinguished based on their size, cargo, and biosynthesis (Thery et al., 2009; Heinen et al., 1999). DC-derived extracellular entities (EVs) are important mediators of the immune response by transporting antigens between DCs and by their ability to prime the immune response independently of parental DCs (Segura et al., 2005; Thery et al., 2002; Wakin et al., 2011). Due to these immunomodulatory effects and other properties unique to EVs, DC-derived EVs (dexosomes) are a preferred choice for off-the-shelf cell-based immunotherapy. Specifically, because EVs are subcellular entities with no known direct ability to lyse cells, the risk of graft-versus-host disease is reduced.In addition, producing a sufficient number of DCs for cell-based vaccination can be technically challenging, and chemotherapy involving the recruitment of these cells negatively impacts the patient's quality of life. Dexosomes, however, offer a novel modality because they can arise from DCs with relatively fewer antigen-specific responses. Finally, while cell-based vaccines are often hindered by the immunosuppressive tumor microenvironment, dexosomes, being subcellular entities, may be unaffected by such a tumor microenvironment.

[0098] In our attempts to utilize the therapeutic potential of dexosomes, our previous research (Halpert et al., 2016) and this study have demonstrated that the immunostimulatory effects of dexosomes are suppressed by the presence of the immunosuppressive molecule CTLA-4. We previously confirmed that CTLA-4 expression is upregulated in mature DCs and subsequently packaged into dexosomes, which are then released into the extracellular space. The uptake of these dexosomes by bystander DCs resulted in a decrease in surface B7 in the bystanders in a manner dependent on the CTLA-4 receptors CD80 and CD86. Furthermore, when DCs treated with CTLA-4 siRNA were co-cultured with autologous T cells, CD88 was reduced compared to DCs treated with non-targeting siRNA (NT-siRNA). + CD25 + IFNγ + It significantly increases the proportion of T cells, and at the same time, Treg(CD4 + CD25 + FoxP3 +This reduced the frequency of occurrence of ) . In a B16 mouse melanoma model, antitumor immunity and survival rates were significantly elevated in recipients of DC vaccines treated with CTLA-4 siRNA compared to recipients of DC vaccines treated with NT-siRNA. These pieces of evidence suggest that CTLA-4, and effectively dexosomes, are key mediators of the DC-driven CD8 T cell response in vitro and in vivo (Halpert et al., 2016).

[0099] In this specification, the inventors apply these prior findings to the effector function of T cells, particularly in the generation of effective type 1 (T1) immunity, and CTLA-4 + To further enhance this, we investigate the direct action of dexosomes. Since synapses between T cells and DCs are critically important for generating a robust immune response, and therefore proximity between DCs and T cells is also critically important, we have developed CTLA-4 + We hypothesized that dexosomes can directly regulate the T1 response to suppress the generation of abnormal programs that destroy tissue. This was based on the inventor's human monocyte-derived dendritic cell model, the conditional knockout mouse (cKO) model (CTLA-4 fl / fl CD11c Cre Using ), and a mouse tumor model, the inventors have identified CTLA-4 + We have demonstrated that dexosomes suppress the generation of a robust T1 immune response, which suggests potential applications in the design of DC-based immunotherapies and in understanding pathological conditions related to the T1 response.

[0100] CTLA-4 levels in dendritic cells (DCs) are regulated by in vitro maturation conditions. In a steady state, the majority of human DCs reside in tissues, with only a relatively small number detectable in peripheral blood. Therefore, collecting tissue-present DCs for therapeutic and research purposes is often only possible in conditions requiring tissue biopsy, bone marrow, or the collection of an entire organ. Thus, our efforts to elucidate the biological characteristics of this leukocyte population, which is difficult to collect in humans, have primarily utilized the in vitro production of monocyte-derived DCs (MoDCs) isolated from peripheral blood. Regarding the full therapeutic potential of DCs, past limitations to therapeutic success should be addressable by identifying previous impairments and finding ways to circumvent them (Laureano et al., 2022). For this purpose, our previous research identified in vitro conditions under which moDCs can polarize toward a phenotype that promotes either a Th1 response or a Th2 response. These moDCs can also mature in a manner that drives a T cell response that is not selective for either the Th1 or Th2 response (hereinafter referred to as Th0 DCs). Within this paradigm, we confirmed that Th1 DCs are characterized by elevated expression of IL12A and IL12B, while Th2 DCs express very little IL12A, low levels of IL12B, but significantly elevated levels of IL13 and CTLA4 (Figures 6A and 6C). Th0 DCs, on the other hand, express intermediate levels of IL12A, IL12B, and CTLA4, but express very little IL13.

