Extracellular vesicles from activated CAR-T cells

Abstract

The present invention provides extracellular vesicles (EVs) derived from T cells expressing chimeric antigen receptors (CARs), pharmaceutical compositions comprising these vesicles, and their use in treating cancer, wherein the T cells are specifically activated with antigens specifically bound by CARs. In particular, the present invention illustrates EVs derived from activated T cells, pharmaceutical compositions comprising these EVs, and their use in treating cancers (e.g., ovarian cancer and breast cancer) that overexpress HER2, wherein the activated T cells express CARs that specifically bind to HER2 cancer antigens.

Description

Technical Field

[0001] The present invention relates to extracellular vesicles derived from activated T cells expressing chimeric antigen receptors, a pharmaceutical composition containing the extracellular vesicles, and use thereof in treating cancer. Background Art

[0002] CAR T cells are engineered T cells that express a chimeric antigen receptor (CAR) that recognizes a specific tumor-associated antigen (TAA) that distinguishes cancer cells from healthy cells. In the past few years, CAR T cells have been used as an immunotherapy for several different tumor diseases, especially leukemias and lymphomas. Several methods are used to transduce or transfect T cells with CAR ex vivo. These methods can include the introduction of DNA or RNA using viral vectors or other methods. As a result, the transfected T cells contain the genomic sequence of a specific protein and present or express the receptor. After recognizing the TAA, the CAR T cell is stimulated and can effectively kill its target cell.

[0003] As an effective treatment modality, there are several adverse events that may be associated with CAR T-cell therapy, such as cytokine release syndrome (CRS) and life-threatening cytokine storm. Side effects associated with CRS may include hypotension, hypoxia, high fever, and neurological disorders. Another major challenge is to overcome the tumor microenvironment so that this treatment can be applied to treat solid tumors, not just hematological malignancies.

[0004] Extracellular vesicles (EVs) are membrane vesicles secreted by different types of cells, including blood cells. EVs can be divided into three subgroups: (I) exosomes, which have a diameter of 30-100 nm and come from endosomal compartments; (II) microvesicles, which have a diameter of 100 nm-1 μm and are released from the cell surface by "encapsulation"; and (III) apoptotic bodies, which have a diameter of 1-5 μm and are released from apoptotic cells. EVs are present in the blood circulation under normal physiological conditions and are elevated in a variety of diseases, such as diabetes and related vascular complications, cardiovascular disease, hematological malignancies, and solid tumors (e.g., breast cancer). Tang et al. (Oncotarget 2015; 6(42): 44179-90) generally discuss different methods for treating cancer using cell and exosome platforms. WO2019 / 128952 describes the preparation method and use of isolated CAR-carrying immune cell exosomes.

[0005] It has been suggested that EVs contain several elements of the parental cell from which they originate, including proteins, DNA fragments, microRNAs, and mRNAs. After release, EVs can interact with target cells through receptor-mediated mechanisms, or they can directly fuse with the plasma membrane of target cells, thereby releasing their contents into recipient cells. Alternatively, EVs can be internalized through endocytosis and release their contents into the cytoplasm of target cells.

[0006] There is an unmet need to develop additional approaches for the safe and effective treatment of cancer. Summary of the invention

[0007] The present invention is based on the observation that isolated extracellular vesicle (EV) populations derived from T cells expressing a chimeric antigen receptor (CAR) activated by exposure to an antigen specifically bound by the CAR provide excellent anti-cancer effects. Populations with medium to large sized EVs were found to have improved apoptotic activity, while populations containing mainly exosomes had weak to moderate activity.

[0008] Therefore, provided herein are compositions and methods based on EVs, providing improved anti-cancer treatments compared to known treatments. According to one aspect, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing chimeric antigen receptors, wherein more than 25% of EVs have a diameter greater than 150 nanometers. In other words, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing chimeric antigen receptors, wherein at least 25% of EVs have a particle size greater than 150nm. The CAR T cells from which EVs are derived / obtained are specifically activated CAR T cells, i.e., activated by tumor-associated antigens specifically bound by CAR. Therefore, isolated EVs are represented as activated EVs. According to some embodiments, at least 29% or at least 30% or at least 35% of EVs have a size greater than 150nm. According to some embodiments, the average size of the isolated activated EVs of the present invention is greater than 140nm. According to other embodiments, the average size of the isolated activated EVs of the present invention is greater than 150nm. According to certain embodiments, the average size of the isolated activated EVs of the present invention is greater than 155nm. According to certain embodiments, the average size of the isolated activated EVs of the present invention is greater than 160 nm. According to some embodiments, the EVs of the present invention present the CAR of the activated CAR T cells from which the EVs originate / derive.

[0009] According to the teachings of the present invention, any CAR specific for a tumor-associated antigen can be used. According to some exemplary embodiments, CAR is selected from anti-HER2 CAR, anti-CD19 CAR and anti-CD38 CAR. According to a more specific embodiment, CAR is N29 CAR. As described below, the isolated EV of the present invention retains the biological and therapeutic properties of the CAR-T cells from which the EV is derived. Therefore, according to some embodiments, the isolated EV having the EV of the present invention is a cytotoxic EV. According to some embodiments, the isolated EV of the present invention expresses CD38, CD3 and / or HLARD antigens on its surface. According to some embodiments, 10%, 20 and / or more than 20% of the isolated EVs of the present invention express CD38, CD3 and / or HLARD antigens, respectively. According to some embodiments, the EV of the present invention may further comprise an exogenously added anticancer agent.

[0010] According to another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an isolated activated EV of the present invention and a pharmaceutically acceptable carrier. According to alternative embodiments, the pharmaceutical composition comprises the EV of the present invention as the only anticancer agent. According to some embodiments, the pharmaceutical composition also comprises another anticancer agent. According to one embodiment, the pharmaceutical composition of the present invention is used to treat cancer. Non-limiting examples of cancers that can be treated using the EV or pharmaceutical composition of the present invention are ovarian cancer, breast cancer, lung adenocarcinoma, gastric cancer, liver cancer, pancreatic cancer, brain cancer, and hematological malignancies. According to some embodiments, the cancer is a cancer that presents a tumor-associated antigen, and the CAR of the CAR T cell from which the EV of the present invention is derived specifically binds to the tumor-associated antigen. According to some embodiments, the pharmaceutical composition of the present invention is administered systemically or intratumorally.

[0011] According to another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an isolated activated EV of the present invention, i.e., an EV derived from an activated T cell (CART cell) expressing a chimeric antigen receptor. According to another aspect, the present invention provides a method for preparing an isolated activated extracellular vesicle of the present invention, wherein the EV is derived from an activated CAR T cell, wherein the method comprises incubating the CAR T cell with a tumor-associated antigen that specifically binds to the CAR under conditions capable of stimulating the T cell, and isolating the obtained activated extracellular vesicle. According to a more specific embodiment, the method for preparing EV of the present invention comprises: (1) incubating the CAR T cell with a tumor-associated antigen that specifically binds to the CAR in a cell culture medium under conditions capable of activating the T cell; (2) separating the CAR T cell from a cell culture medium containing EV; (3) isolating the obtained activated extracellular vesicle, thereby obtaining an isolated activated EV of the present invention, wherein at least 22% or at least 25% of the EV has a size of 150nm or greater. According to some embodiments, the separation of EV comprises centrifugation at a centrifugal force of 8,000×g to 30,000×g for 0.5 to 4 hours. Therefore, according to some embodiments, the method for preparing the EV of the present invention comprises: (1) incubating CAR T cells with tumor-associated antigens that specifically bind to CAR in a cell culture medium under conditions capable of activating T cells; (2) separating the CAR T cells from the cell culture medium; (3) isolating the obtained activated extracellular vesicles by centrifugation at 8,000 to 25,000 × g or 8,000 to 12,000 × g for 30 to 210 minutes, thereby obtaining the isolated activated EV of the present invention, wherein at least 25% or at least 25% of the EVs have a size of 150 nm or greater. According to other embodiments, the present invention provides isolated activated extracellular vesicles prepared by the preparation method of the present invention. In some specific embodiments, the present invention provides isolated activated extracellular vesicles prepared by the following method, comprising (1) incubating CAR T cells with tumor-associated antigens that specifically bind to CAR in a cell culture medium under conditions capable of activating T cells; (2) separating CAR T cells from the cell culture medium; (3) isolating the activated extracellular vesicles obtained by centrifugation at 8,000 to 25,000 × g or 8,000 to 12,000 × g for 30 to 210 minutes. The final prepared EV contains at least 25% of EVs larger than 150 nm in size. According to some aspects, the average size of the isolated EVs is about 140 nm or larger. According to some aspects, the present invention provides extracellular vesicles prepared and isolated according to the preparation method of the present invention. Therefore, in some embodiments, the present invention provides extracellular vesicles that can be obtained by the preparation method of the present invention. The full scope of the present invention will be better understood together with the subsequent drawings, descriptions, examples and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the results of N29 CAR T cells ( Figure 1A ) and untransduced (UT) T cells ( Figure 1B ) Size distribution (diameter in nm) measured by nanoparticle tracking analysis (NTA) of EVs obtained from both T cells incubated with HER2-presenting cancer cells. Figure 1C-1E Data from experiments 5-9 of EV precipitation obtained by 5 different centrifugation protocols as described in the methods section are summarized. In short: Method 1 - centrifugation at 20,000 × g for 60 min. Method 2 - the supernatant obtained from method 1 was further centrifuged at 100,000 × g for 60 min; Method 3 - centrifugation at 10,000 × g for 30 min. Method 4 - the supernatant from method 3 was further centrifuged at 70,000 × g for 110 min. Method 5 - centrifugation at 10,000 × g for 180 min; Sample 1 - N29 superimposed on SKOV; Sample 2 - N29 superimposed on OVCAR; Sample 3 - UT superimposed on SKOV; Sample 4 - UT superimposed on OVCAR; Sample 5 - N29 superimposed on culture medium; Sample 6 - UT superimposed on culture medium; Sample 7 - SKOV superimposed on culture medium; and Sample 8 - OVCAR superimposed on culture medium.

[0013] Figure 1C EV sizes were summarized for all samples and methods; Figure 1D Statistics are shown for samples 1 and 2 separated by methods 1-5.

[0014] Figure 1E The percentage of large EVs (diameter >150 nm) is shown for all samples and methods. Figure 1F Statistics are shown for samples 1 and 2 separated by methods 1-5.

[0015] Figure 1G The number of EVs larger than 150 nm in the preparations obtained by Method 1 and Method 5 is shown.

[0016] Figure 1H -J shows a side scatter (SSC) versus forward scatter (FSC) plot using 0.75 μm beads ( Figure 1H ), EVs from untransduced T cells ( Fig. 1I ) or EVs from T cells transduced with N29 ( Figure 1J ).

[0017] Figure 2 shows the expression of markers on EVs isolated by method 1: Figure 2A-D shows the labeling (percentage) of unstained EVs obtained from: untransduced and unincubated (2A), N29-transduced and unactivated T cells (2B), unactivated N29CAR T cells (2C), and N29 CAR T cells stimulated with SKOV (2D). Figure 2E -H shows isotype control IgGAPC (percent) labeling of EVs obtained from: untransduced and unincubated (2E), N29-transduced and unactivated T cells (2F), unstimulated N29 CAR T cells (2G), and N29 CAR T cells stimulated with SKOV (2H). Fig.2I -L shows anti-CD3-APC (percent) labeling of EVs from: untransduced and unstimulated (2I), N29 transduced and unactivated T cells (2J), unstimulated N29 CAR T cells (2K), and N29 CAR T cells stimulated with SKOV (2L). Anti-CD3 antibodies bind to the T cell receptor (TCR) complex on mature T lymphocytes.

[0018] Figure 2M Membrane antigen expression of CD3, CD38 and HLADR on EV pellets obtained by method 1 (20,000 g, 60 min) and method 2 (100,000 g, 60 min) (n=5-9 experiments) is summarized.

[0019] Figure 3 shows the labeling (percentage) of EVs obtained by method 1 from: unstained non-transduced T cells (3A), unstained N29-GFP transduced CAR (3B), or from isotype control IgG stained non-transduced T cells (3C).

[0020] Figure 4 shows HER2+SKOV cells ( Figure 4A ) or HER2-OVCAR cells ( Figure 4B ) Apoptotic effect of 6 hours exposure to different EV samples obtained in method 1. EVs were obtained from: 1-sample 1 (T cells expressing N29 CART after stimulation with SKOV) 25 μg / well; 2-sample 1, 12.5 μg / well; 3-sample 2 (T cells expressing N29 CART after incubation with OVCAR) 12.5 μg / well; 4-sample 4 (non-transduced T cells incubated with SKOV) 25 μg / well; 5-sample 4 (non-transduced T cells incubated with OVCAR) 25 μg / well.

[0021] Figure 4C Shown are the effects of fresh or thawed EVs from sample 1 or 2, each obtained by method 1 or method 2 (detected by fluorescence microscopy and analyzed by Imaj J software); Figure 4DThe effects of EV exposure for 6 h on SKOV Her2+ cells and OVCAR Her2- cells were summarized;

[0022] Figure 4E The apoptotic effects of eight different EV populations (two different concentrations: 50 μg and 25 μg EVs) obtained by method 1 (dark grey columns) and method 2 (light grey columns) are summarized.

[0023] Figure 5 shows microscopic images of SKOV cells incubated with EVs obtained from different CAR-T cells for 20 hours, as follows: Figure 5A -EVs from N29 CAR-T cells pre-stimulated with SKOV cells; Figure 5B – EVs of anti-CD19 CAR-T pre-stimulated with SKOV cells; Figure 5C – EVs from N29 CAR-T pre-stimulated with Raji cells; and Figure 5D – EVs from anti-CD19 CAR-T preactivated with Raji cells.

[0024] Figure 6 shows images of SKOV cells incubated with EVs obtained from different CAR-T cells for 40 hours, as follows: Fig. 6A – Two images: EVs from N29 CAR-T pre-stimulated with SKOV cells; Figure 6B – EVs from SKOV cell-activated anti-CD19 CAR-T; Figure 6C – Shows the effect of EVs obtained from N29 CAR T stimulated with HER2+ cells on breast cancer HER2-negative cells.

[0025] Figure 7 shows phase images and cells labeled with fluorescent caspase 3 / 7 activity dye and cytotoxicity dye (both for real-time quantification of cell death). EVs isolated from sample 1 or 2 were treated with methods 1, 3, and 5 ( Fig. 7A ) or treated with EVs isolated from sample 1 or 2 using methods 2 or 4, or treated with staurosporine ( Figure 7B ) The image of SKOV cells is recorded in the white dots. The image of SKOV cells was completed by Incucyte.

