Compositions comprising dendritic cell exosomes and methods of use thereof

Abstract

Provided herein are methods for generating extracellular vesicles (EVs) from massively cultured polarized DCs, as well as additional operations of separating CTLA-4 + EV and CTLA-4-EV from each other and recovering the two EV populations in a physically and functionally intact state; also provided is a composition comprising the EV obtained by the method. In addition, the use of CTLA-4-EV in the treatment of cancer, as well as the use of CTLA-4 + EV in the treatment of GVHD and other T cell mediated autoimmune conditions, is also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 501,501, filed May 11, 2023, the entire contents of which are incorporated herein by reference.

[0003] Statement on Federally Funded Research

[0004] This invention was completed with government support, funded by grants from the National Institutes of Health (ANI) under grant numbers AI127387 and AI153326. The government holds certain rights to this invention. Background Technology 1. Technical Field

[0006] This invention generally relates to the fields of immunology and medicine. More specifically, this invention relates to compositions comprising dendritic cell-derived exosomes, methods of their preparation, and methods of use. 2. Background Technology

[0008] Dendritic cells (DCs) are key regulators of adaptive immune responses in mammals. In this role, DCs are able to sense T cells in the surrounding environment. H DCs polarize signals and transmit these signals, thereby influencing the characteristics of downstream T cell responses. However, dendritic cells (DCs) constitute a relatively small cell population in circulating leukocytes. Patients often require very stringent mobilization protocols to generate a sufficient number of cells for therapeutic purposes. Furthermore, DC-based therapies are susceptible to disease-mediated immunosuppression and carry the risk of graft-versus-host disease. Therefore, there is an urgent need to develop compositions and methods that can provide the therapeutic benefits of DCs while avoiding their associated risks. Summary of the Invention

[0009] Therefore, this article provides a therapeutic composition comprising dendritic cell (DC)-derived extracellular vesicles (EVs), requiring a significantly smaller number of DCs than DC-based therapies, thereby reducing the need for mobilization protocols. Because DC-derived EVs are subcellular entities lacking known intrinsic metabolic or signaling capabilities, they are less susceptible to factors such as tumor-mediated immunosuppression, unlike DCs. Furthermore, as subcellular entities, they lack known cell lysis capabilities or mechanisms driving anti-EV immunity, thus allowing for off-the-shelf therapy. Therefore, allogeneic EVs can be delivered without the risk of GVHD or significant host resistance to graft disease. Finally, EVs exhibit excellent stability under storage conditions. Unlike DCs, which must be carefully cryopreserved and thawed to maintain function, EVs remain intact and functional over a wide temperature range (e.g., from 25°C to -80°C).

[0010] This article provides a method for generating extracellular vesicles (EVs) from dendritic cells (DCs), the method comprising (i) causing DCs to convert into T cells. H 1 polarization or T H 2. Maturation of polarization direction, and (ii) separation of EVs secreted by said polarized DCs. This causes DCs to align with T... H 1. Polarization orientation maturation may include maturing the DCs in the presence of IL-12, or loading the DCs with lysates and mRNA formulations from the same cell type. This allows the DCs to polarize towards T... H 2. Polarization direction maturation may include maturing the DCs in the presence of Staphylococcus aureus enterotoxin B (SEB). The dendritic cells may be mononuclear dendritic cells. The immature dendritic cells may be mononuclear-derived dendritic cells. T H A 1-polarized DC can be IL-12 + IL-13 低 and CTLA-4 低 T H A 2-polarized DC can be IL-13 + CTLA-4 + and IL-12 低 T H A 1-polarized DC can be IL-12 + CADM1 + IL-13 低 and CTLA-4 低 T H A 2-polarized DC can be IL-13 + CTLA-4 + CD172a + and IL-12 低 As used in this article, "lo" or "low" expression refers to an expression level measured by qRT-PCR that is at most 50% of the corresponding expression level in the oppositely polarized DC, for example, T H The expression level of IL-13 in polarized dendritic cells is T H IL-13 expression levels in 2 polarized dendritic cells are up to 50%.

[0011] The method may further include separating the secreted EVs into CTLA-4. + and CTLA-4 - Group. The CTLA-4 mentioned above. - The group is formed by consuming CTLA-4 + EV obtained. The CTLA-4 + The EV population was obtained through positive selection using bead-bound anti-CTLA-4 antibodies.

[0012] This document provides compositions comprising dendritic cell-derived extracellular vesicles (EVs). The dendritic cell-derived EVs can be obtained by the methods described herein. The EVs may be CTLA-4. - Or CTLA-4 + The EV can be derived from T. H 1-polarized DC or T H A dual-polarized DC. The diameter of the EV can be approximately 30-200 nm. The EV can be CD63. + CD81 + and HLA-DR + .

[0013] This article provides information on CTLA-4 + EV and CTLA-4 - A method for separating EVs from each other to recover the two EV populations in a physically and functionally intact state, the method comprising separating EVs from mature DC cell culture supernatant using PEG precipitation and extracting CTLA-4 using bead-bound anti-CTLA-4 antibody. + EV, thereby enabling CTLA-4 + and CTLA-4 - The EVs are separate from each other.

[0014] This article provides induced adaptive T H 1 polarization and CD8 + A method for T cell activation, the method comprising reacting the T cells with CTLA-4 - DC-derived EV contacts. The CTLA-4 - DC-derived EVs can be obtained according to the methods described herein. The T cells can be present in a bulk PBMC population or as isolated primary T cells. The T cells can be present in the subject, in which case the method includes administering the EVs to the subject.

[0015] This article provides a method for treating a patient's condition, the method comprising administering CTLA-4 to the patient. - DC-derived EVs. The disease can be a viral infection or cancer. The DC-derived EVs can be administered intratumorally, intravenously, peritumorally, subcutaneously, or intraperitoneally. The method may further include administering an immune checkpoint inhibitor to the patient. The immune checkpoint inhibitor may be a CTLA-4 antagonist, such as ipilimumab, pembrolizumab, or nivolumab.

[0016] This article provides a method for suppressing adaptive TH 1. A method for polarizing and activating T cells, the method comprising reacting the T cells with CTLA-4 + DC-derived EV contact. The T cells may be CD4. + T cells or CD8 + T cells. The EV can be derived from T cells. H 2-polarized DC. The CTLA-4 + DC-derived EVs can be obtained according to the methods described herein. The T cells can be present in a large number of PBMCs or can be isolated primary T cells. The T cells can be present in the subject's body, in which case the method includes administering the EVs to the subject.

[0017] This article provides a method for treating an autoimmune condition in a patient, the method comprising administering CTLA-4 to the patient. + DC-derived EVs. The autoimmune condition can be a T-cell-mediated autoimmune condition. The autoimmune condition can be GVHD following HSCT. The DC-derived EVs can be administered intratumorally, intravenously, peritumorally, subcutaneously, or intraperitoneally.

[0018] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while indicating preferred embodiments of the invention, the detailed description and specific examples are merely illustrative, as 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.

[0019] Brief description of the attached figures

[0020] The accompanying drawings, which are part of this specification, are included herein to further illustrate certain aspects of the invention. The following one or more drawings, along with a detailed description of specific embodiments, will help to better understand the invention.

[0021] Figure 1A-1B . Figure 1A : via T H 1. T cells stimulated by polarized DCs compared to those stimulated by T cells H T cells stimulated by 2-polarized DCs show CD8 + CD161 + Expression was upregulated, while the levels of T cell exhaustion markers PD-1 and Tim3 were downregulated. Figure 1B Added derivative of T H EVs of 2-polarized DCs can reduce IFN-γ expression and increase T H 1-polarized DC presensitized CD8 + CD161 + Tim3 expression on T cells.

[0022] Figure 2 Total DC-derived exosomes were isolated as CTLA-4. + and CTLA-4 - Partially, and co-cultured with total non-adhesive allogeneic PBMCs for 7 days under the conditions shown, followed by flow cytometry analysis under the conditions shown. (Compared with CTLA-4) - PBMCs co-cultured with exosomes increased CD8 + CD161 expression in the cell population, while CTLA-4... + Exosomes tend to suppress CD161 expression. (This is in contrast to CTLA-4.) - CD8 co-cultured exosomes + CD161 + Cells significantly upregulated the expression of granzyme B and CD25, while CTLA-4 expression was significantly higher. + CD8 co-cultured exosomes + CD161 + The cells mainly maintain low expression of granzyme B and are CD25 negative.

[0023] Figures 3A-3B DC's CTLA-4 + The effect of EV secretion on downstream T cell responses. Figure 3A: Only low levels of CTLA-4 secreted. + Or does not secrete CTLA-4 + EV's T H Only polarized dendritic cells can generate CD8. + NK1.1 + T cells. 250,000 polarized dendritic cells (DCs) were adoptively transferred into mice treated with the TLR-7 agonist imiquimod as adjuvant. Results showed that only DCs secreting low levels of CTLA-4 could generate detectable levels of CD8. + NK1.1 + Highly cytotoxic effector memory T cells. Only those treated with CTLA-4. 低 T H In mice with adopted DC 1 polarization, CD8 was observed + NK1.1 + The number of T cells increased tenfold. n = 5-10 mice / group. Figure 3B: CTLA-4 高 Microvesicles inhibit IFN-γ in vitro + CD8 + The generation of T cells. This will be derived from T cells. H CTLA-4 in the supernatant of 2-polarized DC culture 高 Microvesicles added to T HIn a co-culture of polarized DCs and autoreactive T cells, these CTLA-4 were added. 高 Microvesicles are sufficient to enable CD8 + IFN-γ + The number of reactive cells decreased by 50%. p<0.001, so a one-way ANOVA with Tukey post-hoc test was used.

[0024] Figures 4A-4C CTLA-4 - EV promotes T H 1. T cell polarization, while CTLA-4 + EVs suppress T cells T H 1. Polarization. The DC used for exosome isolation is T. H Two polarized DCs (loaded with heterologous class I and II peptides). All EVs derived from these DCs were added to non-adherent PBMCs derived from the ESR amber layer in culture. Depending on the specific circumstances, total EVs or isolated CTLA-4 were used. - EV. Figure 4A Day 2, total CD4 + Cells were analyzed. Figure 4B Day 2, total CD8 + Cells were analyzed. Figure 4C Day 6, total CD8 + Cell analysis was performed. Flow cytometry analysis was conducted on day 6 after the addition of IL-2 on day 5. Prior to flow cytometry staining, large quantities of PBMCs were stimulated for 5 hours with PMA + iomycin + brefidobacterium A. For staining on day 6, PBMCs were stored overnight at 4°C and stained the following day. Statistical analysis: One-way ANOVA with Tukey post-hoc test was used. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; p≤ 0.0001.