[0101] Of particular interest is the role of moDC-derived dexosomes in this Th polarization paradigm, as their immunomodulatory effects, while not fully elucidated, have been considerably documented (Segura et al., 2005; Thery et al., 2002; Wakim et al., 2011). Therefore, the inventors have enhanced their previous findings through miRNASeq analysis of Th0 and Th2 dexosomes, aiming to identify differences in miRNA cargo within dexosomes, thereby deepening our understanding of the differential effects of dexosomes on T cell responses. By analyzing vesicles from three independent donors, the inventors identified 69 miRNA species that appeared differentially in Th0 dexosomes compared to Th2 dexosomes (Figure 6B). The protein cargo carried by dexosomes is another important element in elucidating the role of dexosomes in different Th polarizations. Therefore, the inventors have found that the dexosomes of moDC are CTLA-4 + It was estimated that a subset of CTLA-4 would be included, and that its levels would vary based on the polarization state of the parent DC. For this purpose, the inventors extracted CTLA-4 from bulk dexosome isolates of immature moDCs or Th2 moDCs. + Dexosomes were immunoprecipitated and then subjected to Western blot analysis. CTLA-4 + Subset and CTLA-4 neg Both subsets contained the extracellular vesicle marker tetraspanin CD63, but CTLA-4 negThe inventors confirmed that only a subset was positive for ubiquitinated proteins (Figure 1E). This helps to understand the differences in biosynthesis among these dexosome subsets. Ubiquitination has been shown to drive the packaging of proteins into multivesicular bodies (MVBs), which are destined to be released as extracellular vesicles by the identification and translocation of such cargo by ESCRT proteins (Babst et al., 2002; Odorizzi et al., 1998; Saksena et al., 2007). Therefore, the inventors' findings are relevant to CTLA-4 + This demonstrates that proteins within the dexosome fraction are packaged independently of the ESCRT pathway. Next, the inventors present CTLA-4 as a more efficient means to deepen our understanding of the origin and effects of these dexosome subsets. + Fractions and CTLA-4 neg We attempted to determine the entire proteome of the fraction. Therefore, we investigated CTLA-4 derived from bulk Th2 dexosome isolates. + Dexosomes and CTLA-4 neg Mass spectrometry of dexosomes was performed. Notably, the inventors found that HLA class II Moeti, HSP90AB1, CD81, ITGAM (CD11b), and ITGAX (CD11c) were CTLA-4 + Enriched in dexosomes, HLA class I, CD82, CD36 (and its soluble ligand CD5L), and CD44 are CTLA-4 neg We observed that it was not enriched in dexosomes (Figure 6D). This finding was also reproduced in flow cytometry analysis of HLA class II (HLA-DR) expression (Figure 6F). In addition, we confirmed by cryo-electron microscopy that CTLA-4 + Physical characteristics of the EV fraction and CTLA-4 neg We noted a significant difference between the physical characteristics of the EV fraction and the other components. CTLA-4 negThe fraction constituted a potentially heterogeneous population of vesicles, which were mainly composed of vesicles with a diameter of approximately 200 nm or more. These spherical vesicles were characterized by high electron-density membranes and lumens (Figure 6H). The inventors also noted the previously described phenomenon (Gallart-Palau et al., 2015; Milasan et al., 2016) where smaller vesicles exist encapsulated within larger vesicles. Meanwhile, CTLA-4 + Dexosomes are consistently spherical vesicles about 30 nm in size, and this is their CTLA-4 neg Compared to its counterpart, the electron density was significantly higher. Overall, the inventors of CTLA-4 + This indicates that vesicles are a subset of dexosomes with unique miRNA, proteomics, and physical profiles. Furthermore, CTLA-4 in the dexosome environment + Vesicle production could be regulated by in vitro culture conditions.