[0026] FIG8 shows magnified microscopic images of SKOV cells after exposure to two different concentrations of EVs from sample 1 or EVs from sample 2 isolated by five methods for 4 days. Figures 8A-8E : Sample 1, 50 μg, methods 1-5 respectively; Figure 8F-8J : Sample 1, 25 μg, methods 1-5 respectively; Figure 8K-8O : Sample 2, 50 μg, methods 1-5 respectively; Figure 8P-8T : Sample 2, 25 μg, methods 1-5 respectively; and Figure 8U– Staurosporine treatment.

[0027] FIG. 9 shows magnified microscopic images of OVCAR cells after exposure to 2 different concentrations of EVs from sample 1 or EVs from sample 2 isolated by 5 methods for 4 days. Figures 8A-8E : Sample 1, 50 μg, methods 1-5 respectively; Figure 8F-8J : Sample 1, 25 μg, methods 1-5 respectively; Figure 8K-8O : Sample 2, 50 μg, methods 1-5 respectively; Figure 8P-8T : Sample 2, 25 μg, methods 1-5 respectively; and Figure 8U – Staurosporine treatment.

[0028] Fig.10 Shown are the kinetics of incorporation of a fluorescent caspase 3 / 7 activity marker into SKOV cells following exposure to EVs from samples 1 and 2 obtained by methods 1, 3, and 5. Treatments are listed as follows: 1 - method 1, sample 1, 50 μg; 2 - method 1, sample 1, 25 μg; 3 - method 3, sample 1, 50 μg; 4 - method 3, sample 1, 25 μg; 5 - method 5, sample 1, 50 μg; 6 - method 5, sample 1, 25 μg; 7 - method 1, sample 2, 50 μg; 8 - method 1, sample 2, 25 μg; 9 - method 3, sample 2, 50 μg; 10 - method 3, sample 2, 25 μg; 11 - method 5, sample 2, 50 μg; 12 - method 5, sample 2, 25 μg.

[0029] Fig.11 Shown are the kinetics of incorporation of a fluorescent caspase 3 / 7 activity marker into SKOV cells after exposure of the cells to different EVs from samples 1 and 2 obtained by methods 2 or 4 or after exposure to staurosporine. The treatments are listed as follows: 1 - method 2, sample 1, 50 μg; 2 - method 2, sample 1, 25 μg; 3 - method 4, sample 1, 50 μg; 4 - method 4, sample 1, 25 μg; 5 - staurosporine 1:100; 6 - method 2, sample 2, 50 μg; 7 - method 2, sample 2, 25 μg; 8 - method 4, sample 2, 50 μg; 9 - method 4, sample 2, 25 μg.

[0030] Fig.12 Total caspase activity after 4 days of incubation of EVs from samples 1 and 2 (2 concentrations) isolated by methods 1-5 is summarized.

[0031] Fig.13 Shown is the viability / proliferation of SKOV cells incubated for 40 h with EVs derived from sample 1 or sample 3 purified by method 1 , as measured by the XTT method after 48 h.

[0032] Fig.14The cytotoxic effect of activated EVs on MDA231 HER2+ cells is shown. EVs were obtained from N29 superimposed on MDA231 HER2+ cells (N29 on MDA132 HER2+) or non-transduced T cells superimposed on MDA231 HER2+ cells. MDA231 HER2+ cells were exposed to 3 different dilutions of EVs and measured by CytoTox96 cytotoxicity assay after 48 hours. DETAILED DESCRIPTION

[0033] The present invention relates to a population of extracellular vesicles (EVs), wherein the EVs are derived from activated T cells expressing a chimeric antigen receptor (CAR), wherein at least 25% of the EVs have a size of 150 nm or greater.

[0034] Surprisingly, it was found that EV preparations exhibited outstanding cytotoxic effects on ovarian and breast HER2-positive cancer cells, wherein the EVs were derived from T cells activated by ovarian cancer cells expressing HER2-specific N29 CAR and presenting HER2 (denoted as N29 CAR T), and wherein the EV population contained more than 25% EVs with a size greater than 150 nm. It was further shown that freezing EVs did not lose the effect. Therefore, EVs can be frozen and thawed before use, thereby facilitating handling. This superior cytotoxic effect was not observed for EVs derived from CAR T cells that were not pre-stimulated or incubated with non-related antigens (such as CD19, which are associated with HER2-negative hematological cancers or cancer cells). In addition, the effect of EVs containing 20% ​​or less of a size greater than 150 prepared from specifically activated N29 CAR T cells was much weaker. It was further found that the physical properties of EVs obtained from pre-stimulated CAR T cells and control preparations, such as size distribution and the presence of certain antigenic markers, were different. It is understood that the EVs derived from stimulated N29 CAR T cells with specific characteristics described in this application are merely proof of concept. EVs from T cells containing CARs specific for other tumor-associated antigens or EVs obtained from T cells containing multiple such CARs can be used according to the teachings of the present invention, as long as the EVs have the characteristics as described in this application, in particular the described size.

[0035] According to one aspect, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing chimeric antigen receptors, wherein at least 22% of the EVs have a diameter greater than 150nm. According to some embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing chimeric antigen receptors, wherein at least 22% of the EVs have a size greater than 150nm. According to other embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing chimeric antigen receptors, wherein at least 25% of the EVs have a size greater than 150nm. In particular, embodiments of the present invention relate to isolated activated EVs derived from CAR T cells activated by CAR-mediated stimulation prior to EV separation.

[0036] The terms "extracellular vesicle" and "EV" are used interchangeably herein and refer to a cell-derived vesicle comprising a membrane surrounding an internal space. Typically, the diameter of an extracellular vesicle ranges from 30nm to 1000nm, and may contain various cargo molecules within the internal space, displayed on the outer surface of the extracellular vesicle and / or across the membrane. The cargo molecules may include nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. The term extracellular vesicle also includes the terms "exosomes" and "microvesicles". The terms "exosomes" and "nanovesicles" are used interchangeably herein and refer to EVs with diameters between 30 and 100nm. The term "microvesicle" used herein refers to EVs with diameters between 100 and 1000nm. Typically, EVs may contain at least a portion of the molecular contents of the cells from which they are derived, such as lipids, fatty acids, polypeptides, polynucleotides, proteins and / or carbohydrates. According to the teachings of the present invention, at least 25% of EVs have a size of 150nm or more. Alternatively, at least 25% of EVs have a size of 150nm or more.

[0037] The extracellular vesicles of the present invention are mostly spherical, and the terms "size", "particle size" and "particle size" used herein refer interchangeably to the diameter of the extracellular vesicle or the longer diameter of the extracellular vesicle. The size of the EV of the present invention can be determined using any known particle size measurement method. A non-limiting example is nanoparticle tracking analysis (NTA).

[0038] According to another embodiment, the isolated activated EVs are microvesicles. According to a further embodiment, the isolated activated EVs are a combination of small vesicles and large vesicles.

[0039] According to some embodiments, at least 10% or at least 15% of the isolated activated EVs have a size between 150 and 300 nm. According to some embodiments, at least 27% of the activated EVs have a particle size of 150 nm or more. According to one embodiment, at least 28% of the activated EVs have a size of 150 nm or more. According to one embodiment, at least 28% of the activated EVs have a size greater than 150 nm. According to one embodiment, at least 29% of the activated EVs have a size of 150 nm or more. According to one embodiment, at least 30% of the activated EVs have a size of 150 nm or more. According to one embodiment, at least 32% of the activated EVs have a size of 150 nm or more. According to one embodiment, at least 35% of the activated EVs have a size of 150 nm or more. According to another embodiment, at least 40% of the activated EVs have a size greater than 150 nm. According to another embodiment, at least 42% of the activated EVs have a size greater than 150 nm. According to yet another embodiment, at least 45% of the activated EVs have a size greater than 150 nm. According to another embodiment, at least 50% of the activated EVs have a size greater than 150 nm. According to another embodiment, at least 55% of the activated EVs have a size greater than 150nm. According to some embodiments, at least 60% of the activated EVs have a size greater than 150nm. According to some embodiments, at least 65% of the activated EVs have a size greater than 150nm. According to some embodiments, at least 70% of the activated EVs have a size greater than 150nm. According to some embodiments, 25% to 70% of the activated EVs have a size greater than 150nm. According to other embodiments, 25% to 35% of the activated EVs have a size greater than 150nm. According to certain embodiments, 25% to 45% of the activated EVs have a size greater than 150. According to some embodiments, 30% to 70% of the activated EVs have a size greater than 150nm. According to one embodiment, 35% to 65% of the activated EVs have a particle size greater than 150nm. According to another embodiment, 35% to 45% of the activated EVs have a size greater than 150nm. According to yet another embodiment, 32% to 65% of the activated EVs have a size greater than 150nm.

[0040] The term "X nm or more" also includes the terms "greater than X nm", "X nm or more" and can be replaced by them in any embodiment of the present invention.

[0041] According to some embodiments, at least 0.2%, or at least 0.5%, or at least 0.8% of the isolated activated EVs have a size greater than 300 nm, such as between 300 and 600 nm. According to some embodiments, about 0.3% to about 3% of the isolated activated EVs have a size between 300 and 500 nm.

[0042] According to some embodiments, the average size of activated EVs is 130 nm or more, as measured by nanoparticle tracking analysis (NTA). According to other embodiments, the average size of activated EVs is 132 nm or more. According to other embodiments, the average size of activated EVs is 135 nm or more. According to other embodiments, the average size of activated EVs is 137 nm or more. According to other embodiments, the average size of activated EVs is 140 nm or more. According to other embodiments, the average size of activated EVs is greater than 140 nm. According to other embodiments, the average size of activated EVs is 142 nm or more. According to other embodiments, the average size of activated EVs is 145 nm or more. According to other embodiments, the average size of activated EVs is 147 nm or more. According to other embodiments, the average size of activated EVs is 150 nm or more. According to other embodiments, the average size of activated EVs is 152 nm or more. According to other embodiments, the average size of activated EVs is 155 nm or more. According to other embodiments, the average size of activated EVs is 160 nm or more. According to other embodiments, the average size of activated EVs is greater than 160 nm. According to other embodiments, the average size of activated EVs is 162 nm or greater. According to other embodiments, the average size of activated EVs is 165 nm or greater. According to other embodiments, the average size of activated EVs is greater than 170 nm.

[0043] According to some embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from pre-stimulated T cells (CAR T cells) expressing a chimeric antigen receptor, wherein at least 25% of the EVs have a size greater than 150 nm and the EVs have an average size of 135 or more. According to other embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor, wherein at least 25% of the EVs have a size greater than 150 nm and the EVs have an average size of 140 or more. According to some embodiments, at least 25% of the EVs have a size greater than 150 nm and the EVs have an average size of 140 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 155 nm or more, 160 nm or more, or 170 nm or more. According to some embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor, wherein at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 147nm or greater. According to some embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor, wherein at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 155nm or greater. According to some embodiments, at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 160nm or greater. According to some embodiments, at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 170nm or greater. According to some embodiments, at least 29% of EVs have a size greater than 150nm and the EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, or 170nm or more. According to some embodiments, at least 29% of EVs have a size greater than 150nm and the EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 162nm or more, 170nm or more. According to some embodiments, at least 29% of EVs have a size greater than 150nm and the EVs have an average size of 147nm or more. According to some embodiments, at least 29% of EVs have a size greater than 150nm and the EVs have an average size of 150nm or more. According to some embodiments, at least 29% of EVs have a diameter size greater than 150nm and the EVs have an average size of 155nm or more.According to some embodiments, at least 29% of EVs have a size greater than 150nm and EVs have an average size of 160nm or more. According to some embodiments, at least 29% of EVs have a size greater than 150nm and EVs have an average size of 165nm or more. According to some embodiments, at least 30% of EVs have a size greater than 150nm and EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 170nm or more. According to some embodiments, at least 32% of EVs have a size greater than 150nm and EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 170nm or more. According to some embodiments, at least 35% of EVs have a size greater than 150nm and the EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 170nm or more. According to some embodiments, at least 40% of EVs have a size greater than 150nm and the EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 162nm or more, 170nm or more. According to some embodiments, at least 40% of EVs have a size greater than 150nm and the EVs have an average size of 147nm or more. According to some embodiments, at least 40% of EVs have a size greater than 150nm and the EVs have an average size of 155nm or more. According to some embodiments, at least 40% of EVs have a size greater than 150nm and the EVs have an average size of 160nm or more. According to some embodiments, at least 40% of the EVs have a size greater than 150nm and the EVs have an average size of 165nm or more. According to some embodiments, at least 40% of the EVs have a size greater than 150nm and the EVs have an average size greater than 170. According to some embodiments, at least 40% of the EVs have a size greater than 150nm and the EVs have an average size of 170nm or more. According to some embodiments, at least 43% of the EVs have a size greater than 150nm and the EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 170nm or more.According to some embodiments, at least 46% of EVs have a size greater than 150nm and EVs have an average size of 140nm or more, 145nm or more, 147nm or more, 150nm or more, 155nm or more, 160nm or more, 170nm or more. According to some embodiments, at least 46% of EVs have a size greater than 150nm and EVs have an average size of 147nm or more. According to some embodiments, at least 50% of EVs have a size greater than 150nm and EVs have an average size of 147nm or more or 150nm or more or 155nm or more. According to some embodiments, at least 60% of EVs have a size greater than 150nm and EVs have an average size of 150nm or more or 155nm or more or 160nm or more.

[0044] According to some embodiments, at least 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 3% are larger than 300 nm in size.

[0045] According to some embodiments, the ratio between EVs with a size greater than 150 nm and EVs with a size less than 150 nm is 2:8 to 8:8. According to some embodiments, the ratio between EVs with a size greater than 150 nm and EVs with a size less than 150 nm is about 2:8, about 3:8, about 4:8, about 5:8, about 6:8, about 7:8, or about 1:1. According to some embodiments, the ratio between EVs with a size greater than 150 nm and EVs with a size less than 150 nm is about 1:3, about 2:3, or about 1:1. According to some embodiments, at least 25% of the EVs have a size greater than 150 nm, the EVs have an average size of 132 nm or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 155 nm or greater, 160 nm or greater, or 170 nm or greater, and the ratio between EVs with a size greater than 150 nm and EVs with a size less than 150 nm is from 1:4 to 1:1 or about 1:4, about 1:3, about 2:3, or about 1:1.

[0046] According to some embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of the EVs are larger than 155nm, or larger than 160nm in size.

[0047] The terms "derived from" and "originating from" are used interchangeably herein to refer to extracellular vesicles produced within, by, or from a particular cell, cell type, or cell population (e.g., T cells), particularly from CAR T cells.

[0048] As used herein, the terms "parent cell", "production cell" and "origin cell" include any cell from which extracellular vesicles are obtained and isolated. The term also includes cells that share the protein, lipid, sugar or nucleic acid components of the extracellular vesicle. For example, "parent cell" or "production cell" includes cells that serve as a membrane source for extracellular vesicles. The term "origin CAR T cell" then refers to the CAR T cell from which the EV is derived.