[0025] Figures 5A-5E CTLA4 - EVs can upregulate cytotoxic CD8 + CD161 + Cellular level and function. Mature DCs in the presence of IL-12 will transform into T cells. H 1. Cell polarization, while mature DCs in the presence of SEB will polarize towards T cells. H 2. Cell polarization. From T H 1 polarization and T H Separate CTLA-4 from 2 polarized DCs + and CTLA-4 -EV. CD45RA with all four EV groups separated from the magnetic beads. + Primary allogeneic T cells were co-cultured for five days, after which the T cell population was "re-stimulated" with the same EV population. Flow cytometry analysis was performed on day 13 of culture. Figure 5A ) will be from T H EVs isolated from the supernatant of polarized DCs were co-cultured with primary T cells. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + CD161 + The levels of IFNγ and granzyme B were detected in the cell population. Figure 5B ) will be from T H EVs isolated from the supernatant of polarized DCs were co-cultured with primary T cells. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + CD161 + The levels of IFNγ and granzyme B were detected in the cell population. Figure 5C CD8 + CD161 + Expression levels of IFN-γ and CD25 in T cells. (Top image: From T cells...) H EV separation in a 1-polarized DC. Below: From T H EVs are separated in a 2-polarized DC. Figure 5D Total CD8 + Expression levels of granzyme B and CD25 in T cells. (Top image: Expression levels of granzyme B and CD25 in T cells) H EV separation in a 1-polarized DC. Below: From T H EVs are separated in a 2-polarized DC. Figure 5E Total CD8 + Expression levels of IFN-γ and CD25 in T cells. (Top image: From T cells...) H EV separation in a 1-polarized DC. Below: From T H Separate EVs in a 2-polarized DC. Regardless of T H 1-polarized DC or T H Separation in a 2-polarized DC, CTLA-4 + and CTLA-4 - No functional differences were observed among the EV groups.

[0026] Figure 6A-6G Maturation conditions regulate the expression of CTLA-4 in extracellular vesicles of human monocyte-derived dendritic cells (moDCs). Human monocytes were isolated by CD14-positive immunomagnetic bead sorting and then cultured with IL-4 and GM-CSF for 6 days. On day 6, immature DCs matured and polarized as described. 48 hours later, mature DCs were characterized as follows: Figure 6A qRT-PCR of IL-12a, IL-12b and IL-13; EVs were characterized as follows: Figure 6EWestern blot analysis; Figure 6B miRNA sequencing; Figure 6C qRT-PCR of CTLA-4; Figure 6G Cryo-electron microscopy; Figure 6D Mass spectrometry analysis and ( Figure 6F Flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. p ≤ 0.01; p ≤ 0.001; p ≤ 0.0001.

[0027] Figures 7A-7L. EVs reflect the parental DC phenotype in polarized T cell responses. (Figure 7A) Allogeneic T cells were co-cultured with CFSE-labeled, Th-polarized, and mature DCs for 5 days. EV uptake (CFSE positive) was detected by flow cytometry. Allogeneic T cells were co-cultured with CFSE-labeled mature Th0-polarized DCs for 3 days, followed by flow cytometry analysis to measure (Figures 7B-7C) EV uptake by CD4 and CD8 T cells and (Figures 7D-7G) differential cytokine expression in CD4 and CD8 T cells based on EV uptake. Figure 7H The schematic diagram details the experimental design of the data shown in Figures 7I-7L, in which allogeneic T cells were co-cultured with CellTrace far-red (CTFR)-labeled Th0 polarized DCs and EVs from the culture supernatant of CFSE-labeled Th1 polarized DCs. On day 3, flow cytometry analysis was performed to detect (Figure 7I) EV uptake and (Figure 7J-L) T cell effector molecule expression. Statistical analysis was performed using paired t-tests or one-way ANOVA, combined with Holm-... ídák Multiple comparison test. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; p≤ 0.0001.

[0028] Figures 8A-8F CTLA-4 + EVs inhibit IFNγ expression in T cells. (Figure 8A) Allogeneic T cells were co-cultured with CFSE-labeled Th0 polarized dendritic cells (DCs) in the presence or absence of anti-CTLA-4 antibody (clone: ​​BNI3), and EV uptake was detected by flow cytometry. (Figures 8B-8D) Allogeneic T cells were co-cultured with EVs derived from total Th2 DCs or EVs that had been de-CTLA-4 removed using immunomagnetic beads. +Subpopulations of these EVs were cultured together. T cell effector molecule expression was analyzed by flow cytometry on day 6 (Figures 8E-8F). CFSE-labeled dendritic cells (DCs) were co-cultured with T cells in the presence of anti-CTLA-4 antibody, and their uptake of DC-derived dexosomes was detected. Statistical analysis was performed using one-way ANOVA with Dunn post-hoc test. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; p≤ 0.0001.

[0029] Figures 9A-9D The loss of CTLA-4 in dendritic cells (DCs) drives T-cell activation in mice and leads to lethal consequences. Figure 9A B16-Flt3L tumors were implanted into C57BL / 6 mice, and the mice were sacrificed on day 12. Spleen cells were isolated. CTLA-4 expression in monocytes and dendritic cell subsets was analyzed by flow cytometry. Figures 9B-9C Analysis of wild-type (WT) C57BL / 6 or CTLA-4 by flow cytometry fl / fl CD11c cre Activation status of spleen T cells and CTLA-4 expression in mice (cKO). Figure 9D H&E staining was performed on lymphoid and non-lymphoid tissues of WT or cKO mice. White arrows indicate areas of leukocyte infiltration.

[0030] Figure 10 The loss of CTLA4 in EVs promotes anti-tumor T-cell responses. In the survival curves, these lines, from top to bottom, represent the survival rates of OVA siCTLA4, OVA siNT, IL-12 siNT, and PBS at day 30. Detailed Implementation

[0031] Dendritic cells (DCs) are key regulators of adaptive immune responses in mammals. In this role, DCs are able to sense T cells in the surrounding environment. H DCs transmit polarization signals and this information downstream, thereby influencing the characteristics of downstream T cell responses. DCs transmit this information to T cells by secreting exosomes or extracellular vesicles (EVs). H 2-polarized dendritic cells preferentially secrete EVs carrying the immune regulatory checkpoint molecule CTLA-4, while T cells... H 1. Polarized dendritic cells (DCs) preferentially secrete EVs lacking this protein. This article provides a method for generating EVs from cultures of polarized DCs, and the method for combining CTLA-4+ EVs with CTLA-4... -Additional operations involving the separation of EVs from each other and the recovery of both EV populations in a physically and functionally intact state. CTLA-4 during in vitro culture of T cells. + EVs can suppress adaptive T H 1 polarization and CD8 + T cell activation, while CTLA-4 - EVs can induce adaptive T H 1 polarization and CD8 + T cell activation. CTLA-4 - EV can also induce the generation of a key CD8. + T cell memory populations, characterized by CD161 expression. These effects were observed in primary T cells isolated from large quantities of PBMCs and magnetic beads. Large-scale production and direct administration of these EVs are expected to promote CD8. + The establishment of T-cell responses can be used to treat cancer or to treat autoimmune conditions, including post-HSCT GVHD, by suppressing activated T cells. Therefore, this article also provides information on CTLA-4. - The use of EVs in cancer treatment, and CTLA-4 + Use of EVs in the treatment of GVHD and other T cell-mediated autoimmune conditions.

[0032] I. Definition

[0033] As used in this specification, "a" or "an" may refer to one or more types. As used in the claims herein, when used in conjunction with the word "comprising," the word "a" or "an" may refer to one or more types.

[0034] The term “or” as used in the claims means “and / or” unless specifically indicated as referring only to alternatives or where alternatives exclude each other, but the disclosure supports the definition of referring only to alternatives and “and / or”. As used herein, “another” may refer to at least a second or more.

[0035] Throughout this application, the term “about” is used to indicate that numerical values ​​include inherent variations in apparatus error, and that inherent variations in the method are used to identify differences between numerical values, subjects of study, or values ​​within 10% of a specified numerical value.

[0036] As used herein, with respect to a specific component, "substantially free" means that the particular component was not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the specified component resulting from any unplanned contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the amount of the specified component is undetectable using standard analytical methods.

[0037] The term “substantially” should be understood as meaning that a method or composition comprises only the prescribed steps or materials, and that these steps or materials do not substantially affect the essential characteristics of the methods and compositions.

[0038] "Treatment" or "treatment" includes: (1) suppressing the disease of a subject or patient who is experiencing or exhibiting the pathology or symptoms of a disease (e.g., preventing further development of the pathology and / or symptoms); (2) improving the disease of a subject or patient who is experiencing or exhibiting the pathology or symptoms of a disease (e.g., reversing the pathology and / or symptoms); and / or (3) causing any measurable reduction in the severity of the disease of a subject or patient who is experiencing or exhibiting the pathology or symptoms of a disease.

[0039] "Prevention" or "avoidance" includes: (1) suppressing the development of the disease in a subject or patient who may be at risk of and / or susceptible to the disease but has not yet developed or exhibited any or all of the pathology or symptoms of the disease, and / or (2) delaying the development of the pathology or symptoms of the disease in a subject or patient who may be at risk of and / or susceptible to the disease but has not yet developed or exhibited any or all of the pathology or symptoms of the disease.

[0040] As used herein, the terms "patient" or "object" refer to a living mammal, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or object is a primate. Non-limiting examples of human patients include adults, adolescents, infants, and fetuses.

[0041] When used in the specification and / or claims, the term "effective" means sufficient to achieve the desired, desired, or anticipated result. In the context of treating a patient or subject with a compound, "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" means an amount sufficient to treat or prevent a disease when the compound is administered to the subject or patient to treat or prevent the disease.

[0042] II. Extracellular vesicles of dendritic cells

[0043] Methods for isolating, culturing, and presensitizing dendritic cells from which DC-derived EVs can be isolated are well known in the art. For example, U.S. Patent 8,728,806 (the entire contents of which are incorporated herein by reference) provides a detailed method for providing presensitized dendritic cells. In some aspects, the dendritic cells used according to embodiments are isolated from an object to be treated by the methods described herein. In other aspects, the dendritic cells may be derived from different objects, such as HLA-matched donors. In some aspects, the dendritic cells are derived from a dendritic cell bank with a well-defined HLA typing.

[0044] Methods for isolating cell populations rich in dendritic cell precursors and immature dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, dendritic cell precursors and immature dendritic cells can be separated by collecting heparinized blood, performing hemocytosis or leukocyte separation, preparing leukocyte layers, rosette method, centrifugation, density gradient centrifugation (e.g., using Ficoll (such as FICOLL-PAQUE®), PERCOLL® (colloidal silica particles coated with non-dialyzable polyvinylpyrrolidone (PVP) (15-30 mm in diameter)), sucrose, differential cell lysis, filtration, etc. In some embodiments, leukocyte populations can be prepared, for example, by collecting blood from the subject, removing fibrinogen to remove platelets, and lysing red blood cells. In other words, leukocyte populations can be prepared, for example, by defibrinating blood samples to remove platelets and lysing red blood cells. Dendritic cell precursors and immature dendritic cells can optionally be enriched, for example, using PERCOLL® gradient centrifugation, to obtain mononuclear dendritic cell precursors. Alternatively, dendritic cell precursors can be selected by CD14 screening of G-CSF-mobilized peripheral blood. In yet another aspect, mononuclear dendritic cell precursors can also be selected by size using a differential centrifugation program (sometimes called elutriation).