[0102] Both parental dendritic cells (DCs) and their dexosomes similarly polarize T cell responses. A growing body of evidence suggests that dexosomes possess the ability to drive T cell responses. Understanding the mechanisms governing this immunomodulatory action has so far focused on the dexosomal ability (paracrine) to suppress or enhance DC maturation, and their ability to crossdress DCs with the antigens they carry. In this role, the dexosomal action on T cells is indirect and relies on the presence of other cells, including B cells, NK cells, and DCs. MHC-II + ICAM-1 +Further evidence exists suggesting that dexosomes can interact with T cells via LFA-1 on T cells, leading to effective TT antigen presentation. However, the effects of differently polarized dexosomes on the T cell response remain unclear. Furthermore, the efficacy of dexosomes in the presence of DCs in the opposite polarized state, a situation that may be encountered when administered as therapy in vivo, is also unknown. For this purpose, we first attempted to determine whether there are differences in the level of T cell uptake of differently polarized dexosomes. We matured and polarized DCs in the form of Th0, Th1, or Th2, and stained them with carboxyfluorescein succinimimidyl ester (CFSE) so that the dexosomes produced by these DCs could be recognized by CFSE, and that T cell uptake of these dexosomes could be tracked based on CFSE positivity. Mature polarized DCs were then cultured with unlabeled allogeneic T cells for 3 days, followed by flow cytometry analysis. The inventors observed similar levels of CFSE positivity (dexosome uptake) in T cells cultured with Th0 dexosomes and T cells cultured with Th1 dexosomes (Figure 7A). On the other hand, those cultured with Th2 dexosomes showed significantly lower vesicle uptake (Figure 7A). Next, the inventors attempted to confirm whether dexosome uptake causes changes in the phenotype of T cells, particularly in the expression of effector molecules. Therefore, the inventors cultured T cells with CFSE-labeled Th0 polarized DCs for 3 days, followed by flow cytometry analysis. By utilizing DCs with an undetermined Th (Th0) in this manner, it became possible to simultaneously evaluate Th1 and Th2 effector molecules in relation to the state of dexosome uptake. The inventors used CD4 + Cell fraction and CD8 + Similar levels of dexosome uptake were observed in the T cell fraction (Figures 7B, 7C). CFSE + CD8 +T cells expressed significantly higher levels of IFNγ and lower levels of granzyme B (Figures 7D, 7E). CFSE + CD4 + The T cells expressed higher levels of IFNγ and IL-4, mirroring the Th-undetermined nature of Th0 DCs (Figures 7F, 7G). Finally, the inventors investigated whether dexosomes could influence the T cell response independently of parental DCs, particularly in the presence of non-parental DCs. To this end, allogeneic T cells were cultured with CFSE-labeled Th1 dexosomes and CellTrace Far Red (CTFR)-labeled Th0 DCs (Figure 7H). Consistent with previous results, the majority of T cells did not take up dexosomes. However, among cells that took up some kind of dexosome, those that took up Th1 dexosomes were more frequently observed than those that took up Th0 dexosomes or both Th1 and Th0 dexosomes (Figure 7I). CD8 + T cell-mediated expression of GrzB, and CD4 + T cells and CD8 + IFNγ expression by T cells is linked to Th1 dexosomes. + The levels were significantly higher than in T cells (Figure 7J-7L). Interestingly, the uptake of both Th0 and Th1 dexosomes was additive in terms of cytokine expression. Taken together, Th1 dexosomes alone can drive the immunostimulatory phenotype of parental DCs, regardless of the presence of non-parental DCs.