[0049] The terms "purify", "purify", "purified", "isolate", "isolate" and "isolated" are used interchangeably herein and refer to the state of a population of extracellular vesicles (e.g., a number of known or unknown amounts and / or concentrations) that has undergone one or more purification / isolation processes, for example, to select desired extracellular vesicles, or to remove or reduce residual biological products and / or remove unwanted extracellular vesicles, such as removing EVs of a specific size. According to one embodiment, the ratio of EVs to remaining parental cells is at least 2, 3, 4, 5, 6, 8 or 10 times higher, or in certain advantageous embodiments at least 50, 100, 1000 or 2000 times higher than in the starting material. According to some embodiments, the ratio is a weight ratio. In some advantageous embodiments, the term "isolated" has the meaning of being substantially cell-free or cell-free, and can be replaced by it.

[0050] The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein and refer to an engineered receptor composed of heterologous domains, which include at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain capable of activating T.

[0051] The extracellular part or domain of CAR includes an antigen binding domain and an optional spacer or hinge region. The antigen binding domain of CAR targets and specifically binds to an antigen of interest, such as a tumor-associated antigen (TAA). The targeting region may include a full-length heavy chain, a Fab fragment, or a single-chain variable fragment (scFvs). The antigen binding domain may be from the same species or a different species as the species in which CAR will be used. In one embodiment, the antigen binding domain is a scFv.

[0052] The extracellular spacer or hinge region of CAR is located between the antigen binding domain and the transmembrane domain. The extracellular spacer may include, but is not limited to, an Fc fragment of an antibody or its fragment or derivative, a hinge region of an antibody or its fragment or derivative, a CH2 region of an antibody, a CH3 region of an antibody, an auxiliary protein, an artificial spacer sequence, or a combination thereof.

[0053] The term "transmembrane domain" refers to a CAR region that spans the plasma membrane or bridges the plasma membrane. The transmembrane domain of the CAR of the present invention is a transmembrane region of a transmembrane protein, an artificial hydrophobic sequence, or a combination thereof.

[0054] The term "intracellular domain" refers to the intracellular part of CAR, which includes an activation domain capable of activating T cells and an optional additional costimulatory domain. The intracellular domain can be the intracellular domain of a T cell receptor (e.g., a zeta chain associated with a T cell receptor complex) and / or can include a stimulation domain or a portion thereof of other receptors (e.g., TNFR superfamily members), such as an intracellular activation domain (e.g., comprising an activation motif (ITAM) based on immunoreceptor tyrosine), an intracellular costimulatory domain, or both. According to some embodiments, the costimulatory domain is selected from the costimulatory domains of CD28, 4-1BB, OX40, iCOS, CD27, CD80, and CD70. According to one embodiment, the costimulatory domain is the costimulatory domain of CD28. According to another, the costimulatory domain is the costimulatory domain of 4-1BB.

[0055] The term "specific binding" or "specificity" with respect to the antigen binding domain of a CAR or antibody or fragment thereof refers to an antigen binding domain that recognizes and binds to a specific antigen, but does not substantially recognize or bind to other molecules in the sample. The term includes an antigen binding domain that binds to the antibody recognition portion (epitope) of its antigen with high affinity, and does not bind to other unrelated epitopes with high affinity.

[0056] In some embodiments, CAR can be specific for tumor-associated antigens. The terms "tumor-associated antigen" and "TAA" are used interchangeably herein and refer to any antigen found in or on tumor cells at a concentration significantly higher than that on normal cells. According to any one of the above embodiments, the CAR of CAR T cells specifically binds to tumor-associated antigens (TAA). According to the teachings of the present invention, any CAR bound to TAA can be used. According to one embodiment, TAA is HER2. According to another embodiment, the CAR of CAR T cells specifically binds to tumor-associated antigens selected from CD19, CD38 and CD24. Other non-limiting examples of CAR that can be used are CARs for antigens selected from MUC1, mesothelin, PSCA, EGFR, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, HER2, ROR1, CD70, CD38, CD138, CD24 and CD19.

[0057] As used herein, the term "T cell" refers to a lymphocyte that expresses a T cell receptor and participates in a variety of cell-mediated immune responses, as is known in the art. T cells may include CD4+T cells, CD8+T cells, and natural killer T cells. The term includes genetically modified T cells, such as cells transduced with nucleic acids such as DNA or RNA, optionally using a vector. The term "CAR T cell" refers to a T cell expressing CAR. In certain embodiments, the present invention relates to a CAR T cell comprising a CD8+T cell population.

[0058] As described above, activated EVs are considered to contain at least a portion of the molecular contents of the parental cells. According to some embodiments, the activated EVs of the present invention contain chimeric antigen receptors (CARs) of origin CAR T cells (in at least one subset of the EV population disclosed herein). According to one embodiment, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, or at least 25% of the activated EVs of the present invention contain chimeric antigen receptors (CARs) of origin CAR T cells. According to one embodiment, CAR is present on the outer membrane of EV. According to some embodiments, at least 10% of the isolated activated EVs of the present invention contain chimeric antigen receptors. According to some embodiments, at least 15% of the isolated activated EVs of the present invention contain chimeric antigen receptors. According to some embodiments, at least 18% of the isolated activated EVs of the present invention contain chimeric antigen receptors. According to some embodiments, at least 20% of the isolated activated EVs of the present invention contain chimeric antigen receptors. According to some embodiments, at least 3%, at least 5%, at least 10% of the isolated activated EVs of the present invention present chimeric antigen receptors. According to some embodiments, at least 15% of the isolated activated EVs of the present invention present a chimeric antigen receptor. According to some embodiments, at least 25% or at least 30% of the isolated activated EVs of the present invention contain a chimeric antigen receptor. According to some embodiments, 10% to 90% of the EVs present a CAR. According to some embodiments, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 60%, 20% to 60%, 20% to 50%, 15% to 45%, 15% to 40% of the EVs present a CAR. According to some embodiments, the CAR is an anti-HER2 CAR. According to one embodiment, the CAR is an N29 CAR.

[0059] The terms "HER2" and "human HER2" are used interchangeably herein and refer to a protein called human epidermal growth factor receptor 2, receptor tyrosine-protein kinase erbB-2, also known as CD340 (differentiation cluster 340), proto-oncogene Neu, Erbb2 (rodent) or ERBB2 and having an extension number EC2.7.10.1. The term "anti-Her2" or "αHer2" refers to the antigen binding domain of a CAR, or an antigen binding domain of an antibody that specifically binds to human Her2.

[0060] According to some embodiments, the EV of the present invention originates from a CAR T cell, wherein the CAR specifically binds to HER2 (anti-HER2 CAR). According to one embodiment, the anti-HER2 CAR comprises three complementary determining regions (CDRs) of a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and three complementary determining regions (CDRs) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2. According to one embodiment, the CAR that specifically binds to HER2 is an N29 CAR, as known in the art, such as described in Globerson-Levin A et al., Molecular therapy, 2014; 22 (5); 1029-38. In general, N29 is a monoclonal antibody that specifically binds to the human HER2 receptor, and the N29 CAR comprises the scFv of the N29 antibody as an antigen binding domain. According to some embodiments, the CAR T cell expresses the N29 CAR (N29 CAR T cell). According to one embodiment, the activated EV is derived from an activated N29 CAR T cell. According to some embodiments, the N29 CAR has an amino acid sequence of SEQ ID NO: 3. According to other embodiments, the N29 CAR is encoded by the DNA sequence SEQ ID NO:4.

[0061] According to any of the above embodiments, the CAR T cells are activated CAR T cells. The terms "pre-stimulated", "pre-activated", "stimulated" and "activated" with respect to CAR T cells are used interchangeably herein and refer to CAR T cells that have been incubated and thus stimulated by tumor-associated antigens that are specifically bound by CAR. In addition, the term refers to the state of T cells that provide CAR-mediated stimulation before EV separation. Such activated CAR T cells may also be expressed as "specifically activated CAR T cells" and EVs obtained from activated CAR T cells are expressed as activated EVs, although in some embodiments the term activation may be omitted with respect to EVs. As is known in the art, activation is achieved by incubating CAR T cells with specific tumor-associated antigens for a time sufficient to activate T cells.

[0062] According to some embodiments, incubation is carried out for 3 to 96, 6 to 72 or 12 to 48 hours, for example 24 hours.Activation can be, for example, accompanied by induced cytokine production, IL-2, IL-5, IL-8, IL-12, IL-17, IL-21, MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), MIG (CXCL9), IP10 (CXCL10), irregular chemokine (fractalkine) (CX3CL1), G-CSF, GM-CSF, Flt-3L, IL-1Rα, and / or TNFα levels increase, receptors such as CD25 (IL-2 receptor) and CD71 (transferrin receptor) expression increase, costimulatory molecules such as CD26, CD27, CD28, CD30, CD154 or CD40L and CD134 expression increase, and detectable effector function. With respect to T cells, "activated" may also have the meaning of a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation.

[0063] The terms "stimulated" and "activated" with respect to EVs refer to EVs obtained from the above-mentioned activated CAR T cells. Compared with natural, non-activated EVs (EVs obtained from non-activated CAR T cells), the activated EVs described herein generally exhibit improved properties (e.g., the anti-cancer properties of their parent CAR T cells). As described above, activated EVs may include the contents or partial contents of their parent CAR T cells. Therefore, in some embodiments, EVs contain or express elevated levels of cytokines and / or receptors, as in their parent activated CAR T cells. Without wishing to be bound by a particular theory or mechanism of action, activated EVs can be distinguished from unactivated EVs by the presence of surface markers and / or intracellular markers. For example, but not limited to, activated EVs may contain or express increased levels of T cell activation markers, such as CD25 and / or CD137 (41-BB), CD3, CD38, and HLARD.

[0064] According to some embodiments, at least 15% of the EVs of the present invention express the CD3 antigen on their outer membrane. According to some embodiments, at least 20%, at least 22%, at least 24%, at least 25%, or at least 28% of the EVs of the present invention express the CD3 antigen on their outer membrane. According to some embodiments, at least 25% of the EVs of the present invention express the CD3 antigen on their outer membrane.

[0065] According to some embodiments, at least 20% of the EVs of the invention express the HLARD antigen on their outer membrane. According to some embodiments, at least 22%, at least 24%, at least 25%, or at least 28% of the EVs of the invention express the HLARD antigen on their outer membrane. According to some embodiments, at least 25% of the EVs of the invention express the HLARD antigen on their outer membrane.

[0066] According to some embodiments, at least 10% of the EVs of the present invention express the CD38 antigen on their outer membrane. According to some embodiments, at least 8%, at least 12%, at least 15%, at least 18%, or at least 20% of the EVs of the present invention express the CD38 antigen on their outer membrane. According to some embodiments, at least 25% of the EVs of the present invention express the CD38 antigen on their outer membrane.

[0067] According to some embodiments, at least 15% of the EVs of the present invention express the CD3 antigen on their outer membrane, at least 20% of the EVs of the present invention express the HLARD antigen on their outer membrane, and at least 8% of the EVs of the present invention express the CD38 antigen on their outer membrane.

[0068] According to the teachings of the present invention, activated EVs are those obtained from CAR T cells activated by incubation with their corresponding TAAs, i.e., TAAs to which CAR specifically binds and thus activates T cells. According to one embodiment, CAR-T cells are incubated with TAAs for 6 to 48 or 12 to 36 hours. In other embodiments, CAR-mediated stimulation is provided to T cells no more than 24 hours before EV collection, such as 18, 12 or 6 hours before EV separation. According to certain exemplary embodiments, T cells have been activated by cells (e.g., tumor cells or antigen presenting cells) presenting TAAs. According to certain exemplary embodiments, T cells are activated by TAAs expressed or presented by entities such as liposomes, or by TAAs attached to the surface of entities such as flat plates. According to certain exemplary embodiments, T cells are activated by the surface of TAAs that present CAR-specific binding. According to one embodiment, TAA is HER2 and CAR is N29 CAR. According to another embodiment, CAR is N29 CAR and CAR T cells are incubated with ovarian cancer cells expressing HER2. According to one embodiment, ovarian cancer cells are SKOV cells. According to another embodiment, the CAR is an N29 CAR and the CAR T cells are incubated with HER2-positive breast cancer cells. According to another embodiment, the CAR is an N29 CAR and the CAR T cells are incubated with HER2 antigens. According to some embodiments, the EV is derived from activated N29 CAR T cells. According to one embodiment, the EV is derived from N29 CAR T cells activated with HER2-positive ovarian cancer cells. According to one embodiment, the EV is derived from N29 CAR T cells activated with SKOV cells. According to one embodiment, the EV is derived from N29 CAR T cells activated with HER2 breast cancer cells. According to one embodiment, the EV is derived from N29 CAR T cells activated with primary HER2-positive cancer cells. According to one embodiment, primary HER2-positive cancer cells are obtained from cells of a subject with the cancer. According to some embodiments, the T cells are CD8+T cells. According to other embodiments, the T cells are CD4+T cells. According to yet another embodiment, the CAR T cells are a combination of CD4+ and CD8+CAR T cells.