[0045] Dendritic cell precursors and immature dendritic cells may optionally be prepared in a closed aseptic system. As used herein, the term "closed aseptic system" or "closed system" refers to a system that minimizes or eliminates contact with non-sterile ambient air, recirculated air, or other non-sterile conditions. Closed systems for isolating dendritic cell precursors and immature dendritic cells typically exclude operations such as density gradient centrifugation in open tubes, cell transfer in open air, and cell culture in tissue culture plates or unsealed culture flasks. In a typical embodiment, the closed system allows for the aseptic transfer of dendritic cell precursors and immature dendritic cells from an initial collection container to a sealable tissue culture container without exposure to non-sterile air.

[0046] In some embodiments, monocyte dendritic cell precursors can be separated by adhering to a monocyte binding matrix. For example, a group of leukocytes (e.g., leukocytes separated by leukoablation) can be brought into contact with a monocyte dendritic cell precursor adhesion matrix. When the leukocyte group is in contact with the matrix, the monocyte dendritic cell precursors within the leukocyte group preferably adhere to the matrix. Other leukocytes (including other potential dendritic cell precursors) have a reduced binding affinity to the matrix, thereby preferentially enriching the monocyte dendritic cell precursors on the matrix surface.

[0047] Suitable matrices include those with a large surface area to volume ratio. Such matrices can be granular or fibrous, for example. Suitable granular matrices include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other microbeads suitable for protein adsorption. Suitable fibrous matrices include microcapillaries and microvilli. Granular or fibrous matrices generally allow the elution of adherent monocyte-dendritic cell precursors without significantly reducing the viability of the adherent cells. Granular or fibrous matrices can be substantially poreless to facilitate the elution of monocyte-dendritic cell precursors or dendritic cells from the matrix. A “substantially poreless” matrix is ​​one in which at least a majority of the pores are smaller than the cells, minimizing cell retention within the matrix.

[0048] The adhesion of monocyte dendritic cell precursors to the matrix can optionally be enhanced by the addition of a binding medium. Suitable binding media include monocyte dendritic cell precursor media (e.g., AIM-V®, RPMI1640, DMEM, X-VIVO15®, etc.), supplemented with, for example, one or any combination of the following: cytokines (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4)), plasma, serum (e.g., human serum, such as autologous or allogeneic serum), purified proteins (e.g., serum albumin), divalent cations (e.g., calcium and / or magnesium ions), and other molecules that facilitate the specific adhesion of monocyte dendritic cell precursors to the matrix or prevent the adhesion of non-monocyte dendritic cell precursors to the matrix. In some embodiments, the plasma or serum may be heat-inactivated. The heat-inactivated plasma may be homologous to or heterologous to leukocytes.

[0049] After monocyte-dendritic cell precursors adhere to the matrix, the unattached leukocytes need to be separated from the monocyte-dendritic cell precursor / matrix complex. Any suitable method can be used to separate the unattached cells from the complex. For example, the mixture of unattached leukocytes and the complex can be allowed to settle, and then the unattached leukocytes and culture medium can be decanted or drained. Alternatively, the mixture can be centrifuged, and the supernatant containing the unattached leukocytes can be decanted or drained from the settled complex.

[0050] Isolated dendritic cell precursors can be cultured in vitro to achieve differentiation, maturation, and / or expansion. As used herein, "isolated immature dendritic cells," "dendritic cell precursors," "T cells," and other cells refer to cells that have been artificially removed from their native environment and are therefore not natural products. Isolated cells may exist in purified, semi-purified, or non-native environments. In short, in vitro differentiation typically involves culturing dendritic cell precursors or cell populations containing dendritic cell precursors in the presence of one or more differentiating agents. Suitable differentiating agents may be, for example, cell growth factors (e.g., cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and / or combinations thereof). In some embodiments, monocyte-derived dendritic cell precursors differentiate into monocyte-derived immature dendritic cells.

[0051] Dendritic cell precursors can be cultured and differentiated under suitable culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, etc. Tissue culture media can be supplemented with serum, amino acids, vitamins, cytokines (such as GM-CSF and / or IL-4), divalent cations, etc., to promote cell differentiation. In some embodiments, dendritic cell precursors can be cultured in serum-free media. Such culture conditions may optionally exclude any animal-derived products. A typical combination of cytokines 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). After the dendritic cell precursors differentiate into immature dendritic cells, their phenotype is similar to that of skin Langerhans cells. Immature dendritic cells are typically CD14-positive. - and CD11c + They do not express or express low levels of CD80 and CD83, and are able to capture soluble antigens through a special endocytosis process. Immature DCs express high levels of CD86.

[0052] Immature dendritic cells mature into mature dendritic cells (DCs). Mature DCs lose their ability to take up antigens and upregulate the expression of co-stimulatory cell surface molecules and various cytokines. Specifically, mature DCs express higher levels of MHC class I and II antigens than immature DCs, and mature DCs are typically identified as CD80-positive. + CD83 + CD86 + and CD14 - Increased MHC expression leads to increased dendritic surface antigen density, while upregulation of co-stimulatory molecules CD80 and CD86 enhances T cell activation signals through their co-stimulatory counterparts (such as CD28) on T cells.

[0053] For example, mobilized (e.g., granulocyte colony-stimulating factor (G-CSF) mobilized monocytes) from peripheral blood progenitor cells can be separated by centrifugation at 450 × g for 20 min on a Histopaque-1077 (Sigma, St. Louis, Missouri, USA) gradient. After separation, CD14 can be separated using a magnetic separation column containing CD14 microbeads, according to the manufacturer's instructions (Miltenyi Biotec). + MNC). CD14 + Cells can be cultured for 6 days in a humidified incubator at 37°C and 5% CO2, at a concentration of 2×10⁻⁶. 6 Cells / mL were cultured in AIM-V medium (Invitrogen, Carlsbad, California, USA) supplemented with 10% human AB serum (Atlanta Biologicals, Lawrenceville, Georgia, USA), 50 μg / mL streptomycin sulfate (Invitrogen), 10 μg / mL gentamicin sulfate, 2 mM L-glutamine (Invitrogen), 50 ng / mL GM-CSF (Amgen, Thousand Oaks, California, USA), and 10 ng / mL IL-4 (R&D Systems, Minneapolis, Minnesota, USA). On day 3, the medium was removed and replaced with an equal volume of fresh medium. After 6 days of culture in GM-CSF and IL-4, immature dendritic cells (DCs) were collected using enzyme-free cell dissociation buffer. Flow cytometry showed that immature DCs exhibited CD11c... + CD80 - CD83 - CD86 + CD209 + and HLA-DR + The phenotype.

[0054] Immature dendritic cells (DCs) can be loaded with a single recombinant protein and / or any vector that transduces the expression of the same protein. For example, immature DCs can be loaded with tumor lysates, tumor mRNA, or both. If loading with lysate, an exemplary protocol is as follows: immature DCs are loaded with 5 × 10⁻⁶ cells of cell lysate diluted four times. 6 Cells were cultured at a concentration of [number] cells / mL at 37°C for 3 hours. After lysate loading, cells were collected by centrifugation, washed once to remove residual lysates, and then cultured at 1–2 × 10⁻⁶ cells / mL. 6Cells were matured at a density of 100 cells / mL in AIM-V medium (Invitrogen, Carlsbad, California, USA) for 36–48 hours. This medium was supplemented with 10% human AB serum (Atlanta Biologicals, Lawrenceville, Georgia, USA), 50 μg / mL streptomycin sulfate (Invitrogen), 10 μg / mL gentamicin sulfate, 2 mM L-glutamine (Invitrogen), 50 ng / mL GM-CSF (Amgen, Thousand Oaks, California, USA), 10 ng / mL IL-4 (R&D Systems, Minneapolis, Minnesota, USA), and supplemented with 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 PG). (Sigma) (See Decker et al., Vaccine 24:3203-3216, 2006). To stimulate dendritic cells (DCs) to migrate towards T... H 2. Polarization, with further supplementation of the culture medium, for example, 10 ng / mL SEB (see Halpert et al., Stem Cells and Development 25:774-787, 2016). T H Two-polarized dendritic cells (DCs) exhibited low levels of IL-12 expression and high levels of IL-13, GATA3, CTLA-4, and SIRP-α (CD172a) expression, as determined by qPCR. To stimulate DCs to polarize into T cells... H 1. Polarization, for example, by further supplementing the culture medium with 1-2 ng / mL IL-12. T H 1-polarized DCs exhibited low levels of IL-13 and CTLA-4 expression and high levels of IL-12 and CADM1 expression, which were determined by qPCR.

[0055] For mRNA loading, an exemplary electroporation procedure is as follows: immature DCs are subjected to 4 × 10 7 A concentration of 1 cell / mL was suspended in Viaspan solution (Barr Laboratories, Pomona, NY, USA) and mixed with tumor mRNA to achieve a concentration of 1 μg mRNA / 10 6Cells were then incubated on ice for 10 minutes in electroporation cuvettes with a 0.4 cm gap (Bio-Rad, Hercules, California, USA). Electroporation was then performed using a GenePulser Xcell (Biorad) at 300 V, 150 μF, and Ω=∞. Immediately after electroporation, cells were introduced into culture medium and treated in the same manner as DCs loaded with lysates. The viability of DCs after electroporation was typically higher than 75%.

[0056] Dual-loaded DCs can receive lysates and mRNA preparations from the same cell type, thereby inducing T... H 1. Polarization. When simultaneously loaded with mRNA and lysate, the cell may receive the lysate first and then the mRNA, or vice versa. Preferably, when simultaneously loaded with mRNA and lysate, the cell receives the mRNA first and then the lysate to avoid exposing the mRNA to any RNases that may be present in the lysate.

[0057] After load and maturation, DC will exhibit CD11c behavior. + CD80 + CD83 + CD86 ++ CD209 + and HLA-DR ++ Notably, markers involved in co-stimulation or antigen presentation (CD80, CD83, CD86, HLA-DR) were upregulated during maturation, while markers involved in cell adhesion (CD11c, CD209) were downregulated.

[0058] The mature dendritic cells described in this invention can be prepared (i.e., matured) by contacting immature dendritic cells with an effective amount or concentration of a nucleic acid composition and a homologous cell antigen composition. The effective amount of the nucleic acid composition is typically up to, at least, or about 0.01, 0.1, 1, 5, 10 to 10, 15, 20, 50, 100 ng or mg of nucleic acid per culture dish or per cell, including all values ​​between these ranges. The effective amount of the cell lysate antigen composition is typically up to, at least, or about 0.01, 0.1, 1, 5, 10 to 10, 15, 20, 50, 100 ng or mg of protein per culture dish or per cell. In some cases, cell lysate antigens ranging from 0.001 ng tumor antigen / cell to 1 μg cell lysate antigen / million cells may be used. The cell lysate antigen components may optionally be heat-inactivated, irradiated, or treated (e.g., exposed to proteases) before contact with the dendritic cells (DCs).