[0103] CTLA-4 +is a marker representing dexosomes that inhibit the type 1 immune response. One of the key differences observed in our previous efforts to identify differences between Th1 moDCs and Th2 moDCs was in the expression of extracellular vesicle-borne CTLA-4 between these two DC types (Halpert et al., 2016). In particular, Th2 DCs expressed CTLA-4 significantly higher than Th1 DCs. In this context, CTLA-4 was shown to reduce the expression of the costimulatory molecule B7 by mediating dexosome uptake (paracrine) by neighboring DCs. Notably, when CTLA-4 was absent in moDCs, co-culturing with allogeneic T cells resulted in CD8 + T cell activation and IFNγ expression were suppressed. Subsequent experiments confirmed that loss of CTLA-4 in the BMDC vaccine significantly improved survival and tumor clearance in a mouse melanoma model. Based on these observations and the known role of CTLA-4 as an important mediator of the T1 T cell response, we have proposed that CTLA-4 plays a role in the T cell response. + We attempted to investigate the role of dexosomes. For this purpose, we isolated dexosomes from moDCs advancing to Th2, and then used antibody-bound immunomagnetic beads to perform CTLA-4 + The fraction was depleted. Subsequently, allogeneic T cells produced CTLA-4 neg Depleted dexosomes or CTLA-4 neg It was cultured with any of the total dexosomes, including CTLA-4. neg The dexosomes contained are CD4 + T cells and CD8 + It significantly suppressed IFNγ expression in T cells (Figures 8A-8D), while CTLA-4 neg Depleted dexosomes rescued IFNγ expression. In particular, IL-4 and granzyme B expression were affected in the opposite direction. Specifically, CTLA-4 + Dexosomes are CD4 + T cells and CD8 +The expression of IL-4 and granzyme B by each T cell was promoted. Next, the inventors attempted to confirm whether CTLA-4 mediates the interaction between dexosomes and T cells, as has been observed in DCs. For this purpose, the inventors cultured CFSE-labeled DCs with T cells in the presence of anti-CTLA-4 (clone: ​​BNI3). CD4 + CD8 is also involved in the uptake of dexosomes by T cells. + No differences were observed in the uptake of dexosomes by T cells (Figures 8E, 8F). Overall, CTLA-4 + The dexosome fraction suppressed the T1 T cell response, but CTLA-4 itself was not required for the interaction between T cells and dexosomes.

[0104] CTLA-4 in dendritic cells (DCs) is necessary for maintaining immune homeostasis. In humans and mice, the DC classification includes not only moDCs but also plasmacytoid DC subsets and conventional DC subsets. Our research to date has focused on in vitro differentiated moDCs, but since these DC subsets play crucial roles in the initiation, maintenance, and termination of the immune response, it is important to clarify the role of CTLA-4 in DCs in the in vivo environment. Therefore, we attempted to clarify the CTLA-4 expression profiles of separate DC subsets in a steady state. Using flow cytometry analysis, we observed the highest levels of CTLA-4 expression in T1 conventional DCs (cDC1). CTLA-4 expression was significantly lower and similar in plasmacytoid DCs (pDCs), type 2 conventional DCs (cDC2), and moDCs, while monocytes expressed the lowest levels of CTLA-4 (Figure 9A). Next, the inventors investigated the role of CTLA-4 in the suppression of DC-mediated immune responses, specifically focusing on CD11c + Cells do not express CTLA-4 (CTLA-4 fl / fl CD11c CreConditional knockout (cKO) mice were generated. In these mice, the loss of CTLA-4 led to immunodysregulation, characterized by elevated levels of activated T cells in the spleen (Figures 9B, 9C) and decreased frequency of splenic Treg occurrence. Compared to wild-type mice, the tissue structures in the thymus, spleen, and Peyer's patches of cKO mice were severely disrupted, and the leukocyte occupancy in these tissues was also significantly reduced (Figure 9D). In addition, the inventors noted the absence of pancreatic tissue and the reduced number of Peyer's patches in cKO mice. Leukocyte infiltration was observed in the lungs and livers of cKO mice, and alveolar space dilation was observed in cKO mice. Ultimately, the cKO mice suffered from stunted growth and died within 6 weeks.