[0069] According to some embodiments, the present invention provides extracellular vesicles (EVs), wherein the EVs are derived from activated N29 CAR T cells, wherein at least 20% of the EVs have a size greater than 150nm. According to some embodiments, the present invention provides extracellular vesicles (EVs), wherein the EVs are derived from activated N29 CAR T cells, wherein at least 22% of the EVs have a size greater than 150nm. According to some embodiments, the present invention provides extracellular vesicles (EVs), wherein the EVs are derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150nm. According to some embodiments, at least 27% of the activated EVs have a size of 150nm or greater. According to one embodiment, at least 28% of the activated EVs have a size of 150nm or greater. According to one embodiment, at least 29% of the activated EVs have a size of 150nm or greater. According to one embodiment, at least 30% of the activated EVs have a size of 150nm or greater. According to one embodiment, at least 32% of the activated EVs have a size of 150nm or greater. According to one embodiment, at least 35% of the activated EVs have a size of 150nm or greater. According to another embodiment, at least 40% of the activated EVs have a size greater than 150nm. According to another embodiment, at least 42% of the activated EVs have a size greater than 150nm. According to yet another embodiment, at least 45% of the activated EVs have a size greater than 150nm. According to another embodiment, at least 50% of the activated EVs have a size greater than 150nm. According to another embodiment, at least 55% of the activated EVs have a size greater than 150nm. According to yet another embodiment, at least 60% of the activated EVs have a size greater than 150nm. According to yet another embodiment, at least 65% or at least 65% of the activated EVs have a size greater than 150nm. According to some embodiments, 22% to 70% or 25% to 70% of the activated EVs have a size greater than 150nm. According to other embodiments, 25% to 35% of the activated EVs have a size greater than 150nm. According to certain embodiments, 25% to 45% of the activated EVs have a size greater than 150. According to some embodiments, 30% to 70% of the activated EVs have a size greater than 150nm. According to one embodiment, 35% to 65% of the activated EVs have a size greater than 150nm. According to other embodiments, the average size of the activated EVs is 135nm or greater. According to other embodiments, the average size of the activated EVs is 140nm or greater. According to some embodiments, the average size of the activated EVs is 145nm or greater. According to certain embodiments, the average size of the activated EVs is 150nm or greater. According to one embodiment, the average size of the activated EVs is 155nm or greater, 160nm or greater, or 165nm or greater.According to one embodiment, the average size of the activated EVs is greater than 160nm or greater than 170nm. According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 22%, at least 25%, at least 29%, at least 30%, at least 32%, at least 35%, at least 37%, at least 40%, at least 43%, at least 45%, or at least 46%, at least 50%, at least 55%, at least 60%, or at least 65% of the EVs have a particle size greater than 150nm and the average size of the EVs is selected from 132 or more, 135nm or more, 137nm or more, 140nm or more, 142nm or more, 145nm or more, 147nm or more, 150nm or more, 152nm or more, 155nm or more, 160nm or more, 162nm or more, 165nm or more, 167nm or more, and 170nm or more. According to some embodiments, the present invention provides isolated EVs derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150 nm and the EVs have an average size selected from 132 or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 142 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 152 nm or greater, 155 nm or greater, 160 nm or greater, 162 nm or greater, 165 nm or greater, and greater than 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 29% or at least 30% of the EVs have a size greater than 150 nm and the EVs have an average size selected from 132 or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 142 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 152 nm or greater, 155 nm or greater, 160 nm or greater, 162 nm or greater, 165 nm or greater, and greater than 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 35% of the EVs have a size greater than 150 nm and the EVs have an average size selected from 132 or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 142 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 152 nm or greater, 155 nm or greater, 160 nm or greater, 162 nm or greater, 165 nm or greater, and greater than 170 nm.According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 22% of the EVs have a size greater than 150nm and the EVs have an average size of 135 or more. According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 135 or more. According to other embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 140 or more. According to some embodiments, at least 29% of the EVs have a size greater than 150nm and the EVs have an average size of 147nm or more. According to some embodiments, at least 29% of the EVs have a size greater than 150nm and the EVs have an average size of 150nm or more. According to some embodiments, at least 29% of the EVs have a size greater than 150nm and the EVs have an average size of 155nm or more. According to some embodiments, at least 29% of the EVs have a size greater than 150nm and the EVs have an average size of 160nm or more. According to some embodiments, at least 29% of the EVs have a size greater than 150nm and the EVs have an average size of 165nm or more. According to some embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150nm and the EVs have an average size of 147nm or more. According to some embodiments, at least 35% of the EVs derived from activated N29 CAR T cells have a particle size greater than 150nm, and the EVs have an average size greater than 150nm or 155nm or more or 160nm or more. According to some embodiments, at least 40% of the EVs derived from activated N29 CAR T cells have a particle size greater than 150nm, and the EVs have an average size of 155nm or more or 160nm or more or 165nm or more. According to some embodiments, at least 45% or at least 60% of EVs derived from activated N29 CAR T cells have a particle size greater than 150 nm, and the EVs have an average size of 155 nm or more, or 160 nm or more, or 165 nm or more. According to some embodiments, at least 55% of EVs derived from activated N29 CAR T cells have a particle size greater than 150 nm, and the EVs have an average size of 155 nm or more, or 160 nm or more, or 165 nm or more. According to some embodiments, at least 60% of EVs derived from activated N29 CAR T cells have a size greater than 150 nm, and the EVs have an average size of 155 nm or more, or 160 nm or more, or 165 nm or more.According to some embodiments, at least 25% of the EVs have a size greater than 150 nm, the EVs have an average size of 132 nm or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 155 nm or greater, 160 nm or greater, or 170 nm or greater, and the ratio between EVs with a size greater than 150 nm and EVs with a size less than 150 nm is from 1:4 to 1:1 or about 1:4, about 1:3, about 2:3, or about 1:1. According to some embodiments, at least 25% or at least 30% of the activated EVs of the present invention comprise N29 CAR. According to some embodiments, at least 15% of the EVs derived from activated N29 CAR T cells express CD3 antigen on their outer membrane. According to some embodiments, at least 20% of the EVs derived from activated N29 CAR T cells express HLARD antigen on their outer membrane. According to some embodiments, at least 8% of the EVs derived from activated N29 CAR T cells express the CD38 antigen on their outer membrane. According to some embodiments, at least 20% of the EVs of the present invention express the CD3 antigen on their outer membrane, at least 20% of the EVs of the present invention express the HLARD antigen on their outer membrane, and at least 10% of the EVs of the present invention express the CD38 antigen on their outer membrane. According to some embodiments, N29 CAR T is activated by incubation with HER2-positive cancer cells. According to some embodiments, HER2-positive cancer cells are selected from ovarian cancer cells expressing HER2, breast cancer cells expressing HER2, and primary cancer cells expressing HER2. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles derived from N29 CAR-T cells, wherein the T cells are incubated with ovarian cancer cells expressing HER2 for 12 to 36 hours, wherein the EVs are isolated within 24 hours after incubation. According to another embodiment, N29 CAR-T cells are incubated with breast cancer cells expressing HER2 for 12 to 36 hours, wherein the EVs are isolated within 24 hours after incubation. According to some embodiments, EVs are isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after incubation. According to other embodiments, EVs are isolated within 1, 2, 3, 4, 5, 6, or 7 days after incubation. For example, T cells can be incubated at a ratio of T cells to target cells of 1.5:1 to 3:1, such as 2:1. According to one embodiment, T cells are incubated with target cells at a ratio of 15:1 to 1:5, 10:1 to 1:4, 8:1 to 1:3, 5:1 to 1:2, or 3:1 to 1:1 T cells to target cells.

[0070] According to any of the above embodiments, the activated EV is a cytotoxic EV, that is, it exhibits target-specific (e.g., tumor-directed) cytotoxicity. According to one embodiment, the activated EV exhibits cytotoxic activity against cancer cells. According to a further embodiment, the activated EV of the present invention exhibits cytotoxic activity specific to cancer cells (e.g., those cells that exhibit or express the TAA targeted by the CAR of the parental cell). According to some embodiments, the cytotoxic activity is apoptosis.

[0071] Unexpectedly, it was found that the isolated activated EVs of the present invention can exert excellent anti-tumor effects even in the absence of exogenously added anti-cancer agents or payloads. Therefore, in other embodiments, the present invention relates to the isolated activated EVs of the present invention that do not contain any exogenous anti-cancer agents.

[0072] According to another embodiment, the activated EV also comprises an anticancer agent. When referring to a compound, an agent, a part or a composition, the terms "anticancer", "antitumor" and "antitumor" are used interchangeably herein and refer to a compound, a drug, an antagonist, an inhibitor or a modulator having anticancer properties or inhibiting or preventing the growth, function or proliferation of cells, especially tumor cells and / or causing the ability to destroy cells, especially tumor cells. According to some embodiments, the anticancer agent is selected from chemotherapeutics, radioisotopes, toxins, cytokines such as interferons, and antagonists targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, the anticancer agent is a chemotherapeutic agent. As used herein, the term "exogenous anticancer agent" refers to an anticancer agent loaded into an EV after the EV is separated from a T cell.

[0073] The present invention provides a preparation, formulation or composition comprising a plurality of isolated activated EVs according to the present invention. According to one embodiment, the composition is a pharmaceutical composition. Therefore, according to another aspect, the present invention provides a pharmaceutical composition comprising isolated EVs derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier, wherein at least 20% of the EVs have a particle size greater than 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising isolated activated EVs derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier, wherein at least 22% of the EVs have a size greater than 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising isolated activated EVs derived from activated T cells (CAR T cells) expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier, wherein at least 25% of the EVs have a size greater than 150 nm.

[0074] Each of the embodiments related to the isolated activated EVs described in any of the above aspects also applies to this article.

[0075] According to any aspect or embodiment of the invention, the term "at least X% of EVs have a size greater than 150 nm" may be replaced with the term "on average X% or more of EVs have a size greater than 150 nm". Therefore, according to some embodiments, the present invention provides isolated activated extracellular vesicles (EVs) derived from activated T cells expressing a chimeric antigen receptor (CAR T cells), wherein an average of 22% or more of EVs have a particle size greater than 150 nm. According to some embodiments, an average of 25% or more, 30% or more, 35% or more, or 40% or more of EVs have a particle size greater than 150 nm.

[0076] As used herein, the term "pharmaceutical composition" refers to a composition comprising a therapeutic agent (e.g., the activated and isolated EVs of the present invention) formulated together with one or more pharmaceutically acceptable carriers.

[0077] The term "therapeutically effective amount" of EVs is an amount of EVs that will have the intended therapeutic effect when administered to a subject. The therapeutic effect is not necessarily produced in a single administration and may only appear after a series of doses are administered. Therefore, a therapeutically effective amount can be administered in one or more administrations. The precise effective amount required for a subject will depend on, for example, the size, health and age of the subject, the nature and extent of the cognitive impairment, the therapeutic agent or combination of therapeutic agents selected for administration, and the mode of administration. The effective amount in a given situation can be readily determined by a skilled person by routine experimentation.

[0078] The preparation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be prepared using methods known in the art to provide a rapid, continuous or delayed release of the active ingredient after being applied to a mammal. For example, the preparation can be selected from ointments, granules, lotions, liniments, lemonade, aromatic waters, powders, syrups, eye ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, spirits, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules. According to one embodiment, the pharmaceutical composition is a liquid composition. According to another embodiment, the composition is an injectable composition.

[0079] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients such as lactose, starch or talc or liquid carriers such as water, fatty oils or liquid paraffin.

[0080] Other carriers or excipients that can be used include, but are not limited to, materials derived from animal or plant proteins, such as gelatin, dextrin, and soy, wheat, and psyllium seed proteins; gums such as gum arabic, guar gum, agar, and xanthan gum; polysaccharides; alginates; carboxymethylcellulose; carrageenan; dextran; pectin; synthetic polymers such as polyvinylpyrrolidone; polypeptide / protein or polysaccharide complexes such as gelatin-arabic gum complexes; sugars such as mannitol, glucose, galactose, and trehalose; cyclic sugars such as cyclodextrins; inorganic salts such as sodium phosphate, sodium chloride, aluminum silicate; and amino acids and derivatives thereof having 2 to 12 carbon atoms, such as, but not limited to, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-hydroxyproline, L-isoleucine, L-leucine, and L-phenylalanine. Each possibility represents a separate embodiment of the present invention.

[0081] Solutions or suspensions used in parenteral, intradermal or subcutaneous applications typically include the following ingredients: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol (or other synthetic solvents), antibacterial agents (such as benzyl alcohol, methyl parabens), antioxidants (such as ascorbic acid, sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetates, citrates, phosphates) and agents for adjusting tonicity (such as sodium chloride, glucose). The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, disposable syringes or multiple dose glass or plastic vials.

[0082] Pharmaceutical compositions suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the composition substantially isotonic with the blood of the intended recipient. For example, such compositions may also include water, alcohol, polyols, glycerol, and vegetable oils. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Such compositions preferably include a therapeutically effective amount of the compounds of the invention and / or other therapeutic agents, and a suitable amount of carrier to provide a form suitable for administration to a subject.

[0083] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to animals or humans. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards required by government drug regulatory agencies (e.g., U.S. Food and Drug Administration (FDA) Office of Biologics standards).

[0084] The terms "enriched" or "enriched" are used interchangeably and refer to a composition comprising a higher content and / or concentration of extracellular vesicles than the initial composition. In some embodiments, the composition or pharmaceutical composition of the present invention is an enriched composition, i.e., the amount and / or concentration of extracellular vesicles is higher than the initial amount and / or concentration obtained after EV purification. In some embodiments, the concentration of activated EVs is 1.1, 1.5, 2, 3, 5, 10, 50, 100, 500, or 1000 times higher than the starting material.

[0085] According to one embodiment, the pharmaceutical composition comprises activated and isolated EVs derived from activated CAR T cells. According to one embodiment, CAR T cells are activated by incubation with TAAs that specifically bind to CAR. According to one embodiment, CAR T cells are incubated with TAAs for 1 to 96 hours. According to some embodiments, CAR T cells are incubated with TAAs for 8 to 48 hours or 12 to 36 hours. According to one embodiment, TAA is HER2 and CAR is N29 CAR. According to another embodiment, CAR is N29 CAR and CAR T cells are incubated with ovarian cancer cells presenting HER2. According to a further embodiment, CAR is N29 CAR and CAR T cells are incubated with breast cancer cells presenting HER2. According to one embodiment, ovarian cancer cells are SKOV cells. According to a further embodiment, CAR is N29 CAR and CAR T cells are N29 CAR T cells incubated with primary HER2-positive cells.

[0086] According to some embodiments, the present invention provides a pharmaceutical composition comprising isolated activated EVs derived from activated T cells expressing anti-HER2 CAR and a pharmaceutically acceptable carrier, wherein at least 22 or at least 25% of the EVs have a size greater than 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising isolated activated EVs derived from activated N29CAR T cells and a pharmaceutically acceptable carrier, wherein at least 25% of the EVs have a size greater than 150 nm.

[0087] According to some embodiments, the T cells are selected from CD4+T cells, CD8+T cells, and combinations thereof. According to one embodiment, the CAR T cells are activated with HER2, for example with ovarian or breast cancer cells presenting HER2.

[0088] According to some embodiments, the pharmaceutical composition does not contain any additional anti-tumor agent. In other embodiments, the present invention relates to a pharmaceutical composition comprising the isolated activated EV of the present invention as the only anti-cancer agent.

[0089] The terms "substantially free," "essentially free," "free of," "not including," and "not comprising" are used interchangeably and refer to a composition that does not include, contains, or comprises a particular component, e.g., the composition comprises less than 0.1 wt%, less than 0.01 wt%, or less than 0.001 wt%. In some embodiments, the term "free of" means a composition that comprises trace amounts of a component that is not included, e.g., trace amounts of a component used in a purification process.

[0090] According to other embodiments, the composition further comprises an additional anticancer agent. According to some embodiments, the anticancer agent is selected from chemotherapeutics, radioisotopes, toxins, cytokines such as interferons, and antagonists targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, the anticancer agent is a chemotherapeutic agent. In other embodiments, the additional anticancer agent is a CAR T cell, wherein the CAR of the T cell is different from the CAR of the CAR T cell from which the EV originates.

[0091] According to some embodiments, the pharmaceutical composition is a cell-free composition.

[0092] According to any of the above embodiments, the pharmaceutical composition of the present invention is used to treat cancer.