[0059] Immature dendritic cells (DCs) are typically exposed to effective amounts of nucleic acid and cell lysate antigen compositions for at most, at least, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 minutes, several hours, or several days. Immature DCs can be cultured and matured under suitable maturation conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, etc. Tissue culture media can be supplemented with amino acids, vitamins, cytokines (such as GM-CSF and / or IL-4), divalent cations, etc., to promote cell maturation.

[0060] Dendritic cell maturation can be monitored using methods known in the art. Cell surface markers can be detected using methods familiar in the art, such as flow cytometry and immunohistochemistry. Cytokine production can also be monitored (e.g., by ELISA, FACS, or other immunoassays). Whether or not pre-sensitized, dendritic cell precursors, immature dendritic cells, and mature dendritic cells can be cryopreserved after binding to the antigen for future use. Cryopreservation methods are well known in the art. For example, U.S. Patent No. 5,788,963, the entire contents of which are incorporated herein by reference.

[0061] To isolate EVs from mature DCs, the cell culture supernatant was centrifuged sequentially at 400×g for 10 minutes and then at 2000×g for 30 minutes at room temperature to remove precipitated cells and cell debris from the formulation. The EVs in the resulting supernatant were precipitated using PEG precipitation. For example, the resulting supernatant was incubated with pre-chilled total exosome separation reagent (Invitrogen, catalog number: 4478359) for 16–18 hours. Subsequently, the mixture was centrifuged at 10,000×g for 1 hour at 4°C. The resulting precipitate was resuspended in PBS buffer and incubated with anti-CTLA-4 antibody bound to magnetic beads at 4°C for 16–18 hours to promote CTLA-4 absorption by applying a strong magnetic field. + EV separation / removal. Complete CTLA-4 + EV recovery was achieved by elution with high-salt buffer at room temperature for 2 hours. The isolated EVs ranged in diameter from 30 to 200 nm, as measured by cryo-electron microscopy. Regardless of CTLA-4 state, the EVs exhibited CD63... + CD81 + and HLA-DR + The phenotype.

[0062] III. Composition and Processing Method

[0063] Some aspects of the implementation involve CTLA-4 isolated from mature dendritic cells. + Or CTLA-4 -The provision of EVs. Specifically, for the development of CD8 inhibitors for cancer treatment. + T cells should be given CTLA-4. - EV. On the other hand, CTLA-4 should be administered to suppress activated T cells in order to treat T cell-mediated autoimmune conditions, such as post-HSCT GVHD. + EV.

[0064] In the treatment of T-cell-mediated autoimmune conditions, such conditions that can be treated using 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, shotgun retinochoroidal disease (BSR), and sympathetic ophthalmia). In addition, examples of autoimmune conditions 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, Sidnam's chorea, myasthenia gravis, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, Hennech-Schönlein purpura, post-streptococcal nephritis, erythema nodosum, aortitis, Addison's disease, and so on. Osteopathy, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpassinomyces syndrome, thromboangiitis obliterans, primary biliary cirrhosis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrotic alveolitis.

[0065] In the treatment of cancer, the cancers that can be treated using the methods described herein include, but are not limited to, bladder cancer, leukemia, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gingival cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. In some respects, cancer can be a tumor, malignant tumor; carcinoma; undifferentiated carcinoma; giant cell / spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma associated with familial adenomatous polyposis; solid carcinoma; malignant carcinoid; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; anaerobic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; breast Head and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland adenocarcinoma; sebaceous gland adenocarcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystic adenocarcinoma; papillary cystic adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cystic adenocarcinoma; 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 carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androgen blastoma; Sertoli cell carcinoma; malignant Ledich's cell tumor; malignant lipocytoma; malignant paraganglioma; malignant Extramammary paraganglioma; Pheochromocytoma; Balloon sarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Malignant melanoma in giant nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müller's tube mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner's tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma; Malignant ovarian tumor Goiter; Choriocarcinoma; Malignant mesonephric tumor; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Cortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pineal tumor; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibroblastic astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma;Ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia. In other respects, the cancers mentioned are brain cancer (e.g., glioma), prostate cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), acute myeloid leukemia (AML), melanoma, renal cell carcinoma, or chronic lymphocytic leukemia.

[0066] In some respects, the EVs used in these methods are suitably contained in pharmaceutically acceptable carriers. These carriers are non-toxic, biocompatible, and selected to not adversely affect the biological activity of the EVs. EVs can be formulated for local delivery (i.e., to a specific site of the body, such as skeletal muscle or other tissues) or systemic delivery. Suitable carriers for parenteral delivery such as injection, infusion, or perfusion, as well as for local delivery, include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solution, glucose solution, Hanks' solution, or propylene glycol. Additionally, sterile fixative oils can also be used as solvents or suspension media. For this purpose, any biocompatible oil, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids such as oleic acid are used in the preparation of injectable formulations.

[0067] Solutions of the pharmaceutical compositions can be prepared in water, with appropriate mixing of surfactants (such as hydroxypropyl cellulose). The compositions disclosed herein may contain glycerol, liquid polyethylene glycol, and mixtures thereof. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0068] In some respects, administration of pharmaceutical compositions in the form of injections is advantageous, and the injections can be liquid solutions or suspensions. Typical compositions for this purpose contain pharmaceutically acceptable carriers. For example, the compositions may contain less than, equal to, or more than 10 mg, 25 mg, 50 mg, or up to about 100 mg of human serum albumin per milliliter of phosphate-buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients (including salts), preservatives, buffers, etc.

[0069] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, physiological saline, and parenteral carriers (such as sodium chloride, Ringer's glucose solution, etc.). Intravenous infusion carriers include fluids and nutritional supplements. Preservatives include antibacterial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of a pharmaceutical composition are routinely adjusted according to well-known parameters.

[0070] In other respects, pharmaceutical compositions containing EVs may include classic pharmaceutical formulations. In some embodiments, the composition may be administered to the subject by any method known to those skilled in the art. Examples include administration via intravenous injection, intradermal injection, arterial injection, intraperitoneal injection, intralesional injection, intratumoral injection, peritumoral injection, intramuscular injection, subcutaneous injection, intracystic injection, mucosal injection, injection, infusion, continuous infusion, catheter administration, or any other method known to those skilled in the art or any combination of the above methods. According to some aspects, pharmaceutical compositions containing EVs may be administered via any commonly used route, as long as the route reaches the target tissue. Administration may be via in situ injection, intradermal injection, subcutaneous injection, intramuscular injection, intratumoral injection, peritumoral injection, intraperitoneal injection, or intravenous injection. Such compositions are generally administered in a pharmaceutically acceptable form containing physiologically acceptable carriers, buffers, or other excipients. In some aspects of the embodiments, the EV is administered to a lymphatic tissue site near the lesion cell population in the subject. The lymphatic tissue site may be lymphatic tissue draining the tissue surrounding the lesion cell population. For example, in some embodiments, EV is administered to lymph nodes draining tissue surrounding the lesion cell population. In some embodiments, EV is administered to tissue draining into lymphatic tissue sites near the lesion cell population within the subject.

[0071] The effective amount of a pharmaceutical composition depends on the intended target. The term "unit dose" or "dose" refers to a physically discrete unit suitable for use by the subject, each unit containing a predetermined amount of the pharmaceutical composition that is calculated to produce the desired response associated with its administration, i.e., the appropriate route and treatment regimen. Depending on the number of treatments and the unit dose, the amount to be administered depends on the desired protective effect or efficacy.

[0072] The precise dosage of a pharmaceutical composition depends on the judgment of the clinician and varies from person to person. Factors influencing dosage include the patient's physical and clinical condition, route of administration, treatment goals (e.g., symptom relief or disease cure), and the potency, stability, and toxicity of the specific therapeutic substance. In some respects, the actual dose of the composition administered to an animal or patient can be determined by physical and physiological factors such as weight, severity of the condition, type of disease being treated, prior or concurrent treatment interventions, the patient's idiopathic condition, and the route of administration. In any case, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0073] In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% (w / v) of an active agent, such as isolated EV. In other embodiments, the EV may contain about 2% (w / v) to about 75% (w / v) of units, or, for example, about 25% (w / v) to about 60% (w / v) of units, and any range thereof. In other non-limiting examples, the dosage may also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, to about 1000 mg / kg / body weight or more, and any other ranges that may be derived therefrom. In non-limiting examples of the numerical ranges derived herein, about 5 micrograms / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 mg / kg / body weight, etc., may be administered.

[0074] The method may involve administering a composition containing (or comprising) exosomes at concentrations of: about, at least about, or at most 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, 11.5, 1 2.0, 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, 5 9, 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, 255260, 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), micrograms (μg), milligrams (mg), or grams of exosomes, or any available range thereof. The above values ​​may also represent the dose calculated based on the patient's weight, in ng / kg, mg / kg, or g / kg, or any available range thereof.

[0075] Alternatively, the concentration of exosomes in the composition may be 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, or 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, μg / mL, mg / mL, or g / mL, or any available range thereof.

[0076] The composition may be given to a patient (or taken by the patient) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more, or any available range thereof; and may be given 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 any available time range thereof. Specifically, the composition may be administered once, twice, three times, four times, five times, or six times daily (or any available range thereof), and / or as needed by the patient. Alternatively, the composition may be administered every 2, 4, 6, 8, 12, or 24 hours (or any available range thereof), or may be administered by the patient themselves. In some embodiments, the patient is treated with the composition for a specified period or dose after the onset of symptoms of demyelinating disease.

[0077] IV. Combination Therapy

[0078] To improve the effectiveness of dendritic cell-derived EV therapy, it may be necessary to combine these compositions with other effective drugs for treating the target disease.

[0079] As a non-limiting example, cancer treatment may employ the pre-sensitized dendritic cell composition of this embodiment in combination with other anticancer agents. "Anticancer" agents refer to those capable of negatively impacting cancer in a subject's body, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, shrinking tumor volume, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses against cancer cells or tumors, preventing or inhibiting cancer progression, or prolonging the lifespan of cancer patients. More generally, the combined amount of these other compositions should be sufficient to kill or inhibit cell proliferation. The process may include simultaneously contacting cells with an anticancer peptide or nanoparticle complex and one or more agents or factors. This can be achieved by contacting cells with a single composition or pharmaceutical formulation containing both agents, or by simultaneously contacting cells with two different compositions or formulations, one composition containing a dendritic cell composition and the other containing a second agent.

[0080] Dendritic cell composition treatment can precede or follow other drug treatments, with intervals ranging from minutes to weeks. In embodiments where other drugs and the dendritic cell composition are applied separately to the target, it should generally be ensured that the intervals between each delivery are short so that the drug and the dendritic cell composition can still produce a beneficial combined effect on the cells. In such cases, it may be considered to contact the cells in two ways over about 12-24 hours, more preferably over about 6-12 hours. In some cases, it may be necessary to significantly extend the treatment time, with intervals between separate administrations ranging from 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).

[0081] Various combinations can be used, with dendritic cell-derived MV therapy designated as "A," and a second agent (such as radiotherapy, chemotherapy, or anti-inflammatory drugs) designated as "B." A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A In some embodiments, the dendritic cell therapy of this embodiment will be administered to patients following the general protocol for chemotherapy drug administration, taking into account the toxicity of the carrier (if any). Treatment cycles are expected to be repeated as necessary. Furthermore, it is envisioned that various standard therapies and surgical procedures can be combined with the described high-proliferation cell therapy.