[0105] CTLA-4 + Dexosomes suppress the antitumor immune response in mice. In T cell-EV cultures, the inventors observed a differential T1 response, and therefore attempted to investigate the role of these EV subsets in generating the T1 response in vivo. For this purpose, the inventors used a tumor model (B16-OVA) in which tumor elimination and survival rates are based on effective T1 immunity. The inventors hypothesized that OVA-loaded EVs that progress to type 2 can successfully promote the antitumor response in the absence of CTLA-4, consistent with in vitro observations. Therefore, the inventors isolated EVs from DC cultures that progress to T1, and also isolated EVs from DCs that progress to a type 2 response (T2) and are loaded with OVA protein, and that were treated with siCTLA4 or non-targeted siRNA. B16-OVA-carrying mice were vaccinated with T1 EV, siCTLA4 T2 EV, or siNT T2 EV once their tumors became palpable and measurable with calipers. To clarify the role of each of these EV types in the anti-tumor immune response, tumor growth, survival rates, lung metastasis, and flow cytometry of tumor-infiltrating lymphocytes were analyzed (Figure 10).

[0106] While promising in preclinical models, Phase I and II trials of DC-derived EVs (dexosomes) for melanoma and NSCLC did not meet expectations (Escudier et al., 2005; Munich et al., 2012; Besse et al., 2016; Pujol et al., 2015). Therefore, elucidating the composition, biosynthesis, and function of dexosomes is crucial to unlocking their therapeutic potential, particularly when they are associated with immunosuppressive CTLA-4. Dendritic cells play a vital role in maintaining tolerance to self and symbionts, as well as in initiating robust adaptive immune responses. CTLA-4 - / - The fact that mouse death is mediated by autoimmunity highlights the importance of this molecule, but this does not reveal the role of immune-critical DCs in the biological properties of CTLA-4, nor does it reveal the reverse. Understanding the interaction between these two types of immune-critical mediators is essential to uncovering the underlying critical mechanisms that mediate tolerance loss, vaccine response, and suppression of antitumor immune responses.

[0107] In this specification, the inventors demonstrate that dendritic cells (DCs) express CTLA-4 intracellularly, and that CTLA-4 is also expressed on the surface of extracellular vesicles (EVs) released from DCs. In addition, CTLA-4 expression can be regulated by the culture conditions under which parental DCs are produced. Specifically, DCs that induce a type 1 response (T1) expressed low levels of CTLA-4 and IL-13, in addition to high levels of IL-12. On the other hand, DCs that induce a type 2 response (T2) expressed high levels of CTLA-4 and IL-13, along with low levels of IL-12. When allogeneic T cells were co-cultured with a bulk EV population, a T cell phenotype that replicated the effects of parental DCs was obtained.

[0108] All methods disclosed and claimed herein can be carried out and implemented without excessive experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in relation to certain preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the methods described herein, and to the steps of the methods, or to the order of the steps of the methods, without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that both chemically related and physiologically related active substances may be used in place of the active substances described herein, and the same or similar results may be achieved. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0109] References The following references are incorporated herein by reference to the extent that they provide exemplary procedural details or other details that supplement the details set forth herein. TIFF2026517917000003.tif178146TIFF2026517917000004.tif164146

Claims

1. A method for producing extracellular vesicles (EVs) from dendritic cells (DCs), wherein (i) T H 1-polarization or T H A method comprising (ii) maturing DCs toward either bipolarization, and (ii) isolating EVs secreted by polarized DCs.

2. T H The method according to claim 1, wherein the step of maturing the DC toward 1-polarization includes maturing the DC in the presence of IL-12, or loading the DC with lysates and mRNA preparations derived from the same cell type.

3. T H The method according to claim 1, wherein the step of maturing the DC toward bipolarization includes maturing the DC in the presence of enterotoxin B (SEB) of S. aureus.

4. The method according to any one of claims 1 to 3, wherein the dendritic cells are monocytic dendritic cells.

5. The method according to any one of claims 1 to 4, wherein the immature dendritic cells are monocyte-derived dendritic cells.

6. T H 1-polarized DC, IL-12 + IL-13 lo , and CTLA-4 lo The method according to claim 1.

7. T H The polarized DC is IL-13 + , CTLA-4 + , and IL-12 lo The method according to claim 1, wherein it is such.

8. The secreted EV is CTLA-4 + The group and CTLA-4 neg The method according to any one of claims 1 to 7, further comprising the step of separating into groups.

9. CTLA-4 neg The EV group, CTLA-4 + The method according to claim 8, obtained by the depletion of EVs.

10. CTLA-4 + The method according to claim 8, wherein the EV population is obtained by positive selection using an anti-CTLA-4 antibody conjugated to beads.