[0093] As used herein, the term "treating cancer" should be understood to include, for example, treatment causing: tumor shrinkage; reduction in tumor growth rate; stagnation of tumor size; reduction in the number of metastases; reduction in the number of additional metastases; reduction in cancer invasiveness; reduction in the rate of progression of a tumor from one stage to the next; inhibition of tumor growth in mammalian tissues with malignant cancer; control of the establishment of metastases; inhibition of tumor metastasis formation; regression of established tumors and reduction in cancer-induced angiogenesis, inhibition of cancer cell growth and proliferation, etc. As used herein, the term "treating cancer" should also be understood to include prevention, such as prevention of cancer recurrence after previous treatment (including surgical resection) and prevention of progression of cancer in susceptible individuals (genetically, due to lifestyle, chronic inflammation, etc.). As used herein, "prevention of cancer" should therefore be understood to include prevention of metastasis, such as after surgery or after chemotherapy.

[0094] As used herein, the term "cancer" refers to all types of cancers, tumors or malignancies found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. According to one embodiment, the cancer is a solid tumor. According to one embodiment, the cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma, gastric cancer, breast cancer, melanoma, skin tumors, lymphomas, leukemias, gastrointestinal tumors (including colon cancer, gastric cancer, pancreatic cancer, colon cancer, small intestine cancer), ovarian cancer, cervical cancer, lung cancer, prostate cancer, renal cell carcinoma and / or liver metastasis.

[0095] According to some embodiments, the cancer is a cancer in which cells present an antigen to which the CAR specifically binds. According to some embodiments, the cancer presents the HER2 antigen. According to other embodiments, the cancer presents CD19. According to yet another embodiment, the cancer presents the CD38 antigen.

[0096] According to some embodiments, the cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma, gastric cancer, liver cancer, pancreatic cancer and brain cancer, and hematological malignancies.

[0097] In one embodiment, a pharmaceutical composition comprising isolated activated EVs derived from activated anti-HER2 CAR T cells is used to treat HER2-positive cancer.

[0098] According to some embodiments, the HER2-positive cancer is selected from ovarian cancer, breast cancer, gastric cancer, lung adenocarcinoma, uterine cancer, endometrial cancer, and HER2+ salivary duct carcinoma.

[0099] The terms "HER2-positive" and "HER2+" are used interchangeably herein and refer to cells that overexpress the HER2 antigen.

[0100] According to other embodiments, a pharmaceutical composition comprising an isolated activated EV of the present invention is used to treat HER2-positive ovarian cancer, wherein the EV is derived from an anti-HER2 CAR T cell activated by HER2-specific activation. According to some embodiments, a pharmaceutical composition comprising an isolated activated EV of the present invention derived from activated anti-HER2 CAR T cells is used to treat HER2-positive breast cancer. According to an alternative embodiment, a pharmaceutical composition comprising an isolated activated EV of the present invention derived from activated anti-HER2 CAR T cells is used to treat HER2-positive ovarian cancer. According to some embodiments, the anti-HER2 CAR is N29 CAR. According to some embodiments, a pharmaceutical composition comprising an isolated activated EV of the present invention derived from activated N29 CAR T cells is used to treat breast cancer. According to a further embodiment, a pharmaceutical composition comprising an isolated activated EV of the present invention derived from activated N29 CAR T cells is used to treat ovarian cancer. According to yet another embodiment, a pharmaceutical composition comprising an isolated activated EV of the present invention derived from activated N29 CAR T cells is used to treat lung adenocarcinoma or gastric cancer. According to some embodiments, the pharmaceutical composition comprises isolated activated EVs derived from activated N29 CAR T cells, wherein at least 22%, at least 25%, at least 29%, at least 30%, at least 32%, at least 35%, at least 37%, at least 40%, at least 43%, at least 45% or at least 46%, at least 50% or at least 55% of the EVs have a particle size greater than 150 nm and / or the EVs have an average size selected from 132 or greater, 135 nm or greater, 137 nm or greater, 140 nm or greater, 142 nm or greater, 145 nm or greater, 147 nm or greater, 150 nm or greater, 152 nm or greater, 155 nm or greater, 160 nm or greater, 162 nm or greater, 165 nm or greater, and 170 nm or greater. According to other embodiments, the present invention provides isolated activated EVs derived from activated N29 CAR T cells, wherein at least 25% of the EVs have a size greater than 150 nm and the EVs have an average size of 140 or greater. According to some embodiments, at least 29% of the EVs have a size greater than 150 nm and the EVs have an average size of 150 nm or greater, 155 nm or greater, 160 nm or greater, or 165 nm or greater. According to some embodiments, the ratio between EVs with a particle size greater than 150 nm and EVs with a particle size less than 150 nm is 1:4 to 1:1 or about 1:4, about 1:3, about 2:3, or about 1:1. According to some embodiments, at least 20% of the EVs of the present invention express CD3 antigens on their outer membranes and / or at least 20% of the EVs of the present invention express HLARD antigens on their outer membranes and / or at least 10% of the EVs of the present invention express CD38 antigens on their outer membranes.According to some embodiments, at least 10% or at least 15% or at least 20% of the EVs of the present invention express N29 CAR on their surface. According to another embodiment, N29 CAR-T cells are incubated with breast cancer cells expressing HER2 for 12 to 36 hours, wherein EVs are isolated within 24 hours after incubation. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles derived from N29 CAR-T cells incubated with ovarian cancer cells expressing HER2 for 12 to 36 hours, wherein EVs are isolated within 24 hours after incubation. For example, T cells can be incubated at a ratio of T cells to target cells of 1.5: 1 to 3: 1, for example 2: 1. According to one embodiment, T cells are incubated with target cells at a ratio of T cells to target cells of 15: 1 to 1: 5, 10: 1 to 1: 4, 8: 1 to 1: 3, 5: 1 to 1: 2 or 3: 1 to 1: 1.

[0101] According to some embodiments, activating comprises activating with primary cancer cells obtained from a subject suffering from cancer. According to one embodiment, the primary cancer cells are obtained from cancer tissue of a subject suffering from cancer.

[0102] According to some embodiments, the use comprises thawing the EVs or the pharmaceutical composition comprising the EVs prior to administration.

[0103] The pharmaceutical compositions of the present invention can be administered by any known method.

[0104] The term "giving" or "applying" a substance, compound or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinal, intracerebrally, and transdermally (by absorption, such as through a skin catheter). The compound or agent can also be appropriately introduced by a rechargeable or biodegradable polymer device or other device, such as a patch and a pump, or a formulation that provides an extended, slow or controlled release of the composition. It can also be applied, for example, once, multiple times, and / or over one or more extended periods. According to some embodiments, the composition is applied 1, 2, 3, 4, 5, or 6 times a day. According to some embodiments, the composition is applied 1, 2, 3, 4, 5, or 6 times a month. In some embodiments, administration includes direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer or allows others to administer and / or provides a drug prescription to a patient administers the drug to the patient.

[0105] According to one embodiment, the pharmaceutical composition is formulated as a solution for injection. According to another embodiment, the pharmaceutical composition is administered systemically. According to some embodiments, the pharmaceutical composition is injected, such as intravenously or intramuscularly. According to one embodiment, the pharmaceutical composition is administered topically. According to some embodiments, the pharmaceutical composition is administered intratumorally. According to another embodiment, the pharmaceutical composition is administered near the tumor.

[0106] In some embodiments, the present invention relates to a pharmaceutical composition comprising the isolated activated EV of the present invention as the sole anti-cancer agent for treating cancer.

[0107] According to some embodiments, the pharmaceutical compositions of the present invention are co-administered with additional anti-cancer agents.

[0108] According to some embodiments, the anticancer compound is selected from chemotherapeutic agents, radioisotopes, toxins, cytokines such as interferons, and antagonists targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, the anticancer agent is a chemotherapeutic agent. In other embodiments, the anticancer agent is a CAR T cell.

[0109] According to some embodiments, co-administration of the pharmaceutical composition of the present invention and an additional anti-tumor compound or agent is carried out in a regimen selected from the following: a single combined composition, separate individual compositions administered essentially simultaneously, and separate individual compositions administered under different schedules, and including treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0110] The term "co-administration" includes administration of the first and second agents in a substantially simultaneous manner, such as in a single dosage form, such as a capsule or tablet having a fixed ratio of the first and second amounts, or in multiple dosage forms for each. The agents may be administered in a sequential manner in either order. When co-administration includes administration of each agent separately, the agents are administered close enough in time to produce the desired effect (e.g., complex formation).

[0111] The term "sequential manner" refers to administration of two compounds at different times and optionally in different modes of administration. The agents may be administered in a sequential manner in either order.

[0112] The term "substantially simultaneous manner" refers to administration of the two compounds with only a short time interval between them. In some embodiments, the time interval is in the range of from 0.01 to 60 minutes.

[0113] According to another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering an effective amount of isolated activated EVs of the present invention. According to one embodiment, the method comprises administering a pharmaceutical composition of the present invention. According to one embodiment, the cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma, and gastric cancer. In some embodiments, the present invention provides a method for treating a cancer selected from breast cancer, ovarian cancer, lung adenocarcinoma, and gastric cancer, the method being by administering an effective amount of isolated activated EVs derived from activated CAR T cells expressing N29 CAR, wherein at least 25% of the EVs are greater than 150 nm in size.

[0114] Each of the embodiments related to the isolated activated EVs of the present invention as described in any of the above aspects also apply herein.

[0115] According to another embodiment, the method further comprises co-administering an additional anticancer agent. According to some embodiments, the anticancer agent is a chemotherapeutic agent. According to other embodiments, the anticancer agent is a composition comprising CAR T cells.

[0116] According to another aspect, the present invention provides the use of the EV according to the present invention in the preparation of a medicament for treating cancer.

[0117] According to another aspect, the present invention provides a method for preparing isolated activated extracellular vesicles of the present invention. According to some embodiments, the present invention provides a method for preparing isolated activated extracellular vesicles derived from activated CAR T cells, wherein at least 22% or at least 25% of the EVs have a size greater than 150 nm, wherein the method comprises incubating the CAR T cells with a tumor-associated antigen that specifically binds to the CAR under conditions capable of stimulating the T cells, and isolating the obtained activated extracellular vesicles.

[0118] According to some embodiments, the preparation method comprises (1) incubating CAR T cells with a tumor-associated antigen that specifically binds to the CAR, wherein the incubation is performed in a cell culture medium under conditions capable of activating T cells; (2) separating the T cells from the culture medium; (3) separating EVs from the culture medium by centrifugation at 8,000 g to 30,000 for 0.5 to 4 hours; (4) optionally washing the EVs; and 5) optionally freezing the EVs at a temperature below -60°C, thereby obtaining the EVs of the present invention, wherein at least 22% of the EVs have a size greater than 150 nm. According to one embodiment, at least 25% of the EVs have a size greater than 150 nm.

[0119] According to some embodiments, the separation of T cells from the cell culture medium in step (2) includes separating the cell culture medium from cells and large cell particles. The term "large cell particles" refers to cell particles larger than 1 μm, such as cell debris, organelles, etc. A cell culture medium containing EVs of the present invention is thus obtained. The separation can be performed in one step or in multiple steps. According to some embodiments, the separation of T cells from the culture medium includes the following steps: step (2i) includes centrifuging the culture medium containing activated T cells at 200g to 600g for 5 to 60 minutes and separating / collecting the precipitate, thereby separating the precipitate from the culture medium, and step (2ii) includes centrifuging the cell culture medium (supernatant) obtained in step (2i) at 1000g to 3000g for 10 to 60 minutes, and separating the resulting precipitate from the culture medium. According to some embodiments, the method only includes step (2ii). Therefore, according to some embodiments, step 2 includes centrifuging the cell culture medium obtained from the previous step (step 1) at 1000g to 3000g for 10 to 60 minutes and separating the resulting precipitate from the cell culture medium. The cell culture medium obtained after centrifugation can be expressed as a supernatant.

[0120] According to some embodiments, step (2ii) includes centrifuging at 1000 to 2000g for 10 to 50 minutes, or 10 to 30 minutes or 10 to 20 minutes. According to some embodiments, step (2i) includes centrifuging the culture medium at 200 to 600g or about 400g for 5 to 15 minutes. According to some embodiments, step (2i) includes centrifuging the culture medium at 200 to 600g or about 400g for 5 to 30 minutes. According to some embodiments, separating T cells from the culture medium includes the following steps: step (2i) includes centrifuging the culture medium containing activated T cells at 200g to 600g for 5 to 15 minutes and separating / collecting the precipitate, thereby separating the precipitate from the culture medium, and step (2ii) includes centrifuging the cell culture medium obtained in step (2i) at 1000g to 3000g for 10 to 30 minutes, and separating the resulting precipitate from the culture medium. According to some embodiments, separating T cells from culture medium containing EVs comprises centrifugation at about 400 g for 5 to 15 minutes, and centrifuging the supernatant at about 1500 x g for 10 to 20 minutes and collecting the supernatant for EV purification.

[0121] According to some embodiments, the method comprises only step (2ii). In the absence of step (2i), (2ii) comprises centrifuging the cell culture medium obtained from step (1). According to other embodiments, the method may further comprise other steps before step 3, wherein the centrifugal force does not exceed 5,000 g.

[0122] Steps (2i), (2ii) or a combination thereof should be short enough to pellet cells and large cell particles, but not the EVs of the invention. Therefore, in some embodiments, a short centrifugation in step (2i) may be preferred.

[0123] Alternatively, separation of the cell culture medium containing the EVs of the invention from cells and large cell particles is a continuous process performed as known in the art, for example by microfluidic systems, hollow fiber bioreactor technology (Whitford W. et al. www.GENengnews.com 2015), nanoscale separation arrays (Wunsch BH. Nature nanotechnology 2016), or magnetic nanowires (Lim JJ Nanobiotechnology. 2019).

[0124] According to some embodiments, the T cells obtained in step (2i) or step (2i) can be recycled, i.e., used to further prepare the EV of the present invention. Therefore, the cells collected in step (2i) or step (2i) are incubated with a tumor-associated antigen that specifically binds to the CAR in a cell culture medium under conditions that activate the T cells to start the process, and then separated from the culture medium in step (2) as described above. The number of cycles in which CAR T cells are used to prepare the EV of the present invention is limited only by the ability of the T cells to produce the EV of the present invention having all the above-mentioned characteristics.

[0125] The noun and gerund of the term "incubation" are used interchangeably herein and refer to the process of contacting or exposing CAR T cells to a desired entity under conditions capable of stimulating T cells. According to one embodiment, the CAR T cells are incubated with the TAA for at least 1 hour. According to another embodiment, the incubation is at least 6, 12, 18 or 24 hours. According to another embodiment, the incubation is 1 to 96 hours. According to some embodiments, the incubation lasts 6 to 84, 12 to 72, 18 to 60, 24 to 48 or 30 to 32 hours. According to another embodiment, the incubation is 6 to 48, 12 to 42, 18 to 36 hours. According to another embodiment, the incubation is 20 to 30 hours. In other embodiments, the incubation is performed for about 24 hours.