[0082] A. Chemotherapy

[0083] Cancer therapy also includes a variety of combination therapies. In some aspects, the dendritic cell-derived MV composition of the embodiments is administered (or formulated) in combination with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor, such as 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 inhibitor. Non-limiting examples of protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, and sorafenib. Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, Rapamycin apamycin), temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0084] Other combination chemotherapy drugs include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridine derivatives such as benzodopa, carboquone, meturedopa, and uredopa; and ethyleneimine and melamine derivatives, including atratamine, triethylenemelamine, and trietylenephosphoramide. Triethylenethiophosphoramide and trimethylolmelamine; acetogenins, especially bullatacin and bullatacinone; camptothecin derivatives, including the synthetic analog topotecan; bryostatin; callystatin; CC-1065, including its synthetic analogs adozelesin, carzelesin, and bizelesin; cryptophytin derivatives, especially cryptophytin-1. 1) and cryptophycin 8; dolastatin; duocarmycin, including synthetic analogs KW-2189 and CB1-TM1; eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustard drugs, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, and prednimustine. Trofosfamide and uracilmustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics, including calicheamicin, especially calicheamicin γII and calicheamicin ωII; dynemicins, including dynemicin A; bisphosphonates, Examples include clodronate; esperamicin; neocarzinostatin chromophore and related chromophore proteins; aclacinomycin; actinomycin; authramycin; azaserine; bleomycin; cactinomycin; carabicin; carminomycin; and cazinofeline. rzinophilin; chromomycin; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin, including morpholine-duxorubicin, cyanomorpholine-duxorubicin, 2-pyrrololine-duxorubicin and deoxyduxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin;Mitomycin derivatives, such as mitomycin C; mycophenolic acid; nogalamycin; olivomycin; peplomycin; potfiromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin; tubercidin; ubenimex; and others. Nosstatin; zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine. Ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine (Ara-C), dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone, dromostanolone propionate, epistanol, and mepiston. Mepitiostane and testolactone; anti-adrenergic drugs, such as mitotane and trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;Edatraxate; Deofamine; Colchicine; Diaziquone; Elformithine; Elliptinium Acetate; Epothilone; Etoglucid; Gallium Nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansine and other maytansine-like compounds. Ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex. ccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes, especially T-2 toxin, verracurin A, roridin A, and anguidine (triene biotoxins). Enes; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactal; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; taxanes, such as paclitaxel and docetaxel; gemcitabine;6-Thioguanine; mercaptopurine; platinum coordination compounds, 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 inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyltransferase inhibitors, transplatinum; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing drugs. In some embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, this embodiment may be used in combination with Gleevec (e.g., Gleevec may be administered to the patient at about 400 to about 800 mg / day). In some embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.

[0085] B. Immunotherapy

[0086] Those skilled in the art will understand that immunotherapy can be used in combination with or incorporating the methods of this embodiment. In the context of cancer treatment, immunotherapy typically relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. For instance, an immune effector can be a specific antibody against certain markers on the surface of tumor cells. The antibody itself can act as a therapeutic effector or can recruit other cells to actually influence cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and act as a target only. Alternatively, the effector can be a lymphocyte carrying a surface molecule that can interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0087] In one aspect of immunotherapy, tumor cells must possess certain markers that can be targeted (i.e., not present on most other cells). Many tumor markers exist, and any one of them may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy combines anticancer effects with immunostimulatory effects. Immunostimulatory molecules are also present, 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.

[0088] Immunotherapy currently under investigation or in use includes immune adjuvants, such as Mycobacterium bovis (B. bovis). Mycobacterium bovis ), Plasmodium falciparum ( 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, antiganglioside GM2 and anti-p185. It is anticipated that one or more anticancer therapies can be used in conjunction with the antibody therapies described herein.

[0089] In some implementations, immunotherapy can be an immune checkpoint inhibitor. This includes immune checkpoint on signals (e.g., co-stimulatory molecules) or off signals. Immune checkpoint targeting blockade can target immune checkpoint proteins including adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (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), and programmed death ligand 1. (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T cell immune receptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activation V domain Ig inhibitor (VISTA, also known as C10orf54) and 4-1BB (CD137). Specifically, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0090] Immune checkpoint inhibitors can be drugs, such as small molecules, recombinant forms of ligands or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-264, 2012; both incorporated herein by reference). Known immune checkpoint protein inhibitors or analogues thereof can be used, particularly chimeric, humanized, or human antibodies. Those skilled in the art will recognize that aliases and / or equivalent names may be used for certain antibodies mentioned in this disclosure. In the context of this disclosure, such class names and / or equivalent names may be used interchangeably. For example, lambrolizumab is also known by the aliases and equivalent names MK-3475 and pembrolizumab.

[0091] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a particular aspect, the PD-L2 binding partner 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. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists known in the art for use in the methods provided herein, such as those described in U.S. Patent Application Publication Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, are all incorporated herein by reference.

[0092] 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 nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding moiety 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 is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO. ® This is the anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambolizumab, and KEYTRUDA. ® SCH-900475 is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0093] Another immune checkpoint protein that can be targeted using the methods described herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as a “switch” when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is similar to the T-cell costimulatory protein CD28, both of which bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also present in regulatory T cells and may be important for their function. Activation of T cells via T-cell receptors and CD28 leads to increased CTLA-4 expression; CTLA-4 is an inhibitory receptor for the B7 molecule.

[0094] 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods described herein can be generated using methods known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. Anti-CTLA-4 antibodies that can be used in the methods described herein are disclosed, for example: U.S. Patent No. 8,119,129; PCT Publications WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly known as ticilimumab); 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 number 2505 (antibody CP-675206); and Mokyr wait, (1998) Cancer Res , 58:5301-5304. The teachings in each of the foregoing disclosures are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding CTLA-4 may also be used. Humanized CTLA-4 antibodies are described, for example, in International Patent Applications WO2001 / 014424, WO2000 / 037504 and U.S. Patent No. 8,017,114; all of which are incorporated herein by reference.

[0095] 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, WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs 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 competitively binds to and / or binds to the same CTLA-4 epitope as the antibody described above. In yet another embodiment, the antibody has at least about 90% amino acid sequence identity in the variable region with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules used to modulate CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, as well as the immunoadhesin described in U.S. Patent No. 8,329867, which is incorporated herein by reference.

[0096] Another immune checkpoint protein that can be targeted in the methods provided herein is lymphocyte activation gene 3 (LAG-3), also known as CD223. The Genbank accession number for the complete human LAG-3 protein sequence is NP-002277. LAG-3 is present on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an "off" switch when it binds to class II MHC on the surface of antigen-presenting cells. Inhibition of LAG-3 activates both effector T cells and 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art can be used. Exemplary anti-LAG-3 antibodies are relatlimab (also known as BMS-986016) or its antigen-binding fragments and variants (see, for example, WO 2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, for example, WO 2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in WO 2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in WO 2017 / 220569.

[0097] Another immune checkpoint protein that can be targeted in the methods provided herein is the V-domain Ig inhibitor of T cell activation (VISTA), also known as C10orf54. The GenBank accession number for the complete human VISTA protein sequence is NP_071436. VISTA is present on leukocytes and inhibits T cell effector function. 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human VISTA antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-VISTA antibodies recognized in the art can be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as onvatilimab) (see, for example, WO 2015 / 097536, WO2016 / 207717, WO 2017 / 137830, WO 2017 / 175058). The small molecule CA-170 can also inhibit VISTA; this small molecule selectively targets PD-L1 and VISTA (see, for example, WO 2015 / 033299, WO 2015 / 033301).

[0098] Another immune checkpoint protein that can be targeted in the methods provided herein is indoleamine 2,3-dioxygenase (IDO). The GenBank accession number for the complete human IDO protein sequence is NP_002155. In some embodiments, the immune checkpoint inhibitor is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and navoximod (GDC-0919).

[0099] Another immune checkpoint protein that can be targeted in the methods provided herein is CD38. The GenBank accession number for the complete human CD38 protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CD38 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods disclosed herein can be generated using methods known in the art. Alternatively, anti-CD38 antibodies recognized in the art can be used. An exemplary anti-CD38 antibody is daratumumab (see, for example, U.S. Patent No. 7,829,673).

[0100] Another immune checkpoint protein that can be targeted in the methods provided herein is ICOS, also known as CD278. The GenBank accession number for the complete human ICOS protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human ICOS antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-ICOS antibodies recognized in the art can be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, for example, WO 2016 / 154177, WO 2018 / 187191) and GSK3359609 (see, for example, WO 2016 / 059602).

[0101] Another immune checkpoint protein that can be targeted in the methods provided herein is the T-cell immune receptor (TIGIT) having Ig and ITIM domains. The GenBank accession number for the complete human TIGIT protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human TIGIT antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-TIGIT antibodies recognized in the art can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, for example, WO 2017 / 030823, WO 2016 / 028656).

[0102] Another immune checkpoint protein that can be targeted in the methods provided herein is OX40, also known as CD134. The GenBank accession number for the complete human OX40 protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human OX40 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-OX40 antibodies recognized in the art can be used. An exemplary anti-OX40 antibody is PF-04518600 (e.g., see WO 2017 / 130076). ATOR-1015 is a bispecific antibody against CTLA4 and OX40 (see, for example, WO 2017 / 182672, WO 2018 / 091740, WO 2018 / 202649, WO2018 / 002339).

[0103] Another immune checkpoint protein that can be targeted in the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), also known as TNFRSF18 and AITR. The GenBank accession number for the complete human GITR protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human GITR antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-GITR antibodies recognized in the art can be used. An exemplary anti-GITR antibody is TRX518 (e.g., see WO 2006 / 105021).

[0104] Another immune checkpoint protein that can be targeted in the methods provided herein is T-cell immunoglobulin and mucin domain-3 (TIM3), also known as HAVCR2. The GenBank accession number for the complete human TIM3 protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human TIM3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-TIM3 antibodies recognized in the art can be used. Exemplary anti-TIM3 antibodies include LY3321367 (see, for example, WO2018 / 039020), MBG453 (see, for example, WO 2015 / 117002), and TSR-022 (see, for example, WO 2018 / 085469).

[0105] Another immune checkpoint protein that can be targeted in the methods provided herein is 4-1BB, also known as CD137, TNFRSF9, and ILA. The GenBank accession number for the complete human 4-1BB protein sequence is 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, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human 4-1BB antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of this disclosure can be generated using methods known in the art. Alternatively, anti-4-1BB antibodies recognized in the art can be used. An exemplary anti-4-1BB antibody is PF-05082566 (e.g., utomilumab, see WO 2012 / 032433).