11. A composition comprising dendritic cell-derived extracellular vesicles (EVs) obtained by the method described in any one of claims 1 to 10.

12. The aforementioned EV is CTLA-4 + The composition according to claim 11.

13. The aforementioned EV is T H The composition according to claim 12, derived from bipolarized DC.

14. The aforementioned EV is T H The composition according to claim 12, derived from a unipolarized DC.

15. The aforementioned EV is CTLA-4 neg The composition according to claim 11.

16. The aforementioned EV is T H The composition according to claim 15, derived from a unipolarized DC.

17. The aforementioned EV is T H The composition according to claim 15, derived from bipolarized DC.

18. The composition according to any one of claims 11 to 17, wherein the EV has a diameter of about 30 to 200 nm.

19. The aforementioned EV is CD63 + CD81 + , and HLA-DR + The composition according to any one of claims 11 to 18.

20. CTLA-4 + EV and CTLA-4 neg A method for separating EVs and EV populations from each other in order to recover both EV populations in a physically and functionally intact state, comprising the steps of isolating EVs from the cell culture supernatant of mature DCs using PEG precipitation, and using an anti-CTLA-4 antibody conjugated to beads. + Extract EV, thereby CTLA-4 + EV and CTLA-4 neg The method, comprising the step of separating the EV from each other.

21. Adaptive T H A method for inducing 1. Polarization and activation of T cells, wherein the T cells are CTLA-4 neg The method, which includes the step of bringing it into contact with a DC-derived EV.

22. The T cells are CD8 + The method according to claim 21, wherein the cell is a T cell.

23. The aforementioned T cells are CD4 + The method according to claim 21, wherein the cell is a T cell.

24. The aforementioned CTLA-4 neg The method according to any one of claims 21 to 23, wherein the DC-derived EV is as described in any one of claims 15 to 17.

25. The method according to any one of claims 21 to 24, wherein the T cells are present in the bulk PBMC.

26. The method according to any one of claims 21 to 24, wherein the T cells are isolated naive T cells.

27. The method according to any one of claims 21 to 24, wherein the T cells are present in the subject, and the method includes the step of administering the EV to the subject.

28. A method for treating a disease in patients who require it, CTLA-4 neg The method comprising the step of administering DC-derived EVs to the patient.

29. The method according to claim 28, wherein the disease is a viral infection or cancer.

30. The method according to claim 28 or 29, wherein the DC-derived EV is administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally.

31. The method according to any one of claims 28 to 30, further comprising the step of administering an immune checkpoint inhibitor to the patient.

32. The method according to claim 31, wherein the immune checkpoint inhibitor is a CTLA-4 antagonist.

33. The method according to claim 31 or 32, wherein the immune checkpoint inhibitor is ipilimumab, pembrolizumab, or nivolumab.

34. Adaptive T H 1-polarization and CD8 + A method for inhibiting the activation of T cells, wherein the T cells are CTLA-4 + The method, which includes the step of bringing it into contact with a DC-derived EV.

35. The aforementioned CTLA-4 + The method according to claim 34, wherein the DC-derived EV is as described in any one of claims 12 to 14.

36. The method according to claim 34 or 35, wherein the T cells are present in the bulk PBMC.

37. The method according to claim 34 or 35, wherein the T cells are isolated naive T cells.

38. The method according to claim 34 or 35, wherein the T cells are present in the subject, and the method includes the step of administering the EV to the subject.

39. The aforementioned EV is T H The method according to any one of claims 34 to 38, derived from bipolarized DC.

40. A method for treating autoimmune conditions in patients, and CTLA-4 + The method comprising the step of administering DC-derived EVs to the patient.

41. The aforementioned CTLA-4 + The method according to claim 40, wherein the DC-derived EV is as described in any one of claims 12 to 14.

42. The method according to claim 40 or 41, wherein the autoimmune pathology is a T cell-mediated autoimmune pathology.

43. The method according to any one of claims 40 to 42, wherein the autoimmune pathological condition is post-HSCT GVHD.

44. The aforementioned CTLA-4 + The method according to any one of claims 40 to 43, wherein DC-derived EV is administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally.

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