[0126] In some embodiments, EVs are isolated immediately after incubation. According to some embodiments, EVs are isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after T cells are separated from the culture medium. According to other embodiments, EVs are isolated within 1, 2, 3, 4, 5, 6, or 7 days after T cells are separated from the culture medium. According to some embodiments, EVs are isolated within 24 hours after T cells are separated from the culture medium. According to some embodiments, EVs are isolated within 48 hours after T cells are separated from the culture medium. According to some embodiments, EVs are isolated within 72 hours after T cells are separated from the culture medium.

[0127] According to other embodiments, at the time of purification, the ratio of EV to cells is at least 2, 3, 4, 5, 6, 8 or 10 times higher than that of the initial cells, or alternatively and usually at least 50, at least 100, at least 500 or at least 1000 times higher. According to some embodiments, purification provides substantially cell-free EVs. According to other embodiments, purification provides cell-free EV compositions. According to some embodiments, incubation of CAR T cells with TAAs includes incubation with intact TAAs, a portion of TAAs to which CAR specifically binds (epitopes or portions containing epitopes), or with entities expressing the TAAs (e.g., intact cells, EVs expressing the TAAs, or any carriers such as (liposomes expressing the TAAs)). According to some embodiments, incubation includes incubation with cells presenting the TAAs.

[0128] According to some embodiments, separating EVs (step 3) comprises low-force centrifugation of the culture medium containing EVs. According to some embodiments, separation comprises centrifugation at 8,000g to 30,000g for 0.5 to 4 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 30,000g for 0.5 to 3 hours. According to another embodiment, separation comprises centrifugation at 8,000g to 30,000g for 0.5 to 2 hours. According to yet another embodiment, separation comprises centrifugation at 8,000g to 30,000g for 0.5 to 1.5 hours.

[0129] According to some embodiments, separation comprises centrifugation at 8,000g to 20,000g for 0.5 to 4 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 20,000g for 0.5 to 3 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 20,000g for 0.5 to 2.5 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 20,000g for 0.5 to 2 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 20,000g for 0.5 to 1.5 hours. According to some embodiments, separation comprises centrifugation at 16,000g to 22,000g for 0.5 to 4 hours or 0.5 to 3 hours or 0.5 to 2 hours or 0.5 to 1.5 hours. According to some embodiments, separation comprises centrifugation at about 20,000g for 0.5 to 2.5 hours.

[0130] According to some embodiments, separating comprises centrifugation at 8,000 g to 15,000 g for 0.5 to 4 hours or 0.5 to 3 hours or 0.5 to 2.5 hours or 0.5 to 2 hours or 0.5 to 1.5 hours.

[0131] According to some embodiments, separation comprises centrifugation at 8,000g to 12,000g for 0.5 to 4 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 12,000g for 0.5 to 3 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 12,000g for 0.5 to 2 hours. According to one embodiment, separation comprises centrifugation at 8,000g to 12,000g for 0.5 to 1.5 hours. According to another embodiment, separation comprises centrifugation at 8,000g to 12,000g for 1 to 3 hours. According to yet another embodiment, separation comprises centrifugation at 8,000g to 12,000g for 2 to 4 hours. According to some embodiments, separation comprises centrifugation at 8,000g to 10,000g for 0.5 to 4 hours or 0.5 to 3 hours or 0.5 to 2 hours or 0.5 to 1.5 hours. According to some embodiments, separation comprises centrifugation at 8,000g to 10,000g for 1 to 4 hours, or 1 to 3 hours, or 1 to 2 hours, or 1 to 1.5 hours. According to other embodiments, separation comprises centrifugation at 8,000g to 10,000g for 1 to 4 hours, or 1 to 3 hours, or 1 to 2 hours, or 2 to 4 hours.

[0132] According to some embodiments, separation comprises low force centrifugation. According to some embodiments, separation comprises centrifugation at 15,000g to 25,000g for 0.5 to 4 hours. According to one embodiment, separation comprises centrifugation at 15,000g to 25,000g for 0.5 to 3 hours. According to another embodiment, separation comprises centrifugation at 15,000g to 25,000g for 0.5 to 2.5 hours. According to another embodiment, separation comprises centrifugation at 15,000g to 25,000g for 0.5 to 2 hours. According to yet another embodiment, separation comprises centrifugation at about 20,000g for 0.5 to 1.5 hours.

[0133] According to some embodiments, CAR is an anti-HER2 CAR. According to some embodiments, CAR is an N29 CAR. According to certain embodiments, activation includes activation of anti-HER2 CAR T cells, such as activation of HER2 CAR T cells with ovarian cancer cells (e.g., SKOV cells) presenting HER2. According to some embodiments, activation includes activation of anti-HER2 CAR T cells, such as activation of N29 CAR T cells with breast cancer cells presenting HER2. According to some embodiments, incubation is 9 to 48 hours. According to some embodiments, incubation is 12 to 36 hours. According to some embodiments, incubation is 18 to 36 hours. In some embodiments, activated extracellular vesicles are isolated immediately after CAR T cells are separated from the culture medium or within 24 hours or 48 hours.

[0134] According to some embodiments, the method comprises washing the EV pellet at least once, for example 1, 2, 3 or more times in step (4).

[0135] According to some embodiments, the method further comprises freezing the EVs at a temperature below -60°C (eg, -80°C or below). According to some embodiments, a cryoprotectant buffer may be used to protect the EVs during the freezing process.

[0136] According to some embodiments, the preparation method is method 1 described in the examples. According to some embodiments, the preparation method is method 5 described in the examples. According to some embodiments, the preparation method is method 3 described in the examples.

[0137] Typically, T cells contain CD8 + T cell population. According to some embodiments, the T cells are CD8 + According to other embodiments, the T cells are CD4 + According to yet another embodiment, the CAR T cells are at least CD4 + and CD8 + Combinations of CAR T cells.

[0138] According to some embodiments, the present invention provides isolated activated extracellular vesicles prepared by a method according to any one of the above embodiments. Therefore, the present invention provides isolated activated extracellular vesicles prepared by the following steps: (1) incubating CAR T cells with tumor-associated antigens that specifically bind to CAR under conditions capable of stimulating T cells, preferably for 6 to 96 hours; (2) separating T cells from a culture medium containing EVs by centrifugation at 200 to 600 g for 10 to 30 minutes, and collecting the precipitate; and centrifuging the remaining culture medium at 1000 to 3000 g for 10 to 30 minutes and discarding / collecting the resulting precipitate, thereby obtaining a culture medium containing EVs; (4) separating EVs from the culture medium by centrifugation at 8,000 g to 30,000 for 0.5 to 4 hours; (5) optionally washing EVs; (6) optionally freezing EVs at a temperature below -60 ° C, thereby obtaining EVs of the present invention, wherein at least 22% or at least 25% of the isolated EVs have a particle size of 150 nm or greater. According to some embodiments, separating the T cells from the culture medium comprising EVs comprises centrifugation at about 400 g for 5 to 15 minutes, and centrifuging the supernatant at about 1500 x g for 10 to 20 minutes.

[0139] According to another embodiment, the method further comprises a step of enriching the EV population containing CAR. The enrichment step of CAR-EV can include the use of magnetic beads conjugated with specific antibodies against CAR or anti-CD3 or any other sorting method.

[0140] The terms "comprising", "including", "having", and "containing" are used interchangeably herein and have the meaning of "consisting at least in part of...". When interpreting each statement in this specification that contains the term "comprising", features other than those that begin with the term or the term may also be present. Related terms such as "comprising" should be interpreted in the same manner. The terms "having" and "comprising" may also include the meanings of "consisting of" and "consisting essentially of", and may be replaced by these terms. The term "consisting of" does not include any components, steps or procedures that are not specifically described or listed. The term "consisting essentially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not substantially change the basic and novel characteristics of the claimed composition or method.

[0141] As used herein, the term "about" when referring to a measurable value such as an amount, duration, etc., is intended to encompass variations of + / - 10% or + / - 5%, + / - 1%, or even + / - 0.1% of the specified value.

[0142] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0143] Example

[0144] Materials and methods

[0145] T cell preparation

[0146] Peripheral human blood lymphocytes (PBL) were isolated from the blood of healthy human donors by density gradient centrifugation on Ficoll-Paque (Axis-shield, Oslo, Norway). PBL were activated in non-tissue culture treated 6-well plates pre-coated with purified anti-human CD3 and purified anti-human CD28 for 48 hours at 37°C. Activated lymphocytes were collected and subjected to two consecutive retroviral transductions in non-tissue culture treated 6-well plates pre-coated with RetroNectin supplemented with human IL-2 (100 IU / mL). After transduction, cells were cultured in the presence of 350 IU / mL IL-2 for 24-72 hours. Transduction efficiency was monitored by flow cytometry. Activated but non-transduced (uninfected) cells were included as T cell controls (Eshhar Z, J Immunol Methods 2001, 248: 67-76).

[0147] The N29 CAR targets HER2 expressed on ovarian cancer cells, and the anti-CD19 CAR targets CD19 expressed on malignant hematopoietic cells.

[0148] N29 CAR has a light chain variable region fragment as shown in SEQ ID NO: 2 and a heavy chain variable region fragment as shown in SEQ ID NO: 2. The complete CAR N29 is encoded by the DNA sequence shown in SEQ ID NO: 4 and has an amino acid sequence shown in SEQ ID NO: 3.

[0149] Extracellular vesicle samples

[0150] In the following experiments, unless otherwise stated, extracellular vesicles were obtained from N29 CAR T cells or non-transduced T cells, each of which was incubated or not with target cells: SKOV (HER2+) or OVCAR (HER2-). The cells were incubated for 24 hours. At the end of 24 hours of cell stimulation of target cells, EVs were isolated from the cell culture medium.

[0151] The following sample notices were used in the examples:

[0152] Sample 1. T cells expressing N29 CAR after stimulation with SKOV (HER2+) cells are represented as: N29 superimposed with SKOV;

[0153] Sample 2. T cells expressing N29 CAR after incubation with OVCAR (HER2-) cells are represented as: N29 superimposed with OVCAR;

[0154] Sample 3. Non-transduced T cells incubated with SKOV (HER2+) cells are represented as: UT superimposed with SKOV;

[0155] Sample 4. Non-transduced T cells incubated with OVCAR (HER2-) cells are represented as: UT superimposed with OVCAR;

[0156] Sample 5. The culture medium for N29 CAR T cells is represented as: N29 overlay medium;

[0157] Sample 6. Non-transduced T cell culture medium (no stimulation) is represented as: UT overlay medium;

[0158] Sample 7. Target cell culture medium obtained from SKOV is represented by: SKOV overlay medium; and

[0159] Sample 8. Target cell culture medium obtained from OVCAR cells is denoted as: OVCAR overlay medium.

[0160] In Example 5 below, N29 CAR T cells and anti-CD19 CAR T cells were stimulated (activated) with SKOV or incubated with Raji for 24 hours.

[0161] EVs from each of the above samples were isolated / purified by one of the following methods 1-5.

[0162] Isolation and analysis of extracellular vesicles (EVs)

[0163] Method 1

[0164] The CAR T cell culture medium was collected and centrifuged at 400g for 10 minutes, and the supernatant was further centrifuged at 1500×g for 15 minutes. The supernatant was further centrifuged at 20,000×g (20K×g) for 1 hour. The EV pellet was then aliquoted and frozen at -80°C. The remaining supernatant was further processed according to method 2 (as follows).

[0165] Method 2

[0166] The supernatant obtained in method 1 (after centrifugation at 20,000 xg) was further centrifuged at 100,000 xg for 60 minutes, and then the precipitate (20K-100K xg precipitate) was frozen in aliquots at -80°C.

[0167] Method 3

[0168] The CAR T cell culture medium was collected and centrifuged at 400 g for 5 min, the supernatant was further centrifuged at 1500 × g for 15 min, and then the supernatant was further centrifuged at 10,000 × g for 30 min. The EV pellet was then frozen in aliquots at -80 °C.

[0169] Method 4

[0170] The supernatant from method 3 was further centrifuged at 70,000 xg for 110 min and the pellet was then frozen in aliquots at -80°C.

[0171] Method 5

[0172] The culture medium of CAR-T cells was collected and centrifuged at 400 g for 5 min, the supernatant was further centrifuged at 1500 × g for 15 min, the supernatant was further centrifuged at 10000 × g for 180 min, and the pellet was frozen in aliquots at -80 °C.

[0173] Size assessment

[0174] The size and concentration of EVs were assessed by nanoparticle tracking analysis (NTA), which can measure particles in the range of 50–2000 nm. NTA was performed using a NanoSight NS300 system with a CMOS camera and a 532 nm laser (Malvern Instruments. Malvern, UK), and each sample was measured three times. Since EVs are mostly spherical particles, the size refers to the diameter of the EVs.

[0175] Use 0.7 μm sized beads to set an appropriate size gate for flow cytometric analysis of large EVs. Use fluorescently labeled antibodies to verify the expression of specific antigens.

[0176] Protein assay

[0177] To calculate the protein content in EV samples and use the same amount of protein in each well, total protein detection and quantification was performed by bicinchoninic acid (BCA) colorimetric assay or by Thermo Scientific TM NanoDrop TM To measure EVs.

[0178] Cell viability-XTT assay

[0179] To assess the number of exposed cells and their viability, the metabolic activity of the target cells was analyzed. The ability of CAR T cell-EVs to reduce the tetrazolium salt XTT to an orange formazan compound was measured. The intensity of the dye correlated with the number of viable cells and was monitored by an ELISA reader.

[0180] Cell lines

[0181] MDA231 HER2-positive breast cancer cells (), ovarian cancer cells, (SKOV and SKOV / Luc) (the latter stably expressing the firefly luciferase gene) and pancreatic cancer (CAPAN) are all HER2-positive cell lines and are therefore potential targets for N29 CAR T EV.

[0182] MDA231 HER2-negative, ovarian cancer cells (OVCAR cells), Raji cells, and B lymphocytes from Burkitt's lymphoma, which are all HER2-negative cells and therefore non-target cells for EVs from N29 CAR T cells, were used as control cells.

[0183] Cytotoxic effects of EVs

[0184] The cytotoxic effects of EVs on target cells were observed and recorded by optical microscopy. The non-radioactive cytotoxicity assay (Promega) was used for measurement.