[0106] In some implementations, immunotherapy can be adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated in vitro. T cells used for adoptive immunotherapy can be generated through the expansion of antigen-specific T cells or by genetically engineering and reorienting T cells. The isolation and transfer of tumor-specific T cells has proven to be a successful approach for treating melanoma. New specificity has been successfully generated in T cells through genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signal transduction domains in a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv) containing a monoclonal antibody light chain and variable fragments linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully applied. The signal transduction domains of first-generation CARs are derived from the cytoplasmic region of the CD3ζ or Fc receptor γ chain. CARs have successfully reoriented T cells to target antigens expressed on the surface of tumor cells in various malignancies, including lymphomas and solid tumors.

[0107] In one embodiment, this 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 tumor antigens. An engineered hSDH enzyme may be administered to the patient before and / or concurrently with the adoptive T-cell therapy. Alternatively, autologous and / or allogeneic T cells may be engineered to express the engineered hSDH enzyme.

[0108] C. Radiation therapy

[0109] Other widely used factors that cause DNA damage include commonly known gamma rays, X-rays, and / or radioactive isotopes delivered to tumor cells. Other forms of DNA damage, such as microwave and ultraviolet radiation, are also considered. All of these factors are highly likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens per day for extended periods (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Radioactive isotope doses vary considerably depending on the isotope's half-life, radiation intensity and type, and uptake by tumor cells.

[0110] When applied to cells, the terms “contact” and “exposure” are used herein to describe the process of delivering a therapeutic composition and a chemotherapeutic or radiotherapy agent to or directly juxtaposed with target cells. For example, to achieve cell killing or arrest, two agents are delivered to the cells in an effective combined amount sufficient to kill the cells or prevent their division.

[0111] D. Gene therapy

[0112] In another embodiment, the second treatment is gene therapy, wherein the therapeutic polynucleotide is administered before, after, or simultaneously with the therapeutic composition. Viral vectors for expressing the gene product are well known in the art, including eukaryotic expression systems such as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses (including vaccinia virus), and papillomaviruses (including SV40). Alternatively, administration of the expression construct may also be performed using lipid carriers, such as liposomes or DOTAP:cholesterol vesicles.

[0113] E. Surgery

[0114] Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, therapeutic, and palliative surgeries. Therapeutic surgeries are cancer treatments that can be used in combination with other therapies, such as those described in this article, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0115] Therapeutic surgery includes resection, which involves the physical removal, excision, and / or destruction of all or part of the cancerous tissue. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Moser procedure). Furthermore, this document envisions that the described embodiments can be used in conjunction with the resection of superficial cancers, precancerous lesions, or small amounts of normal tissue. In some aspects, after tumor resection, the dendritic cell composition of this embodiment is administered to the lymphatic tissue draining the original tumor site.

[0116] V. Reagent Kit

[0117] Some embodiments described herein relate to kits, such as diagnostic and therapeutic kits, and kits for preparing and / or delivering exosomes or MVs. For example, a kit may contain one or more pharmaceutical compositions described herein, and optionally include instructions for use. The kit may also contain one or more devices for delivering such compositions. For example, a target kit may contain a pharmaceutical composition and a catheter for directly administering the composition to a patient who has or is at risk of developing demyelinating diseases. In other embodiments, a target kit may contain pre-filled ampoules of isolated exosomes, which may be selectively formulated as pharmaceutical preparations or lyophilized for use with a delivery device. The techniques described herein also include kits for generating MVs from dendritic cells.

[0118] The kit may contain labeled containers. Suitable containers include, for example, bottles, vials, and test tubes. Containers can be made of a variety of materials, such as glass or plastic. The container may contain a composition comprising antibodies effective for therapeutic or non-therapeutic purposes, as described above. The label on the container may indicate that the composition is intended for a specific therapeutic or non-therapeutic purpose, or its use in vivo or in vitro, as described above. In some embodiments, the kit will contain the containers described above, as well as one or more other containers containing materials necessary from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and a package insert with instructions for use.

[0119] VI. Examples

[0120] The following embodiments are included to demonstrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of this disclosure and can therefore be considered as preferred modes constituting their practice. Based on this document, those skilled in the art will understand that various changes can be made to the specific embodiments described herein within the spirit and scope of this document while still yielding the same or similar results.

[0121] Example 1

[0122] In summary, the data in this paper indicate that DCs express and secrete CTLA-4. + and CTLA-4 - Extracellular vesicles. T H CTLA-4 secreted by 1 polarized DCs - EV and CTLA-4 + The EV ratio is higher, while the T H CTLA-4 secreted by 2-polarized DCs - EV and CTLA-4 + The proportion of EVs is low; however, regardless of the DC's T HRegardless of the polarization state, both types of EVs can be recovered, and they appear to have similar functional characteristics, also independent of the DC polarization state. CTLA-4 - EVs can induce T cells to transform into T cells. H 1. It enhances the ability to differentiate polarized phenotypes, induces upregulation of IFN-γ, granzyme B, CD25, and CD161 expression, and increases the CD8 to CD4 ratio in proliferating T cell blasts. CTLA-4 + EV produces the opposite effect, inducing T H 1. Downregulation of polarization and activation markers, and induction of upregulation of depletion markers such as PD-1 and Tim3. These data suggest that CTLA-4 + EVs, or "DC-derived exosomes," can be administered as needed to improve autoimmune conditions, solid organ transplant rejection, or GVHD following HSCT. The difference lies in CTLA-4. - EV / DC-derived exosomes can enhance T when needed. H 1. Administered in cases of immune response, including immunotherapy for chronic viral infections or cancer.

[0123] Example 2

[0124] Dendritic cells (DCs) are key mediators of the immune system, connecting the innate and adaptive branches of the immune response. They not only participate in the first line of defense against pathogens and tumors (innate immunity) but also drive subsequent adaptive immune responses, determining their intensity and nature. The ways in which dendritic cells help modulate adaptive immune responses to persistent attacks is an important area of ​​research, as these mechanisms involve a range of external and internal signals that drive DC differentiation and maturation pathways, specifically related to surface receptors and soluble inflammatory mediators expressed by DCs (Cella et al., 1999; Sallusto et al., 1995; Sallusto et al., 1994). Furthermore, DCs release extracellular vesicles (EVs), which have been reported to influence immune responses in multiple ways (Segura et al., 2005; Thery et al., 2002; Wakim et al., 2011).

[0125] As described in other literature (Thery et al., 2009), extracellular vesicles (EVs) are membrane-bound entities with a diameter of 30–1000 nm that carry payloads and can be released during cellular homeostasis and upon stimulation. These vesicles can be classified based on their size, biogenesis, ultracentrifugation characteristics, and contents (Thery et al., 2009). For example, exosomes (30–150 nm) carry blastocyst proteins and nucleic acids (primarily miRNAs) that are targeted to the endosome membrane, which then invaginates to enclose these payloads, forming multivesicular bodies (MVBs), in which the vesicles reside within the endosome. Once the MVB membrane fuses with the plasma membrane, these vesicles are released into the extracellular space (Babst et al., 2002; Odorizzi et al., 1998; Saksena et al., 2007; Stuffers et al., 2009). Furthermore, the plasma membrane buds outward to form microvesicles (100-1000 nm), extranuclear granules (50-200 nm), and apoptotic bodies (50-500 nm), which can be distinguished based on size, carrier, and biogenetic pathway (Thery et al., 2009; Heinen et al., 1999). Dendritic cell (DC)-derived EVs are key mediators in immune responses through their ability to transfer antigens between DCs and to initiate immune responses independently of parental DCs (Segura et al., 2005; Thery et al., 2002; Wakin et al., 2011). These immunomodulatory functions, along with other EV-specific characteristics, make DC-derived EVs (DC-derived exosomes) an ideal choice for off-the-shelf cellular immunotherapy. Specifically, EVs are subcellular entities and do not possess known direct cytolytic capabilities, thus limiting the risk of graft-versus-host disease. Furthermore, obtaining a sufficient number of dendritic cells (DCs) for cell-based vaccination is technically challenging, and the chemotherapy involved in mobilizing these cells can adversely affect patients' quality of life; DC-derived exosomes offer an alternative that can generate antigen-specific responses with relatively fewer DCs. Finally, because DC-derived exosomes are subcellular entities, they may be unaffected by the immunosuppressive tumor microenvironment, which often hinders cell-based vaccines.

[0126] In exploring the therapeutic potential of DC-derived exosomes, the inventors' past (Halpert et al., 2016) and current research has shown that the presence of the immunosuppressive molecule CTLA-4 inhibits the immunostimulatory effects of DC-derived exosomes. The inventors previously determined that CTLA-4 is upregulated in mature DCs, subsequently packaged into DC-derived exosomes, and released into the extracellular space. Upon observation of these DC-derived exosomes uptake by DCs, the expression of their surface B7 is reduced, a reduction dependent on the CTLA-4 receptors CD80 and CD86. Furthermore, co-culturing DCs treated with CTLA-4 siRNA with autologous T cells significantly increases CD86 expression. + CD25 + IFNγ + The proportion of T cells, and the frequency of Tregs (CD4+) compared to DCs (NT-siRNA) without targeted siRNA treatment. + CD25 + FoxP3 + The antitumor immunity and survival rate of DC vaccine recipients treated with CTLA-4 siRNA were significantly reduced in the B16 mouse melanoma model compared with those treated with NT-siRNA. This evidence suggests that CTLA-4 and DC-derived exosomes are key mediators of DC-driven CD8 T cell responses in vitro and in vivo (Halpert et al., 2016).

[0127] Here, the inventors expanded upon previous research findings and further explored CTLA-4. + The direct impact of DC-derived exosomes on T cell effector function, specifically in the generation of an effective type 1 (T1) immune response. Given that the T cell-DC synapse and the close contact between DCs and T cells are crucial for generating a potent immune response, the inventors hypothesized that CTLA-4... + DC-derived exosomes can directly regulate the T1 response to limit the generation of abnormal tissue destruction programs. This can be achieved using the inventors' human monocyte-derived dendritic cell model and conditional knockout mouse (cKO) model (CTLA-4). fl / fl CD11c Cre ) and mouse tumor models, the inventors proved CTLA-4 + DC-derived exosomes can limit the generation of potent T1 immune responses, which is of great significance for the design of DC-based immunotherapies and the treatment of T1-response-related diseases.

[0128] DC CTLA-4 levels are regulated by in vitro maturation conditionsIn a steady state, most human dendritic cells (DCs) reside in tissues, with relatively few detectable in peripheral blood. Therefore, for therapeutic and research purposes, tissue-resident DCs are typically only obtained in disease states requiring tissue biopsy, bone marrow, or whole-organ harvesting. Consequently, the inventors have primarily relied on in vitro DC generation from monocytes (MoDCs) isolated from peripheral blood to elucidate the biological characteristics of this elusive white blood cell population. To fully realize the therapeutic potential of DCs, the limitations of previous therapeutic successes must be addressed, identifying prior obstacles and methods to circumvent them (Laureano et al., 2022). To this end, the inventors' previous research identified conditions that can polarize moDCs in vitro to promote Th1 or Th2 response phenotypes. These moDCs can also drive T cell responses and mature in a manner unbiased towards Th1 or Th2 responses (hereinafter referred to as Th0 DCs). Under this paradigm, the inventors determined that Th1 DCs are characterized by elevated expression of IL-12A and IL-12B, while Th2 DCs show almost no expression of IL-12A, low IL-12B expression levels, but significantly elevated levels of IL-13 and CTLA-4. Figure 6A and 6C On the other hand, Th0 DCs express moderate levels of IL-12A, IL-12B, and CTLA-4, while IL-13 expression is low or absent.

[0129] Of particular interest is the role of moDC-derived DC-derived exosomes in the Th cell polarization paradigm, as their immunomodulatory effects are well-documented, although the specific mechanisms are not fully elucidated (Segura et al., 2005; Thery et al., 2002; Wakim et al., 2011). Therefore, the inventors extended their previous findings by performing miRNA sequencing analysis on Th0 and Th2 DC-derived exosomes to identify differences in miRNA loads within these DC-derived exosomes, thereby gaining deeper insights into their varying effects on T cell responses. By analyzing vesicles from three independent donors, the inventors identified 69 miRNAs whose expression levels differed between Th0 and Th2 DC-derived exosomes. Figure 6B The protein carriers carried by DC-derived exosomes are another key component in elucidating the role of DC-derived exosomes in Th cell polarization differentiation. Therefore, the inventors previously inferred that moDC DC-derived exosomes contain CTLA-4. + Subpopulations, the levels of which vary based on the polarization state of parental DCs. To this end, the inventors immunoprecipitated CTLA-4 from total DC-derived exosome isolates from immature or Th2-type moDCs. +DC-derived exosomes were analyzed using Western blot analysis. The inventors found that although CTLA-4... + and CTLA-4 - All subsets contain the extracellular vesicle marker tetraspanmembrane protein CD63, but only CTLA-4... - The subsets were positive for ubiquitinated proteins (Figure 1E). This contributes to a deeper understanding of the differences in the biogenesis of these DC-derived exosome subsets. Ubiquitination has been shown to drive protein packaging into multivesicular bodies (MVBs) and the recognition and transport of these carriers by ESCRT proteins, ultimately leading to their release as extracellular vesicles (Babst et al., 2002; Odorizzi et al., 1998; Saksena et al., 2007). Therefore, the inventors' findings suggest that CTLA-4... + Protein packaging in the DC-derived exosome compartments is independent of the ESCRT pathway. Next, the inventors attempted to determine CTLA-4... + and CTLA-4 - The inventors conducted a comprehensive proteomics study of the compartments, using this as a method to more effectively understand the origin and function of these DC-derived exosome subsets. Therefore, they studied CTLA-4 obtained from a large number of Th2 DC-derived exosome isolates. + and CTLA-4 - Mass spectrometry analysis was performed on DC-derived exosomes. Notably, the inventors observed CTLA-4. + The enrichment of HLA class II molecules, HSP90AB1, CD81, ITGAM (CD11b), and ITGAX (CD11c) in DC-derived exosomes, rather than CTLA-4. - Enrichment of HLA class I, CD82, CD36 (and its soluble ligand CD5L), and CD44 in DC-derived exosomes. Figure 6D The results of flow cytometry analysis of HLA class II (HLA-DR) expression were consistent with this. Figure 6F Furthermore, the inventors noted that, according to cryo-electron microscopy, CTLA-4... + and CTLA-4 - The physical properties of the extracellular vesicles differ significantly. Although CTLA-4 - Some may constitute heterogeneous vesicle clusters, but these clusters primarily consist of vesicles with a diameter of approximately 200 nm or larger. These spherical vesicles are characterized by an electron-dense membrane and cavity (Fig. 6H). The inventors have also noted the presence of smaller vesicles encapsulated by larger vesicles, a phenomenon previously reported (Gallart-Palau et al., 2015; Milasan et al., 2016). On the other hand, CTLA-4... +DC-derived exosomes are consistently spherical, with a diameter of approximately 30 nm, and their electronic density is significantly higher than that of CTLA-4. - Corresponding entity. In summary, the inventors proved CTLA-4 + Vesicles are a subset of DC-derived exosomes, possessing unique miRNAs, proteomes, and physical profiles. Furthermore, CTLA-4 is present in the DC-derived exosome microenvironment. + Vesicle production can be regulated by in vitro culture conditions.

[0130] Parental dendritic cells (DCs) and their DC-derived exosomes polarize T cell responses in a similar manner. Increasing evidence suggests that DC-derived exosomes can drive T cell responses. Currently, the understanding of the mechanisms regulating this immunomodulatory effect focuses primarily on the ability of DC-derived exosomes to inhibit or enhance DC maturation (paracrine) and to cross-label their own antigens onto DCs. In this context, the effects of DC-derived exosomes on T cells are indirect and dependent on the presence of other cells, including B cells, NK cells, and DCs. Furthermore, there is other evidence suggesting that MHC-II... + and ICAM-1 + DC-derived exosomes may interact with T cells via LFA-1 on T cells, thereby promoting the efficient presentation of TT antigens. However, the role of DC-derived exosomes with different polarizations in T cell responses remains to be elucidated. Furthermore, the efficacy of DC-derived exosomes during in vivo therapeutic administration is unclear when DCs have opposite polarization states. To address this, the inventors first sought to determine whether there are differences in the uptake levels of DC-derived exosomes with different polarizations by T cells. The inventors matured and polarized DCs into Th0, Th1, or Th2 isotypes and stained them with carboxyfluorescein succinimide (CFSE), thus labeling these DC-derived exosomes with CFSE, allowing for the tracking of T cell uptake of these DC-derived exosomes based on CFSE positivity. Subsequently, mature polarized DCs were co-cultured with unlabeled allogeneic T cells for 3 days and analyzed by flow cytometry. The inventors observed that the CFSE positivity levels (DC-derived exosome uptake) were similar in T cells co-cultured with Th0 and Th1 DC-derived exosomes (Figure 7A). On the other hand, vesicle uptake levels of T cells co-cultured with Th2 DC-derived exosomes were significantly reduced (Figure 7A). Next, the inventors sought to determine whether DC-derived exosome uptake led to alterations in T cell phenotype, specifically the expression of effector molecules. Therefore, the inventors co-cultured T cells with Th0-polarized CFSE-labeled DCs for 3 days and performed flow cytometry analysis. Using this Th cell-nonspecific dendritic cell (Th0) method, the relationship between Th1 and Th2 effector molecules and DC-derived exosome uptake status could be assessed simultaneously. The inventors observed CD4... +and CD8 + The uptake levels of DC-derived exosomes in T cell compartments were similar (Figs. 7B, 7C). CFSE + CD8 + T cells expressed significantly higher levels of IFNγ, but lower levels of granzyme B (Fig. 7D, 7E). CFSE + CD4 + T cells expressed higher levels of IFNγ and IL-4, consistent with the Th-agnostic nature of Th0 DCs (Fig. 7F, 7G). Finally, the inventors investigated whether DC-derived exosomes could influence T cell responses independently of parental dendritic cells, particularly in the presence of non-parental dendritic cells. Therefore, the inventors co-cultured allogeneic T cells with CFSE-labeled Th1 DC-derived exosomes and CellTrace far-red (CTFR)-labeled Th0 DCs. Figure 7H Consistent with previous findings, most T cells did not take up any DC-like exosomes. However, among the T cells that did take up DC-like exosomes, the frequency of T cells taking up Th1 DC-like exosomes was higher than that of T cells taking up Th0 DC-like exosomes or simultaneously taking up Th1 and Th0 DC-like exosomes (Figure 7I). In Th1 DC-like exosomes... + T cells, CD8 + GrzB expression and CD4 expression on T cells + and CD8 + IFNγ expression on T cells was significantly increased (Figures 7J-7L). Interestingly, the uptake of Th0 and Th1 DC-derived exosomes had an additive effect on the expression of these cytokines. In conclusion, even in the presence of non-parental DCs, Th1 DC-derived exosomes can promote the immunostimulatory phenotype of their parental DCs.

[0131] CTLA-4 + Labeled DC-derived exosomes can suppress type I immune responses. The inventors previously worked to elucidate the differences between Th1 and Th2 moDCs, finding one key distinction to be the expression of CTLA-4 carried by extracellular vesicles in these two DC types (Halpert et al., 2016). Specifically, CTLA-4 expression in Th2 DCs was significantly higher than in Th1 DCs. In this context, CTLA-4 was shown to mediate the uptake (paracrine) of DC-derived exosomes by neighboring DCs, leading to decreased expression of the co-stimulatory molecule B7. Notably, the absence of CTLA-4 in moDCs restricts CD8 expression. +T cell activation and IFNγ expression during co-culture with allogeneic T cells. Subsequent experiments determined that in a mouse melanoma model, the absence of CTLA-4 in the BMDC vaccine significantly improved survival and promoted tumor clearance. Given these observations and the known role of CTLA-4 as a key mediator of T1 T cell responses, the inventors sought to investigate the role of CTLA-4 in T cell responses. + The role of DC-derived exosomes. To this end, the inventors isolated DC-derived exosomes from Th2-biased moDCs and then used antibody-bound immunomagnetic beads to remove CTLA-4 from them. + Partially. Subsequently, allogeneic T cells were separately compared with CTLA-4-removed cells. - Some DC-derived exosomes or CTLA-4 not removed - A portion of the total DC-derived exosomes were cultured. Results showed that they contained CTLA-4. - Some DC-derived exosomes significantly inhibited CD4. + and CD8 + T cell IFNγ expression (Figs. 8A-8D), while CTLA-4 was removed - DC-derived exosomes restored IFNγ expression. Notably, IL-4 and granzyme B expression were affected in opposite directions. Specifically, CTLA-4... + DC-derived exosomes promoted CD4 activation. + and CD8 + Expression of IL-4 and granzyme B in T cells. Next, the inventors sought to determine whether CTLA-4 mediates the interaction between DC-derived exosomes and T cells, as observed in DCs. Therefore, the inventors co-cultured CFSE-labeled DCs with T cells in the presence of an anti-CTLA-4 antibody (clone: ​​BNI3). In the presence or absence of anti-CTLA-4 antibody, CD4 expression was observed. + or CD8 + There was no difference in T cell uptake of DC-derived exosomes (Fig. 8E, 8F). Overall, although CTLA-4 itself is not essential for T cell-DC-derived exosome interaction, CTLA-4... + DC-derived exosomes partially limit T1 T cell responses.