[0185] At the end of exposure to EVs, target cell nuclei were stained with Hoechst 33342 staining solution (ABCAM) to indicate total cell number, while apoptosis was measured by ANNEXIN / PI kit (MEBCYTO, MBL, MA, USA) according to the manufacturer's instructions. The cytotoxic effects of EVs on target cells were followed and documented by:

[0186] 1. Optical microscope (ZOE TM Fluorescent Cell Imager, Bio-Rad) and analyzed by Image J software;

[0187] 2. INCUCYTE (Sartorius, Germany), live cell imaging and analysis. The percentage of apoptotic target cells was calculated by the number of cells labeled with PI / total number of nuclei or PI / total cell area. In addition, the number of cells labeled with caspase 3 / 7 or cytotoxic dyes was also measured.

[0188] Statistical analysis

[0189] GraphPad Prism4, Bonferroni's multiple comparisons, one-way ANOVA test and nonparametric MannWhitney t-test.

[0190] Example 1. Characterization of extracellular vesicles obtained in method 1

[0191] Extracellular vesicles were obtained from T cells expressing N29 CAR or non-transduced cells. Representative results of multiple measurements (n=4) of EVs obtained from 3 different CAR T cells.

[0192] The size (diameter) of extracellular vesicles secreted by N29 CAR T cells and non-transduced T cells, both incubated with HER2-expressing breast cancer cells, was measured by NTA analysis and compared. N29 CAR T stimulated with target cells (HER2-positive breast cancer cells) secreted more EVs (~25-fold) than non-transduced EVs incubated with HER2+ breast cancer cells, and their EVs showed different size distributions (Figure 1).

[0193] In addition, non-transduced T cells (10 6 / ml) and SKOV cells (0.5x10 6 / ml) and then 9.54×10 7 ±1.05×10 7 The ratio of cells to EVs was found to be 1:63. In contrast, in target SKOV cells (0.5x10 6 After stimulation with N29 CAR T (10 6 A total of 2.24×10 9 ±1.40×10 8 The cell-to-EV ratio was found to be 1:1490.

[0194] Example 2: Characterization of extracellular vesicles isolated by five methods

[0195] EVs were obtained and characterized from the eight different samples defined above (EVs derived from N29 CAR T cells or EVs from non-transduced cells, each sample incubated or not with SKOV or OVCAR) by separation using five different centrifugation protocols (methods 1-5, as described above in the Materials and Methods section). The results are shown in Figure 1 and Tables 1 and 2.

[0196] The size distribution of EVs is as follows Figure 1C , Figure 1D As shown in Table 1. It was found that the EVs in the 20K×g pellet (obtained by method 1) were significantly larger than the EVs obtained by method 2 (20K-100K×g pellet). This was particularly true for EVs secreted by N29 CAR T stimulated by specific target cells (HER2+SKOV cells; p value <0.01) (which also secreted more EVs, data not shown) and N29 CART incubated with nonspecific cells (HER2-OVAR, p value <0.01) ( Figure 1D). Similarly, the EVs obtained by method 3 were significantly larger than those obtained by method 4. In both cases, the differences were statistically significant (p-value < 0.05). In fact, the EVs obtained by methods 1 and 3 had a larger particle size compared to the other methods (p < 0.001), mainly in CAR T EVs stimulated with target HER2+ cells. No statistical significance was detected for the size of EVs obtained by method 5.

[0197] Table 1: Sizes of EVs obtained from samples 1 and 2 by five different isolation methods

[0198]

[0199] In addition, for both samples 1 and 2, the percentage of EVs larger than 150 nm in the 20K × g pellet (obtained by method 1) was significantly higher than that in the pellet obtained by method 2 (p value < 0.05) ( Figure 1E , 1F and Table 2 ). Comparison between the separation methods showed that more than 40% of the EVs obtained by precipitation of the N29 superimposed SKOV sample by method 1 had a size greater than 150 nm, whereas the precipitation of method 2 (20K-100K × g) contained only about 20% of such EVs. The EV precipitation obtained from sample 1 by method 3 (10,000 × g for 30 min) also contained a high proportion of large EVs, comparable to that obtained by method 1 and much higher than that obtained by method 4.

[0200] Table 2. Percentage of EVs with a size greater than 150 nm

[0201]

[0202] from Figure 1G As can be seen from the figure, method 5 provides a higher yield of EVs larger than 150 nm. Method 1 purified approximately 4.1 × 10 10 (±3.8×10 10 ) EVs, and method 5 purified approximately 3.6×10 11 (±1.1×10 11 ) EVs. Although sample 2 provided more EVs larger than 150 nm, these EVs had no cytotoxic effect on cancer cells, as shown below, and therefore had no therapeutic value.

[0203] For large EV (>300 nm) membrane antigen characterization by flow cytometry, EV size gates were set using 0.75 μm beads, indicating that we measured EVs below 1 μm. Figure 1H Shown is a side scatter (SSC) versus forward scatter (FSC) plot using 0.75 μm beads. The R1 gate characterizes the region of the plot where these beads accumulated. Fig. 1I and Figure 1J Shown are FACS analyses of EVs from sample 1 and sample 2 obtained by method 1. EVs from both samples fell within the R1 gating region set, indicating that we measured EVs below 1 micron.

[0204] Example 3. Characterization of EV antigen expression on EVs using isolation methods 1 and 2

[0205] EVs were analyzed for expression of CD3, an antigen that characterizes T cells, compared to EVs nonspecifically labeled with isotype control IgG or unstained. Figure 2I-2L As can be seen from the figure, compared with the control (nonspecific marker ( Figure 2E-2H )) or unstained EVs ( Figures 2A-2D ), approximately 30% of the measured EVs expressed CD3.

[0206] Expression of CD3, CD38, and HLADR antigens on EVs in the pellets obtained from eight samples using either method 1 or method 2 Figure 2M As shown. CD38 is a glycoprotein present on the surface of leukocytes, and HLADR is an MHC class II cell surface receptor encoded by the human leukocyte antigen complex. The left picture shows the EVs obtained by method 1; the right picture shows the EVs obtained by method 2. It can be seen that the EVs obtained by method 1 (20,000g precipitation) showed significantly higher levels of all three membrane antigens than the EVs obtained by method 2. Specifically, among the EVs obtained by method 2, about 30% of the 20,000g precipitation EVs expressed CD3 and HLADR at about 5% and 12%, respectively ( Figure 2M ). It can be seen that about 20% of the EVs obtained by method 1 expressed CD38.

[0207] In a similar experiment, the green appearance of EVs derived from N29-GFP CAR T cells or non-transduced cells was analyzed. The green color of green fluorescent protein (GFP) in EVs was measured by flow cytometry using a fluorescent laser. The results are shown in Figure 3. As can be seen in this figure, the green appearance of EVs derived from N29-GFP CAR T cells or non-transduced cells was significantly different from that of EVs derived from N29-GFP CAR T cells. Figure 3A ) non-specific markers or from untransduced T cells ( Figure 3C ) compared to EVs obtained from the GFP-stained ( Figure 3B ).

[0208] Example 4. Cytotoxicity of EVs obtained by methods 1 and 2

[0209] According to the experimental arrangement described in Table 3, the cytotoxic effects of EVs purified from eight samples by method 1 or 2 on target cells were investigated.

[0210] Table 3. Experimental arrangement for measuring EV cytotoxicity.

[0211]

[0212] Short-term (6 h) exposure of SKOV cells to two different doses (25 μg / well and 12.5 μg / well total protein) of EV obtained from sample 1 (N29 CAR T stimulated by HER2+ cells) induced massive apoptosis of target cells, as shown in the microscopy images ( Figure 4A Hoechst, a nucleic acid stain, was used for total cell counts; colocalization of ANNEXIN-V (green), PI (red), and Hoechst (blue) was recorded as a merged image.

[0213] EVs obtained from sample 2 (N29 CAR T cells incubated with OVCAR) did not affect the viability of SKOV cells ( Figure 4A Column 4). In addition, OVCAR(HER2-) cell viability was not affected by any EV population after short-term exposure (6 h) ( Figure 4B ).

[0214] Figure 4C The effects of EVs isolated by method 1 and method 2 from samples 1 and 2, which were used fresh (within 24 h of isolation) or after storage at −80 °C, are summarized. The percentage of apoptotic effects induced by these EVs is shown in the left panel for SKOV HER2+ cells and in the right panel for OVCAR HER2- cells. Only EVs purified by method 1 from sample 1 (N29 superimposed on SKOV (HER2+)) induced statistically significant apoptosis in SKOV cells, either fresh (p-value < 0.001) or frozen and thawed (p-value < 0.05), compared to all other EV cell populations. On average, approximately 60% of cells were killed after 6 h of exposure to EVs purified by method 1 from sample 1. EVs purified by method 2 from sample 1 and EVs obtained from sample 2 (N29 CAR T cells incubated with non-target OVCAR cells (regardless of purification method)) showed low toxicity, comparable to the control (no EV). No significant apoptotic effect on OVCAR (HER2-negative cells) was observed for any type of tested EVs. Image analysis was performed by Image J software.

[0215] Figure 4D Representative apoptotic effects of different EV populations (EV 50 μg / well and 25 μg / well) on SKOV Her2+ cells (left) and OVCAR Her2- cells (right) are summarized.

[0216] Only EVs purified from sample 1 using method 1 induced a significant apoptosis rate (approximately 60%) compared to all other EV populations.

[0217] Figure 4E The apoptotic effects of 8 different EV samples obtained by method 1 and method 2 (EV 50 μg / well and 25 μg / well) on SKOVHER2+ cells were summarized.

[0218] Example 5. Cytotoxicity of EVs

[0219] Study Design

[0220] EVs were obtained from T cells transfected with N29 or anti-CD19 CARs. Some CAR T cells were activated with SKOV or Raji cells before EV generation. EVs were purified using method 1. According to Table 4, ovarian cancer SKOV cells were exposed to different samples containing EVs.

[0221] Table 4. Study design

[0222]

[0223] * UT – untransduced;

[0224] SKOV cells were seeded in 96-well plates (10,000 cells / well) and exposed to EV populations obtained from similar numbers of cells for 48 h. After 20 and 40 h of exposure, cells were observed and photographed by optical microscopy (x10, x20) and fluorescence microscopy. The results are shown in Figures 5 (20 h incubation) and 6 (40 h incubation). The morphological changes of SKOV cells can be seen in Figure 5A and Fig. 6A As can be clearly seen in the figure, SKOV cells were exposed to EVs obtained from N29 CART cells activated by SKOV cells for 20 and 40 hours, respectively. Cytotoxic effects included morphological dispersion of cell gap junctions, cell elongation, and cell death. When SKOV cells were exposed to EVs derived from anti-CD19 CAR T cells ( Figure 5B , 5D and 6B), EVs derived from N29 CAR T cells were exposed to nonspecific antigens ( Figure 5C ), nor when EVs derived from N29 CAR T cells were exposed to nonspecific antigens and added to HER2-negative MDA231 cells ( Figure 6C). As can be seen, only EVs from N29 CAR T cells stimulated with a specific antigen, i.e., cells expressing HER2, were cytotoxic to SKOV cells. In contrast, EVs derived from CAR T cells stimulated with a nonspecific antigen showed no cytotoxic effect.

[0225] Example 6. Cytotoxicity of EVs

[0226] SKOV cells were incubated with EVs derived from activated GFP-N29 CAR T cells for 40 h. Overlay of fluorescent and bright field images showed that EVs containing green N29 CARs specifically penetrated into ovarian cancer (SKOV) and stained recipient cells green (data not shown).

[0227] Cell viability / proliferation was measured by the XTT method after 48 h. A significant decrease in cellular metabolism was found in cells exposed to EVs obtained from N29 CAR T cells incubated with SKOVs compared to cells exposed to EVs obtained from untransduced T cells ( Fig.13 )

[0228] In addition, EV cytotoxicity can The assay quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. EVs obtained from N29 CAR T cells activated with MDA-231-HER2-positive breast cancer cells induced 60% and 33% killing of MDA-231-HER2-positive breast cancer cells (in a dose-response manner). In contrast, EVs obtained from UT T cells incubated with MDA-231HER2-positive breast cancer cells induced only 3% killing ( Fig.14 ).

[0229] Example 7

[0230] EVs from samples 1-4 isolated by 5 different centrifugation methods 1-5 were further tested for cytotoxicity against SKOV cells by measuring caspase 3 / 7 activity. SKOV cells were exposed to 2 different concentrations (25 or 50 μg / well) of the different EV populations and recorded every 3 hours for the first 2 days and the following 4 days. The results are shown in Figure 7. Figure 7 shows phase images and colocalization with fluorescent caspase 3 / 7 activity dye and cytotoxic reagent labeled cells (dead cells counted), recorded in white dots. Fig. 7A The effect of EV obtained by methods 1, 3 and 5 is shown. Figure 7B The effect of EV obtained by methods 2 and 4 is shown.

[0231] After 4 days of exposure to 50 μg / well and 25 μg / well EVs obtained from sample 1 (N29 superimposed with SKOV) by methods 1, 3, and 5 (low centrifugal force), a large number of apoptotic cells and incorporation of caspase 3 / 7 activity dyes were found in cells, but not in cells exposed to EVs from sample 2 (N29 superimposed with OVCAR) isolated by the same methods (methods 1, 3, and 5). EVs purified from sample 1 by methods 2 or 4 only caused moderate apoptosis rates. EVs purified from sample 2 by methods 2 or 4 did not affect cell viability. Staurosporine, an ATP-competitive kinase inhibitor, was used to induce apoptosis and served as a positive control for cytotoxic effects.

[0232] In addition, as can be seen in Figure 8, morphological changes were observed in SKOV cells after exposure to EVs from sample 1, which were obtained by 3 low-force centrifugation methods (methods 1, 3, and 5). Fig. 8A , C, and E) and after exposure to low doses of these EVs, cells became elongated, non-adherent apoptotic rounded shapes with large gaps between cells ( Fig.8F , H and J).

[0233] EVs obtained from sample 1 by both vigorous centrifugation methods (methods 2 and 4 at both doses) had only a moderate effect on SKOV Her2+ cells (EV 50 μg / well: Figure 8B and D; EV 25 μg / well: Figure 8G and I) apoptosis rates similar to those induced by staurosporine ( Figure 8U ).

[0234] Cells exposed to EVs isolated by the same method from sample 2 (N29 superposed with OVCAR) did not show this morphological change; cells proliferated and covered 100% of the well area (EV 50 μg / well: Figure 8K -O; EV 25 μg / well: Figure 8P -T).

[0235] In contrast, minimal effects were seen on OVCAR cell morphology (Figure 9). Figure 9U ), OVCAR HER2-negative cells exposed to EVs isolated from sample 1 using the five methods induced small intercellular gaps ( Fig.9A -J). Exposure of cells to EVs obtained from sample 2 by the five methods did not affect the cells and they showed extensive proliferation ( Figure 9K -T).

[0236] Kinetics of incorporation of fluorescent caspase 3 / 7 activity marker in SKOV cells after exposure to EVs obtained from samples 1 and 2 using methods 1, 3, and 5. Fig.10 As shown. It clearly shows that the high dose (50 μg / well) EVs obtained from sample 1 by method 1 induced the highest incorporation of caspase 3 / 7 activity markers. These EVs at a lower dose (25 μg / well) and the two doses of EVs obtained by methods 3 and 5 also showed high caspase 3 / 7 activity marker incorporation, which indicates the rate of cell apoptosis. In contrast, high-dose EVs isolated from sample 2 (N29 superimposed on OVCAR) by methods 1 or 3 induced moderate incorporation of caspase 3 / 7 activity markers. In addition, low concentrations of EVs obtained from sample 2 by methods 1 or 3, and any concentrations of EVs obtained from sample 2 (N29 superimposed on OVCAR) by methods 2 or 4 did not affect SKOV cells and did not induce caspase 3 / 7 activity.

[0237] Summary of results

[0238] Summarizing all the presented results, it can be clearly seen that only the EV populations from sample 1 (N29 superimposed on SKOV) and purified by methods 1, 3 and 5 have a strong toxic (apoptotic) effect on HER2-positive cells (e.g. ovarian cancer cells) and HER2-positive breast cancer cells (MDA231). These populations contain a high content of large EVs, with at least 25% of the EVs having a size of 150 nm. The best results were obtained with EVs purified from sample 1 by method 1. At least 30% of these EVs had a size greater than 150 nm. It can also be seen that the EVs obtained by methods 2 and 4, which contain much smaller EVs (in fact, mainly exosomes), do not produce high apoptotic rates and are definitely not at the same level as the samples with a high content of large EVs.

[0239] Example 8. Scale-up of production.

[0240] To increase the yield of EVs, we use different bioreactor systems. Hollow fiber bioreactors (HFBRs) are increasingly used for EV production. In these dynamic settings, cells are expanded on cylindrical hollow fibers, which can accommodate 100 times more cells than a regular T-flask (M. Lu, Eur. J. Pharm. Biopharm. 2017). Alternative bioreactors that we use are the Quantum Bioreactor Culture System (Terumo BCT) (Mendt et al., JCI Insight. 2018; 3(8): e99263.2018) and the Sartorius Benchtop Bioreactor System.

[0241] Example 9. CAR-T activation

[0242] To facilitate the stimulation of CAR-T cells to make "off-the-shelf" activated EVs derived from CAR-T cells, we stimulated CAR-T cells with antigens coated onto tissue culture plates. This overcomes the need to use target cells, culture them, and side effects that may be caused by remnants or residual components of these target cells. Several binding protocols were tested to enhance the accessibility of antigens to CAR T to form an immune synapse.

[0243] Example 10

[0244] The efficacy of the EVs of the present invention in treating cancer in vivo was tested in blood and solid tumor models. Cancer cell lines were injected into immunodeficient mice. For solid tumor models, we injected cell lines derived from ovarian cancer (SKOV, OVCAR) intraperitoneally or subcutaneously. Alternatively, we used a pancreatic tumor model by subcutaneous or orthotopic injection of pancreatic cancer cells (Capan). Cell lines derived from breast cancer (MDA-MB-231) were injected subcutaneously. For blood cancer models, we injected cell lines derived from lymphoma intravenously.

[0245] EV samples 1-8 were purified from activated CAR T cell cultures by method 11 (20,000g, 60 minutes) or 5 (10,000g, 180 minutes). EV samples were labeled with fluorescent dyes (such as XenoLight DiR) as previously described (Ohno S. Molecular Therapy 2013). Labeled EVs (4-100 μg) were injected intravenously or intratumorally into mice carrying transplanted tumor cells twice a week for 4 weeks. EV location and tumor size were monitored using IVIS 12 and 24 hours after each injection. At the end of 4 weeks, brain, heart, spleen, liver, lung, kidney, small intestine, colon, and tumor tissues were harvested for pathological verification.

[0246] Although the present invention has been described above by way of its preferred embodiments, modifications may be made thereto without departing from the spirit and essence of the present invention as defined in the claims. Sequence Listing <110> Ichilov Technology Co., Ltd. YEDA RESEARCH AND DEVELOPMENT CO., LTD. <120> Extracellular vesicles from activated CAR-T cells <130> SMC-017 PCT <150> IL266153 <151> 2019-04-18 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg 1 5 10 15 Ile Ser Val Thr Cys Lys Ala Ser Gln Asp Val Gly Pro Asn Val Ala 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser 35 40 45 Ala Ser Tyr Leu Tyr Asn Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 50 55 60 Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Asn Val Gln Ser Asp Asp 65 70 75 80 Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Thr Tyr Pro Phe Thr Phe 85 90 95 Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 2 Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Val Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Val Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Thr Ser Tyr Tyr Asp Tyr Asp Lys Val Leu Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Lys Gly 115 120 <210> 3 <211> 387 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 3 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Asp Ile Val Met Thr Gln Ser Pro Lys Phe 20 25 30 Met Ser Thr Ser Val Gly Asp Arg Ile Ser Val Thr Cys Lys Ala Ser 35 40 45 Gln Asp Val Gly Pro Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ser Pro Lys Pro Leu Ile Tyr Ser Ala Ser Tyr Leu Tyr Tyr Gly Val 65 70 75 80 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr 85 90 95 Ile Ser Asn Val Gln Ser Asp Asp Leu Ala Glu Tyr Phe Cys Gln Gln 100 105 110 Tyr Asn Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly Glu 130 135 140 Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser 145 150 155 160 Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Val 180 185 190 Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Val Asp Ser 195 200 205 Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met Leu 210 215 220 Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr 225 230 235 240 Cys Val Thr Ser Tyr Tyr Asp Tyr Asp Lys Val Leu Phe Ala Tyr Trp 245 250 255 Gly Gln Gly Thr Thr Val Thr Val Lys Gly Lys His Leu Cys Pro Ser 260 265 270 Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val 275 280 285 Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile 290 295 300 Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 305 310 315 320 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys Tyr Gln 325 330 335 Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg 340 345 350 Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly 355 360 365 Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys 370 375 380 Pro Pro Gln 385 <210> 4 <211> 1177 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 4 atggatttc aggtgcagat ttcagcttc ctgctaatca gtgcctcagt cataatgtct 60 agaggagata ttgtgatgac ccagtctcca aaattcatgt ccacatcagt aggagacagg 120 atcagcgtca cctgcaaggc cagtcagat gtgggtccta atgtagcctg gtatcacag 180 aaaccagggc aatctcctaa accactgatt tactcggcat cctacctata ttatggagtc 240 cctgatcgct tcacaggcag tggatctggg acagatttct ctctcaccat cagcaatgtg 300 cagtctgatg acttggcaga gtatttctgt cagcaatata acacctatcc gttcacgttc 360 ggaggggca ccaagctgga aatcaaaggg tcgacttccg gtagcggcaa atcctctgaa 420 ggcaaaggtg aggtgcagct ggaggagtct ggtggaggat tggtgcagcc taaagggtca 480 ttgaaactct catgtgcagc ctctggattc accttcaata cctacgccat gaactgggtc 540 cgccaggctc caggaaaggg tttggaatgg attgttcgca taagaagtaa aagtaataat 600 tatgcaacat attatgcga ttcagtgaaa gacaggttca ccatctccag agatgattca 660 caaagcatgc tctatctgca aatgaacaac ttgaaaactg aggacacagc catgtattac 720 tgtgtgactt cttactatga ttacgacaag gtcctgtttg cttactgggg ccaaggaact 780 acggtcaccg tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag 840 cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 900 gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 960 atgaacatga ctccccgccg cccggggccc acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tagatctcaa gtgcgaaagg cagctataac cagctatgag aaatcagatg gtgtttacac gggcctgagc accaggaacc aggagactta cgagactctg 1177. aagcatgaga aaccaccaca gtagctttag actcgag

Claims

1. Isolated activated extracellular vesicles (denoted as activated EVs) derived from stimulated T cells expressing a chimeric antigen receptor (denoted as stimulated CAR T cells), wherein the activated EVs are derived from stimulated anti-HER2 CAR T cells, the EVs have a particle size of 30 nm to 1000 nm, and at least 25% of the EVs have a particle size greater than 150 nm; The activated EV is prepared by a method comprising the following steps: (1) incubating CAR T cells with a tumor-associated antigen that specifically binds to an anti-HER2 CAR in a cell culture medium under conditions that can activate T cells; (2) separating the CAR T cells from the culture medium to obtain a culture medium containing activated EVs; and (3) separating the activated EVs from the culture medium by centrifugation at 8,000 × g to 30,000 × g for 0.5 to 4 hours.

2. The activated EVs of claim 1, wherein at least 30% of the EVs have a particle size greater than 150 nm.

3. The activated EV according to claim 1, wherein the average size of the EV is at least 140 nm.

4. The activated EV according to claim 3, wherein the average size of the EV is at least 160 nm.

5. The activated EV of claim 1, wherein the EV presents a chimeric antigen receptor (CAR) of an activated CAR T cell.

6. The activated EV according to claim 1, wherein the anti-HER2 CAR is N29 CAR, wherein N29 CAR comprises three complementarity determining regions (CDRs) of a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and three CDRs of a heavy chain variable region having an amino acid sequence of SEQ ID NO:

2.

7. The activated EV according to claim 6, wherein the anti-HER2 CAR is N29 CAR, wherein N29 CAR comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:

2.

8. The activated EV according to claim 7, wherein the anti-HER2 CAR comprises the amino acid sequence SEQ ID NO:

3.

9. The activated EV according to claim 6, wherein the EV is derived from N29CAR T cells stimulated by HER2 or cells expressing HER2.

10. The activated EV according to claim 9, wherein the cells expressing HER2 are selected from ovarian cancer cells, breast cancer cells and primary cells of HER2-positive cancer.

11. The activated EV according to any one of claims 1 to 10, wherein the EV is a cytotoxic EV.

12. The activated EV of claim 1, wherein the EV has at least one of the following characteristics: (i) at least 10% of the EVs express CD38 antigen; (ii) at least 20% of the EVs express CD3 antigen; or (iii) at least 20% of the EVs express HLA-RD antigen.

13. The activated EV according to claim 1, further comprising an anticancer agent.

14. The activated EV according to claim 1, which does not contain exogenous anti-cancer agents.

15. A pharmaceutical composition comprising the isolated activated EV according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition according to claim 15, comprising the EV as the only anti-cancer agent.

17. The pharmaceutical composition of claim 16, further comprising an additional anticancer agent.

18. The pharmaceutical composition according to any one of claims 15 to 17, which is formulated as an injection preparation.

19. Use of the isolated activated EV according to any one of claims 1 to 14 in the preparation of a medicament for treating cancer, wherein the cells of the cancer present an antigen that is specifically bound by an anti-HER2 CAR.

20. The use according to claim 19, wherein the EV is derived from a T cell comprising an anti-HER2 CAR and the cancer is a HER2-positive cancer.

21. The use according to claim 20, wherein the HER2-positive cancer is selected from ovarian cancer and breast cancer.

22. The use according to claim 19 or 20, wherein the anti-HER2 CAR is N29 CAR; wherein (i) the N29 CAR comprises 3 complementarity determining regions (CDRs) of a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and 3 CDRs of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2; or (ii) the N29 CAR comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2; or (iii) the N29 CAR comprises an amino acid sequence of SEQ ID NO:

3.

23. The use according to claim 19, wherein the medicament is administered systemically or intratumorally.

24. The use according to claim 19, wherein the medicament comprises the EV as the only anticancer agent.

25. The use according to claim 19, wherein the medicament further comprises an additional anti-cancer agent.

26. The use according to claim 19, wherein the medicament is formulated as an injection preparation.

27. A method for preparing the isolated activated extracellular vesicles according to any one of claims 1 to 14, wherein the isolated activated extracellular vesicles are derived from activated anti-HER2 CAR T cells, the method comprising: (1) incubating CAR T cells with tumor-associated antigens that specifically bind to CAR in a cell culture medium under conditions that can activate T cells; (2) separating CAR T cells from cell culture medium; and (3) isolating the obtained activated extracellular vesicles, wherein the separation comprises centrifugation consisting of low-force centrifugation, wherein the low-force centrifugation comprises centrifugation at 8,000×g to 30,000×g for 0.5 to 4 hours, thereby obtaining isolated activated EVs, wherein at least 25% of the EVs have a size of 150 nm or greater.

28. The method of claim 27, wherein the separation is achieved by centrifugation at 8,000 x g to 15,000 x g for 0.5 to 3 hours.

29. The method of claim 28, wherein the separation is achieved by centrifugation at 15,000 x g to 25,000 x g for 0.5 to 1.5 hours.

30. The method according to any one of claims 27 to 29, wherein the incubation in step (1) comprises incubation for 6 to 96 hours.

31. The method according to any one of claims 27 to 29, wherein the incubation in step (1) comprises incubating the CAR T cells with cells or surfaces presenting tumor-associated antigens to which the CAR specifically binds.

32. The method of claim 30, wherein the incubation in step (1) comprises incubating the CAR T cells with cells or surfaces that present a tumor-associated antigen to which the CAR specifically binds.

33. The method according to any one of claims 27 to 29, wherein step (2) comprises step (2ii), step (2ii) comprising centrifuging the culture medium of the previous step at 1000 g to 3000 g for 10 to 60 minutes and separating the precipitate from the culture medium.

34. The method according to claim 30, wherein step (2) comprises step (2ii), step (2ii) comprising centrifuging the culture medium of the previous step at 1000 g to 3000 g for 10 to 60 minutes and separating the precipitate from the culture medium.

35. The method according to claim 31, wherein step (2) comprises step (2ii), step (2ii) comprising centrifuging the culture medium of the previous step at 1000g to 3000g for 10 to 60 minutes and separating the precipitate from the culture medium.

36. The method according to claim 32, wherein step (2) comprises step (2ii), step (2ii) comprising centrifuging the culture medium of the previous step at 1000 g to 3000 g for 10 to 60 minutes and separating the precipitate from the culture medium.

37. The method of claim 32, wherein the method further comprises a step (2i) before step (2ii), wherein step (2i) comprises centrifuging the culture medium with activated T cells from step (1) at 200 g to 600 g for 5 to 60 minutes and separating the precipitate from the culture medium.

38. A method according to claim 27, wherein the CAR T cell is an N29 CAR T cell and the tumor-associated antigen is HER2, wherein (i) the N29 CAR comprises 3 complementarity determining regions (CDRs) of a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and 3 CDRs of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2; or (ii) the N29 CAR comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2; or (iii) the N29 CAR comprises an amino acid sequence of SEQ ID NO:

3.

39. The method of claim 38, comprising incubating N29 CAR T cells with ovarian cancer cells presenting HER2 for 18 to 36 hours and isolating the obtained activated extracellular vesicles.

40. The method of claim 27, wherein the method further comprises washing the obtained EVs.

41. The method of claim 27, wherein the method further comprises freezing the EV.

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