[0132] DC CTLA-4CTLA-4 is essential for maintaining immune homeostasis. In humans and mice, the DC cell population includes moDCs, plasmacytoid DCs, and conventional DC subsets. Although the inventors' current research has primarily focused on in vitro differentiated moDCs, clarifying the role of DCCTLA-4 in the in vivo environment is crucial, as these DC subsets play key roles in the generation, maintenance, and termination of immune responses. Therefore, the inventors sought to determine the CTLA-4 expression profile of different DC subsets under homeostasis. Flow cytometry analysis revealed the highest CTLA-4 expression level in T1 conventional DCs (cDC1). CTLA-4 expression levels were significantly lower and similar between plasmacytoid DCs (pDCs), type 2 conventional DCs (cDC2), and moDCs, while monocytes showed the lowest CTLA-4 expression level. Figure 9A To clarify the role of CTLA-4 in suppressing dendritic cell (DC)-mediated immune responses, the inventors constructed conditional knockout mice (cKOs) in which CD11c + Cells stop expressing CTLA-4 (CTLA-4) fl / fl CD11c Cre In these mice, the absence of CTLA-4 led to immune dysregulation, characterized by elevated levels of activated T cells in the spleen. Figure 9B The frequency of Treg cells in the thymus, spleen, and spleen was decreased. Compared with wild-type mice, cKO mice showed severe histological disturbances in the thymus, spleen, and Peyer's patch, and a significant reduction in the number of white blood cells in these tissues. Figure 9D Furthermore, the inventors observed pancreatic tissue loss and a reduced number of Painley's spots in the cKO mice. In the lungs and liver of the cKO mice, the inventors observed leukocyte infiltration and alveolar enlargement. Ultimately, the cKO mice exhibited stunted growth and died within 6 weeks.

[0133] CTLA-4 +DC-derived exosomes limit antitumor immune responses in mice. Having observed distinct T1 responses in T-cell-EV cultures, the inventors sought to investigate the role of these EV subsets in generating T1 responses in vivo. To this end, they employed a tumor model (B16-OVA) where tumor clearance and survival depended on effective T1 immunization. The inventors hypothesized that OVA-loaded, type 2-biased EVs could promote successful antitumor responses in the absence of CTLA-4, consistent with in vitro observations. Therefore, the inventors isolated EVs from T1-biased dendritic cell (DC) cultures and OVA-loaded, type 2-biased (T2) DCs treated with siCTLA4 or non-targeted siRNA. Mice carrying B16-OVA tumors were immunized with T1 EVs, siCTLA4T2 EVs, or siNT T2 EVs, respectively, when palpable tumors were present and caliper-measurable. Subsequently, flow cytometry analysis was performed on tumor growth, survival rate, lung metastasis, and tumor-infiltrating lymphocytes to determine the role of each EV type in the anti-tumor immune response. Figure 10 ).

[0134] Despite showing promising results in preclinical models, dendritic cells (DC-derived EVs) have failed to meet expectations in phase I and II clinical trials for melanoma and non-small cell lung cancer (Escudier et al., 2005; Munich et al., 2012; Besse et al., 2016; Pujol et al., 2015). Therefore, clarifying the composition, biogenesis, and function of DC-derived exosomes, specifically their relationship with the immunosuppressive CTLA-4, is crucial to fully realizing their therapeutic potential. Dendritic cells play a key role in maintaining immune tolerance in themselves and commensal bacteria, as well as in initiating robust adaptive immune responses. Although CTLA-4... - / - The autoimmune-mediated lethality in mice highlights the importance of this molecule, but it fails to elucidate the role of key immune DCs in CTLA-4 biology, and vice versa. Understanding the interaction between these two key immune mediators is crucial for identifying important potential mechanisms mediating loss of tolerance, vaccine response, and suppression of antitumor immune responses.

[0135] In this study, the inventors demonstrated that dendritic cells (DCs) express CTLA-4 both intracellularly and on the surface of extracellular vesicles (EVs) released from DCs. Furthermore, CTLA-4 expression can be influenced by the parental DC culture conditions. Specifically, type 1 response-biased DCs (T1) express low levels of CTLA-4 and IL-13, and also high levels of IL-12. On the other hand, type 2 response-biased DCs (T2) express high levels of CTLA-4 and IL-13, while expressing low levels of IL-12. Co-culturing allogeneic T cells with a large number of EVs yields T cell phenotypes similar to those of the parental DCs.

[0136] According to this disclosure, all methods disclosed and claimed herein can be manufactured and performed without improper experimentation. While the compositions and methods of this disclosure have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of this disclosure. More specifically, certain chemically and physiologically relevant reagents can be substituted for the reagents described herein, while obtaining the same or similar results. All such similar substitutions and modifications are obviously considered by those skilled in the art to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0137] References

[0138] The following references provide, to some extent, exemplary procedures or other details that supplement the content described herein, and are incorporated herein by reference.

[0139] Babst, M., Katzmann, DJ, Snyder, WB, Wendland, B. and Emr, SD. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev Cell 3, 283-289 (2002).

[0140] Besse, B. et al. Dendritic cell-derived exosomes as maintenance immunotherapy after firstline chemotherapy in NSCLC. Oncoimmunology 5, e1071008 (2016).

[0141] Cella, M. et al. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med 189, 821-829 (1999).

[0142] Decker et al., Vaccine 24:3203-3216, 2006.

[0143] Escudier, B. et al., Vaccination of metastatic melanoma patients with autologous dendritic cell-derived exosomes: results of the first phase I clinical trial. J Transl Med 3, 10 (2005).

[0144] Gallart-Palau, X. et al. demonstrated that extracellular vesicles are rapidly purified from human plasma by protein organic solvent precipitation (PROSPR). Sci Rep 5, 14664 (2015).

[0145] Halpert, MM, and other dendritic cells secrete cytotoxic T-lymphocyte-associated protein-4, which regulates the T-cell response by downmodulating bystander surface B7. (Stem Cells Dev 25, 774-787 (2016)).

[0146] Heijnen, HF, Schiel, AE, Fijnheer, R., Geuze, HJ, and Sixma, JJ. Activated platelets release two types of membrane vesicles: microvesicles formed by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 94, 3791-3799 (1999).

[0147] Laureano, RS et al. Trial watch: Dendritic cell (DC)-based immunotherapy for cancer. Oncoimmunology 11, 2096363 (2022).

[0148] Milasan, A. et al. Extracellular vesicles are present in mouse lymph and their level differs in atherosclerosis. J Extracell Vesicles 5, 31427 (2016).

[0149] Munich, S., Sobo-Vujanovic, A., Buchser, WJ, Beer-Stolz, D., and Vujanovic, NL. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands. Oncoimmunology 1, 1074-1083 (2012).

[0150] Odorizzi, G., Babst, M. and Emr, SD. Fab1p PtdIns(3)P5-kinase function essential for proteinsorting in the multivesicular body. Cell 95, 847-858 (1998).

[0151] Safety and immunogenicity of MAGE-A3 cancer immunotherapy with or without adjuvant chemotherapy in patients with resected stage IB to III MAGE-A3-positive non-small cell lung cancer (J Thorac Oncol 10, 1458-1467 (2015)).

[0152] Saksena, S., Sun, J., Chu, T. and Emr, SD. ESCRTing proteins in the endocytic pathway. Trends Biochem Sci 32, 561-573 (2007).

[0153] Sallusto, F. and Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte / macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179, 1109-1118 (1994).

[0154] Sallusto, F., Cella, M., Danieli, C., and Lanzavecchia, A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 182, 389-400 (1995).

[0155] ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood 106, 216-223 (2005).

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Claims

1. A method of generating extracellular vesicles (EVs) from dendritic cells (DCs), the method comprising (i) polarizing DCs to a T H 1 polarized or T H 2 polarized directionally matured, and (ii) isolating EVs secreted by the polarized DCs.

2. The method of claim 1, wherein the DCs are matured to T H 1 polarizing direction comprises maturing the DCs in the presence of IL-12, or loading the DCs with lysate and mRNA preparations from the same cell type.

3. The method of claim 1, wherein the DCs are polarized to T H 2 polarizing the DCs to maturity comprises maturing the DCs in the presence of Staphylococcus aureus enterotoxin B (SEB).

4. The method of any one of claims 1-3, wherein the dendritic cells are monocyte dendritic cells.

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

6. The method of claim 1, wherein the T H 1 polarized DCs for IL-12 + , IL-13 低 and CTLA-4 低 .

7. The method of claim 1, wherein the T H 2 polarized DCs for IL-13 + , CTLA-4 + and IL-12 低 .

8. The method of any one of claims 1-7, further comprising isolating the secreted EVs into CTLA-4 + and CTLA-4 - populations.

9. The method of claim 8, wherein the CTLA-4 - EV population is obtained by depleting CTLA-4 + EVs.

10. The method of claim 8, wherein the CTLA-4 + EV populations were obtained by positive selection using bead-bound anti-CTLA-4 antibody.

11. A composition comprising a dendritic cell-derived extracellular vesicle (EV) obtained by the method of any one of claims 1-10.

12. The composition of claim 11, wherein the EV is CTLA-4 + .

13. The composition of claim 12, wherein the EVs are derived from T H 2 polarized DCs.

14. The composition of claim 12, wherein the EVs are derived from T H 1 polarized DCs.

15. The composition of claim 11, wherein the EV is CTLA-4 - .

16. The composition of claim 15, wherein the EVs are derived from T H 1 polarized DCs.

17. The composition of claim 15, wherein the EVs are derived from T H 2 polarized DCs.

18. The composition of any one of claims 11-17, wherein the EVs have a diameter of about 30-200 nm.

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

20. A method of isolating CTLA-4 + EVs from each other and recovering both populations of EVs in a physically and functionally intact state, the method comprising isolating EVs from a culture supernatant of mature DC cells using PEG precipitation and extracting CTLA-4 - EVs using bead-bound anti-CTLA-4 antibodies, thereby isolating CTLA-4 + EVs from each other. + EVs from each other. - EVs from each other.

21. A method of inducing adaptive T H 1. A method of polarizing and T cell activation comprising contacting the T cell with CTLA-4 - DC-derived EVs.

22. The method of claim 21, wherein the T cells are CD8 + T cells.

23. The method of claim 21, wherein the T cells are CD4 + T cells.

24. The method of any one of claims 21-23, wherein the CTLA-4 - The DC-derived EVs are according to any one of claims 15-17.

25. The method of any one of claims 21-24, wherein the T cells are present in a population of PBMCs.

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

27. The method of any one of claims 21-24, wherein the T cells are in a subject, wherein the method comprises administering the EVs to the subject.

28. A method of treating a disease in a patient in need thereof, the method comprising administering to the patient CTLA-4 - DC-derived EVs.

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

30. The method of claim 28 or 29, wherein the DC-derived EVs are administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally.

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

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

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

34. A method of inhibiting adaptive T H 1 polarization and CD8 + T cell activation, the method comprising contacting the T cell with CTLA-4 + DC-derived EVs.

35. The method of claim 34, wherein the CTLA-4 + The DC-derived EVs are according to any one of claims 12-14.

36. The method of claim 34 or 35, wherein the T cells are present in a population of PBMCs.

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

38. The method of claim 34 or 35, wherein the T cells are in a subject, wherein the method comprises administering the EVs to the subject.

39. The method of any one of claims 34-38, wherein the EVs are derived from T H 2 polarized DCs.

40. A method of treating an autoimmune condition in a patient, the method comprising administering to the patient CTLA-4 + DC-derived EVs.

41. The method of claim 40, wherein the CTLA-4 + The DC-derived EVs are according to any one of claims 12-14.

42. The method of claim 40 or 41, wherein the autoimmune condition is a T cell-mediated autoimmune condition.

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

44. The method of any one of claims 40-43, wherein the CTLA-4 + DC-derived EVs are administered intratumorally, intravenously, peritumorally, subcutaneously, and intraperitoneally.

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